CN1293711A - Genes encoding MLO proteins and conferring fungal resistance upon plants - Google Patents

Abstract

This invention describes genes encoding proteins which control resistance of plants to fungal pathogens. The invention also describes transgenic plants resistant to fungal pathogens and methods for making plants resistant to fungal pathogens. The invention further discloses a method to isolate additional genes coding for additional proteins controlling the resistance of plants to fungal pathogens.

Description

The gene that encodes the MLO protein and confers resistance to the plant against the fungus

The present invention describes nucleotide sequences encoding proteins that control plant resistance to fungal diseases. The invention also relates to plants resistant to fungal diseases and to methods for the preparation of plants resistant to fungal diseases.

Fungal diseases cause an estimated $9.1 billion in annual crop losses in the United States, and they are caused by a variety of biologically diverse pathogens. Different methods have traditionally been used to control them. Agriculturally important resistant varieties have been bred to provide multi-level resistance against narrow or larger ranges of pathogen isolates or varieties. However, this work involves the long and laborious process of introducing desired traits into commercial lines through genetic crossing, and due to the risk that pests may evolve to overcome natural plant resistance, continued efforts are required to introduce new resistance traits into commercial products in the chemical line. In addition, fungal diseases have been controlled through the application of chemical fungicides. This approach often yields effective control, but is also associated with the possible development of resistant pathogens and can have negative environmental impacts. Furthermore, in certain crops, such as barley and wheat, it is difficult or impractical to control fungal pathogens with chemical fungicides. Current technologies have enabled a better understanding of the interactions between plants and their pathogens at the molecular level and have partially revealed resistance mechanisms. Much of this molecular characterization work has been carried out on the model plant Arabidopsis thaliana, and has now begun to elucidate resistance mechanisms in commercially important crops.

Powdery mildew is a major disease affecting most plant species and has been extensively studied. They are characterized by white to grayish spots or flakes that grow on plant tissue, corresponding to the fungal mycelium and ocystosis. Powdery mildew is caused by several fungi of the order Erysiphales. For example, Erysiphe graminis causes powdery mildew of cereals and grasses. Although powdery mildew is difficult to control in most crops, there are some barley lines that are resistant to most isolates of the known pathogen. Research work has shown that mutations at a single site, the mlo site, are associated with resistance phenotypes. The mechanism of Mlo resistance has been partially elucidated; it involves the formation of large cell wall appositions called papillae at sites of contact with the pathogen, which contain mainly callose but also carbohydrates, phenols and proteins. In mlo plants, cell wall extrasomes block pathogen entry and thus confer resistance.

Unfortunately, this powerful tool for controlling powdery mildew is limited to barley. Given the problems caused by fungal diseases in agriculture, especially powdery mildew, it appears that there is still a need for new effective methods of controlling these types of pathogens in other crops that are economically attractive to farmers and environmentally acceptable.

The present invention addresses the need for new methods of plant disease control through genetic engineering techniques. More particularly, the present invention relates to methods of controlling powdery mildew, preferably powdery mildew in economically important crops.

The present invention relates to isolated DNA molecules encoding Mlo proteins which confer resistance in plants against fungal pathogens. More specifically, the present invention relates to an Mlo protein comprising a conserved amino acid sequence discovered for the first time by the inventors of the present invention, and to an isolated DNA molecule encoding the Mlo protein. The invention also describes vectors for expressing the DNA molecules of the invention in plants. The invention further relates to transgenic plants comprising any of the DNA molecules of the invention. The invention also describes agricultural products having improved phytosanitary properties, including transgenic plants resistant to fungal pathogens by expression of any of the DNA molecules of the invention. The present invention still further relates to a method of producing plants resistant to fungal diseases by altering the expression in transgenic plants of a protein encoded by an endogenous gene copy corresponding to any one of the DNA molecules of the present invention, or by altering the The activity or stability of the protein encoded by the endogenous gene copy of any DNA molecule of the present invention is carried out. Such transgenic plants are expected to be resistant to pathogens infecting living epidermal cells of plants, especially fungi from the order Erysiphales (also known as Erysiphe), preferably against fungi of the genus Erysiphe that cause powdery mildew, more preferably against Plants of Erysipha graminearum. The present invention further describes the method of isolating the encoded protein having the same or similar function as the protein encoded by the DNA molecule of the present invention and encoding the conserved amino acid sequence listed in the present invention.

The present invention thus provides new and effective methods of controlling fungal diseases in economically important crops, making it possible to reduce the amount of chemicals used on the crops and reduce the risk of emergence of pathogens resistant to the control agents.

The present invention thus provides:

A DNA molecule encoding the Mlo protein that confers resistance to fungal pathogens in plants, wherein said protein comprises at least one amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein Said DNA molecules are preferably cDNA molecules. In a preferred embodiment, the DNA molecule is preferably not from barley but from a dicotyledonous plant or the following plants: wheat, maize, rice, oats, rye, sorghum, sugar cane, millet, milo and palmaceae. In a preferred embodiment, the DNA molecule of the present invention is identical or substantially similar to any one of the nucleotide sequences listed in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or the encoding is identical to that of SEQ ID NO: 7. A Mlo protein that is identical or substantially similar to the Mlo protein listed in ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In a more preferred embodiment, the DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is from wheat. In another preferred embodiment, the DNA molecule of the present invention is combined with any one of listed in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 The nucleotide sequence is identical or substantially similar, or the encoding is any one of the nucleotide sequences listed in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 Mlo proteins that encode the same or substantially similar Mlo proteins. In a more preferred embodiment, the DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 is from Arabidopsis mustard. In another preferred embodiment, the aforementioned DNA molecule is modified such that the activity of the endogenous protein is lost. In a particular embodiment of the invention, said DNA modification results in one, all or a combination of the following changes in the amino acid sequence of the corresponding protein:

- tryptophan (163) changed to arginine

- Proline (396) post-frameshift

-Tryptophan (160) post-frameshift

- Methionine (1) changed to Isoleucine

- Glycine (227) changed to aspartic acid

-Methionine (1) changed to valine

- Arginine (11) changed to tryptophan

- loss of phenylalanine (183), threonine (184)

- Change of valine (31) to glutamic acid

- Serine (32) changed to phenylalanine

- Leucine (271) was changed to histidine.

In a further preferred embodiment, the fungal pathogen preferably infects living epidermal cells, more preferably the fungal pathogen is from the order Erysiphales (also known as Erysiphales), especially from the genus Erysipha, most preferably the fungal pathogen is Erysipha graminis.

In another embodiment, the isolated DNA molecule is a molecule that is antisense to the isolated molecule described above, such as encoding at least one amino acid sequence identical to or substantially similar to that set forth in SEQ ID NO: 1 or SEQ ID NO: 2. A DNA molecule of amino acid sequence Mlo protein, such as a molecule antisense to a cDNA molecule, in particular identical or substantially similar to the DNA molecule shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17, And the encoded Mlo protein is an antisense DNA molecule that is identical or substantially similar to the Mlo protein listed in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18.

The present invention further provides:

A protein comprising at least one amino acid sequence identical or substantially similar to that set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein said protein is an Mlo protein and confers resistance to fungal pathogens in plants. The protein is preferably not from barley, but from a dicotyledonous plant or the following plants: wheat, maize, rice, oats, rye, sorghum, sugar cane, millet, milo and palmaceae. In a preferred embodiment, the protein of the present invention consists of a nucleotide sequence identical or substantially similar to any one of the nucleotide sequences listed in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 Coding, or identical or substantially similar to any one of the Mlo proteins listed in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In a more preferred embodiment, said protein is from wheat. In another preferred embodiment, the protein of the present invention is composed of any one of listed in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 Nucleotide sequence codes with identical or substantially similar nucleotide sequences, or any of those listed in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18 The Mlo proteins are identical or substantially similar. In a more preferred embodiment, the protein is from Arabidopsis. In another preferred embodiment, it is preferred that the fungal pathogen infects living epidermal cells, more preferably the fungal pathogen is from the order Erysiphales (also known as Erysiphales), especially from the genus Erysipha, most preferably the fungal pathogen is Erysiphe graminis. In a further preferred embodiment, the present invention also includes mutant or truncated forms of the proteins encoded by any of the DNA molecules described above.

The present invention further provides:

An expression cassette comprising any of the aforementioned DNA molecules, such as the aforementioned cDNA, wherein said DNA molecule is operably linked to a promoter and a termination signal capable of expressing said DNA molecule in a plant. In preferred embodiments, said expression cassette is heterologous. In more preferred embodiments, the promoter and termination signal are eukaryotic. In a more preferred embodiment, the promoter and termination signal are heterologous with respect to the coding region.

The present invention also provides:

A vector containing any of the above expression cassettes. In a preferred embodiment, the vector is used for the transformation of said expression cassette in plants. In another preferred embodiment, the vector of the present invention is used for the amplification of any one of the aforementioned DNA molecules.

The present invention also provides:

A cell comprising an expression cassette or portion thereof comprising an isolated DNA molecule of the invention, wherein the DNA molecule within said expression cassette is expressible in said cell. In a preferred embodiment, the DNA molecule is not from barley. In another preferred embodiment, the cell is a plant cell. In more preferred embodiments, the expression cassette is stably integrated into the genome of the cell, or contained within an autonomously replicating vector and retained in the cell as an extrachromosomal molecule.

The present invention further provides:

A plant comprising an expression cassette or part thereof having an isolated DNA molecule of the invention. In a preferred embodiment, the DNA molecule is not from barley. In another preferred embodiment, the DNA molecule contained within the expression cassette is expressible in plants. In another preferred embodiment, said DNA molecule is stably integrated into the plant genome, or contained within a self-replicating vector and retained in the cell as an extrachromosomal molecule. In another preferred embodiment, the plant is resistant to a fungal pathogen, preferably a fungal pathogen that infects living epidermal cells, more preferably a fungal pathogen from the order Erysiphales (also known as Erysiphales), especially from the genus Erysipha, most preferably The fungal pathogen is Erysiphe graminis.

The invention also relates to seeds of said plants, wherein the seeds have optionally been treated (eg contacted or coated) and/or packaged, eg placed in a bag with instructions for use.

The present invention also provides:

Agricultural products including plants comprising the isolated DNA molecules of the invention. In a preferred embodiment, said agricultural product is used as feed, food or silage, free of mycotoxins such as aflatoxins produced by fungal pathogens. Thus, said agricultural product has improved phytosanitary properties.

The present invention further provides:

A method for preparing plants resistant to fungal pathogens, comprising the steps of:

a) expressing in a plant an RNA transcript encoded by any of the DNA molecules described above in a "sense" orientation; or

b) express in plants an RNA transcript encoded by any of the above DNA molecules in an "antisense" orientation; or

c) expressing in a plant a ribozyme capable of specifically cleaving a messenger RNA transcript encoded by an endogenous gene corresponding to any of the aforementioned DNA molecules; or

d) express in a plant an aptamer specific for an endogenous protein encoded by a gene corresponding to any one of the aforementioned DNA molecules; or

e) expressing in plants a mutated or truncated form of any of the aforementioned DNA molecules, thereby enabling it to become a dominant negative mutant; or

f) modifying in a plant by homologous recombination at least one chromosomal copy of a gene corresponding to any of the DNA molecules described above; or

g) modifying in a plant by homologous recombination at least one chromosomal copy of a regulatory element of a gene corresponding to any one of the DNA molecules described above.

The present invention further provides:

Plants obtained by any of the above methods, including seeds of said plants, wherein the seeds have optionally been treated (e.g. contacted or coated) and/or packaged, e.g. placed in a bag with Instructions for use. In another preferred embodiment, the plants obtained are resistant to fungal pathogens, preferably fungal pathogens infecting living epidermal cells, more preferably fungal pathogens from the order Erysiphales (also known as Erysiphales), especially from the genus Erysipha , most preferably the fungal pathogen is Erysiphe graminis.

The present invention also provides:

Agricultural products with improved phytosanitary properties obtained by any of the above methods.

The present invention further provides:

A method for isolating a DNA molecule encoding an Mlo protein, comprising the steps of:

a) combining a degenerate oligonucleotide encoding at least 6 amino acids in SEQ ID NO: 1 and a degenerate oligonucleotide complementary to a sequence encoding at least 6 amino acids in SEQ ID NO: 2 with DNA extracted from a plant , mixing under conditions that allow said degenerate oligonucleotides to hybridize to said DNA;

b) amplifying a DNA fragment of said plant DNA, wherein said DNA fragment contains a nucleotide sequence capable of annealing to said degenerate oligonucleotide in step a) at its left and right ends; and

c) Obtaining a full-length cDNA clone containing the DNA fragment of step b).

The present invention also provides:

A method of producing mutated copies of the nucleotide sequences of the invention by "in vitro recombination" or "DNA rearrangement". Mutated copies of the nucleotide sequences of the invention can be used to confer greater resistance to fungal pathogens. In a preferred embodiment, mutated copies of the nucleotide sequences of the invention are used to render plants resistant to a wider range of pathogens. One such method is described as follows:

A method for mutagenizing a DNA molecule of the present invention, wherein said DNA molecule has been cut into double-stranded random fragments of expected size, the method comprising the following steps:

a) adding to the resulting population of double-stranded random fragments one or more single-stranded or double-stranded oligonucleotides, wherein said oligonucleotides comprise regions identical to those of the double-stranded template polynucleotide and regions that are heterologous;

b) denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;

c) incubating the resulting population of single-stranded fragments with a polymerase under conditions that cause said single-stranded fragments to anneal at said identical region sufficient for one member of the pair to form an annealed fragment pair triggering the replication of another member, thereby forming a mutagenized double-stranded polynucleotide; and

d) repeating the second and third steps for at least two more cycles, wherein the mixture produced by the second step in the next cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the next cycle A further mutagenized double-stranded polynucleotide is formed.

definition

An "isolated DNA molecule" is a nucleotide sequence that has been removed from its natural environment by human manipulation and is therefore not a natural product. An isolated nucleotide sequence may exist in purified form, or in a non-native environment such as a transgenic host cell.

A "protein" as defined herein is the complete protein encoded by the corresponding nucleotide sequence, or a protein part encoded by the corresponding part of the nucleotide sequence.

An "isolated protein" is a protein encoded by an isolated nucleotide sequence and thus is not a natural product. An isolated protein may be present in purified form, or in a non-native environment, such as a transgenic host cell, where the protein is not normally expressed or is expressed in a different form or in a different amount than in an isogenic, non-transgenic host cell.

A plant that is "resistant to a fungal pathogen" exhibits no or fewer symptoms of fungal infection caused by the fungus by inhibiting or limiting the ability of the fungal pathogen to grow on the plant. As a result the plants grow better, have higher yields and produce more seeds.

"Protein conferring resistance to fungal pathogens in plants" means proteins involved in the regulation of plant genetic pathways responsible for the resistance of plants to fungal pathogens. The protein may be a positive regulator in which the plant's resistance to the fungal pathogen is enhanced, or the protein may be a negative regulator in which the plant's resistance to the fungal pathogen is suppressed. A special case of a protein that confers resistance to fungal pathogens in plants is the Mlo protein.

"Mlo protein" herein refers to members of a family of proteins (Mlo family) with substantially similar functions and some structural homology in disease resistance pathways. Structural homology can be, eg, that the family members have at least one conserved region.

In the broadest sense, the term "substantially similar" when applied to nucleotide sequences refers to a nucleotide sequence equivalent to a reference nucleotide sequence, wherein the equivalent sequence encodes the same sequence as the reference nucleotide sequence. A polypeptide having substantially the same structure and function, eg, only changes in amino acids that do not affect the function of the polypeptide. Preferably, substantially similar nucleotide sequences encode polypeptides encoded by the reference nucleotide sequence. The percent identity between the substantially similar nucleotide sequence and the reference nucleotide sequence is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99% %.

The term "substantially similar" when used herein for a protein means a protein that is comparable to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, eg, only amino acids that do not affect the function of the polypeptide are changed. When applied to protein or amino acid sequences, the percent identity between a substantially similar protein or amino acid sequence and a reference protein or amino acid sequence is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.

The percent sequence identity is determined using computer software based on a kinetic programming algorithm. Preferred computer software within the scope of the present invention includes BLAST (Basic Local Alignment Search Tool) search software, which is designed to search all available sequence databases regardless of whether the query is protein or DNA. The BLAST 2.0 version of this search tool (Notched BLAST) has been published on the Internet (currently http:∥www.ncbi.nlm.nih.gov/BLAST/). It uses a progressive algorithm that employs partial sequence alignments rather than full sequence alignments, thereby enabling the detection of relationships between sequences that share only discrete regions. Scores assigned in BLAST searches have clear statistical interpretations. The software is preferably run with optional parameters set to default values.

The term "gene" refers to a coding sequence and associated regulatory sequences, wherein the coding sequence is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Additional elements that may be present are, for example, introns.

"Expression" refers to the transcription and/or translation of an endogenous or transgene in a plant. For example, in the case of an antisense construct, expression may simply refer to transcription of the antisense DNA.

"Expression cassette" is used herein to mean a DNA sequence capable of directing the expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest in combination with a termination signal active connection. It usually also contains sequences required for correct translation of the nucleotide sequence. The coding region usually encodes a protein of interest, but may also encode a functional RNA of interest, such as an antisense RNA or an untranslated RNA, such as an antisense RNA, that inhibits expression of a particular gene in a sense or antisense orientation. An expression cassette containing a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to another. An expression cassette may also be one that occurs in nature but has been obtained in a recombinant form useful for heterologous expression. However, generally speaking, the expression cassette is heterologous to the host, that is, the specific DNA sequence in the expression cassette does not naturally exist in the host cell, and must be introduced into the host cell or host cell precursor by transformation. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to certain specific external stimuli. In the case of multicellular organisms, such as plants, the promoter may also be specific for a particular tissue or organ or developmental stage.

"Heterologous" as used herein means "of a different natural or synthetic origin" or to represent a non-natural state. For example, if a host cell is transformed with a nucleic acid sequence from another organism, especially another species, the gene is heterologous to the host cell and also heterologous to the progeny of the host cell carrying the gene. The transforming nucleic acid may comprise a heterologous promoter, heterologous coding sequence or heterologous termination sequence. Alternatively, the transforming nucleic acid may be entirely heterologous, or may comprise any possible combination of heterologous and endogenous nucleic acid sequences. Likewise, heterologous also refers to nucleic acid sequences inserted into and derived from the same native cell type of origin, but in a non-native state, such as a different copy number, or under the control of different regulatory elements.

The term "promoter" refers to a DNA sequence that initiates transcription of the associated DNA sequence. The promoter region also contains elements that act as regulators of gene expression such as activators, enhancers and/or repressors.

"Synthetic nucleotide sequence" is used herein to mean a nucleotide sequence that contains structural features not found in the native sequence. For example, an artificial sequence that more closely approximates the G+C content and normal codon distribution of dicot and/or monocot genes is said to be synthetic.

A regulatory DNA sequence is said to be "operably linked" or "associated" with a DNA sequence encoding an RNA or protein if the two sequences are located such that the DNA regulatory sequence affects the expression of the DNA coding sequence.

"Regulatory element" refers to a sequence involved in the expression of a nucleotide sequence. Regulatory elements comprise a promoter and a termination signal operably linked to a nucleotide sequence of interest. They also generally include sequences required for proper translation of the nucleotide sequence.

"Plant" means any plant or part of a plant, especially a seed plant at any stage of development. These also include cuttings, cell or tissue cultures and seeds. As used herein, the term "plant tissue" includes, but is not limited to, whole plants, plant organs, plant seeds, protoplasts, callus, cell cultures, and any plant cells organized into structural and/or functional units group.

"Plant cell" refers to the structural and physiological unit of a plant, comprising the protoplast and cell wall. Plant cells may be in the form of isolated single cells or cultured cells, or as part of higher organizational units such as plant tissues or plant organs.

"Transformation" is used herein to mean the introduction of a nucleic acid into a cell. In particular, the stable integration of the DNA molecule into the genome of the organism of interest.

A "selectable marker" is a gene whose expression in a plant cell confers a selective advantage on the cell. The selective advantage possessed by cells transformed with a selectable marker gene compared to the growth of non-transformed cells may be due to their ability to grow in the presence of negative selection agents such as antibiotics or herbicides. Transformed cells may also have a selective advantage over non-transformed cells due to their enhanced or novel ability to utilize the added compound as a source of nutrients, growth factors or energy. A selectable marker gene also refers to a gene or combination of genes whose expression in a plant cell confers positive and negative selective advantages on the cell.

A "selectable marker" is conferred by a gene whose expression does not confer a selective advantage on transformed cells, but makes transformed cells phenotype distinct from non-transformed cells.

Sequence Brief Description in Sequence Listing

SEQ ID NO: 1 Conserved amino acid sequence 1

SEQ ID NO: 2 Conserved amino acid sequence 2

SEQ ID NO: 3 Nucleotide sequence of wheat Mlo protein TrMlo1

SEQ ID NO: 4 Protein sequence of TrMlo1

SEQ ID NO: 5 Nucleotide sequence of wheat Mlo protein TrMlo2

SEQ ID NO:6 Protein sequence of TrMlo2

SEQ ID NO: 7 Nucleotide sequence of wheat Mlo protein TrMlo3

SEQ ID NO:8 Protein sequence of TrMlo3

SEQ ID NO: 9 Nucleotide sequence of Arabidopsis Mlo protein CIB10259

SEQ ID NO: 10 Protein sequence of CIB10259

SEQ ID NO: 11 Nucleotide sequence of Arabidopsis Mlo protein CIB10295

SEQ ID NO:12 Protein sequence of CIB10295

SEQ ID NO: 13 Nucleotide sequence of Arabidopsis Mlo protein CIB10296

SEQ ID NO:14 Protein sequence of CIB10296

SEQ ID NO: 15 Nucleotide sequence of Arabidopsis Mlo protein F19850

Protein sequence of SEQ ID NO: 16 F19850

SEQ ID NO: 17 Nucleotide sequence of Arabidopsis Mlo protein U95973

Protein sequence of SEQ ID NO: 18 U95973

SEQ ID NO: 19 Oligonucleotide MLO-1

SEQ ID NO:20 Oligonucleotide MLO-3

SEQ ID NO: 21 Oligonucleotide MLO-5

SEQ ID NO:22 Oligonucleotide MLO-7

SEQ ID NO:23 Oligonucleotide MLO-10

SEQ ID NO: 24 Oligonucleotide MLO-15

SEQ ID NO:25 Oligonucleotide MLO-26

SEQ ID NO: 26 Oligonucleotide MLO-GSP1

SEQ ID NO:27 Oligonucleotide MLO-GSP2

SEQ ID NO: 28 Oligonucleotide ST27

SEQ ID NO:29 Oligonucleotide N37544-1

SEQ ID NO: 30 Oligonucleotide N37544-2

SEQ ID NO: 31 Oligonucleotide T22146-1

SEQ ID NO: 32 Oligonucleotide T22146-2

SEQ ID NO: 33 Oligonucleotide H76041-1

SEQ ID NO: 34 Oligonucleotide H76041-2

SEQ ID NO: 35 Oligonucleotide SAS-1

SEQ ID NO:36 Oligonucleotide SAS-2

SEQ ID NO: 37 Oligonucleotide SAS-3

SEQ ID NO: 38 Oligonucleotide SAS-4

SEQ ID NO:39 Oligonucleotide SAS-5

SEQ ID NO: 40 Oligonucleotide SAS-6

SEQ ID NO: 41 Oligonucleotide SAS-7

SEQ ID NO: 42 Oligonucleotide SAS-8

Preserved strains

All deposited strains are deposited at the Northern Regional Research Center, 1815 Northern University Street, Peoria, Illinois 61604, USA.

The present invention relates to DNA molecules encoding Mlo proteins which confer resistance in plants against fungal pathogens. The inventors of the present invention identified for the first time a conserved amino acid sequence in the Mlo protein. The conserved amino acid sequences of the present invention are conserved among the three Mlo proteins from wheat and the three Mlo proteins from Arabidopsis. These amino acid sequences are also conserved in the two putative Arabidopsis Mlo proteins. The first conserved amino acid sequence listed in SEQ ID NO: 1 includes 13 amino acids. The fourth amino acid in SEQ ID NO: 1 is L, V or I, the fifth amino acid is V or L, and the seventh amino acid is F or L. The thirteenth amino acid in SEQ ID NO: 1 is not I, but preferably T, S or A. The second conserved amino acid sequence listed in SEQ ID NO: 2 includes 14 amino acids. The first amino acid in SEQ ID NO: 2 is not M but preferably I, V, S or G. Its third amino acid is F, L or V, its sixth amino acid is Y or N, its seventh amino acid is A or V, its eighth amino acid is L or I, and its tenth amino acid is amino acid is T or S. The present invention includes isolated Mlo proteins comprising at least one of the conserved amino acid sequences described above and isolated DNA molecules encoding the Mlo proteins. The present invention also includes the isolated Mlo protein containing the conserved sequences listed in SEQ ID NO: 1 and SEQ ID NO: 2. In a preferred embodiment, the isolated DNA molecule encoding the Mlo protein of the invention is a cDNA molecule.

In another embodiment, the DNA molecule encoding the Mlo protein comprising at least one of said conserved amino acid sequences is not from barley. In another embodiment, said DNA molecule is from a dicotyledonous plant or from wheat, maize, rice, oats, rye, sorghum, sugar cane, millet, milo or palmaceae. In a preferred embodiment, the DNA molecule of the present invention is identical or substantially similar to the DNA molecule listed in SEQ ID NO: 3, 5 or 7 or SEQ ID NO: 9, 11, 13, 15 or 17, or the encoding is identical to that of SEQ ID NO: 9, 11, 13, 15 or 17 A Mlo protein identical or substantially similar to any one of the Mlo proteins listed in ID NO: 4, 6 or 8 or SEQ ID NO: 10, 12, 14, 16, 18. The DNA molecule set forth in SEQ ID NO: 3, 5 or 7 is from wheat and encodes the Mlo protein set forth in SEQ ID NO: 4, 6 or 8, respectively. The isolation of this DNA molecule is further described in Example 1. The DNA molecule set forth in SEQ ID NO: 9, 11, 13, 15 or 17 is from Arabidopsis thaliana and encodes the Mlo protein set forth in SEQ ID NO: 10, 12, 14, 16 or 18, respectively. The isolation of this DNA molecule is further described in Example 2.

The DNA molecule of SEQ ID NO: 3 encoding the wheat Mlo protein known as TrMlo 1 was deposited as TrMlo 1 and TrMlo 1-5 strains with accession numbers NRRL B-21948 and NRRL B-21949, respectively. The DNA molecule of SEQ ID NO: 5 encoding the wheat Mlo protein known as TrMlo 2 was deposited as TrMlo 2 and TrMlo 2-5 strains with accession numbers NRRL B-21950 and NRRL B-21951, respectively. The DNA molecule of SEQ ID NO: 7 encoding the wheat Mlo protein known as TrMlo3 was deposited as TrMlo 3 and TrMlo 3-5 lines with accession numbers NRRL B-21952 and NRRL B-21953, respectively. TrMlo1 and TrMlo3 contain the full-length cDNA of the corresponding Mlo gene and also contain part of the corresponding 5' and 3' untranslated regions. TrMlo2 is the longest cDNA clone of the corresponding gene that was repaired. It contains the entire coding region, but lacks the first methionine (start codon) deduced from comparison with TrMlo1 and TrMlo3. TrMlo2 also contains part of the 3' untranslated region of the corresponding gene.

The DNA molecule of SEQ ID NO: 9 encoding the Arabidopsis Mlo protein designated CIB10259 was deposited as strain pCIB10259 under accession number NRRL B-21945. The DNA molecule of SEQ ID NO: 11 encoding the Arabidopsis Mlo protein designated CIB10295 was deposited as strain pCIB10295 under accession number NRRL B-21946. The DNA molecule of SEQ ID NO: 13 encoding the Arabidopsis Mlo protein designated CIB10296 was deposited as strain pCIB10296 under accession number NRRL B-21947. CIB10259, CIB10295 and CIB10296 contain the full-length cDNA of the corresponding Mlo gene and also contain part of the corresponding 5' and 3' untranslated regions. Nucleotide sequences encoding Arabidopsis Mlo protein family members F19850 and U95973 were obtained from Gembank. However, the predicted amino acid sequence was determined for both clones and found to not match the predicted amino acid sequence in Gembank. The Mlo protein assayed by the inventors of the present invention is therefore novel and non-obvious. Both new predicted proteins contain the conserved amino acids listed in SEQ ID NO: 1 and 2, so they and the isolated cDNA encoding them are included in the present invention.

The Mlo protein encoded by the DNA molecule of the present invention confers resistance to a plant against a fungal pathogen, preferably a fungal pathogen capable of infecting living plant epidermal cells, more preferably a fungal pathogen from the order Erysiphales (also known as Erysiphales) (Agrios G.( 1988) Phytopathology, Third Edition, Academic Press Inc., especially at page 271). Preferably, the Mlo protein encoded by the DNA molecule of the invention confers resistance to the plant against a pathogen from the genus Erysiphe, more preferably the fungal pathogen is Erysiphe graminis.

The invention also includes recombinant vectors comprising any of the DNA molecules of the invention. In these vectors, said DNA molecule is preferably contained in an expression cassette containing expression elements for expression of said DNA molecule in a host cell capable of expressing said DNA molecule. Said regulatory elements are usually promoters and termination signals, and preferably also include elements allowing efficient translation of the protein encoded by the DNA molecule of the invention. In preferred embodiments, the expression cassette is heterologous. Said vectors are used to transform an expression cassette containing any of the DNA molecules of the present invention into host cells. In a preferred embodiment, the expression cassette is stably integrated into the DNA of the host cell. In another preferred embodiment, the expression cassette is preferably contained in a vector capable of replicating in the host cell and retained in the host cell as an extrachromosomal molecule. In a further preferred embodiment, the DNA molecule of the invention is amplified in a host cell using said extrachromosomal replication molecule. In a preferred embodiment, said host cell is a microorganism, such as a bacterium, especially E. coli. In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, an insect cell or a plant cell.

In a further embodiment, the DNA molecules of the invention are modified by introducing random mutations using techniques known as in vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370:389-391 (1994) and US Patent 5,605,793, hereby incorporated by reference. Generate millions of mutated copies of the nucleotide sequence based on the original nucleotide sequence described herein and recover variants with improved properties, such as increased resistance to fungal pathogens or resistance to a wider range of pathogens . The method includes forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide containing a nucleotide sequence of the present invention, wherein the template double-stranded polynucleotide has been cut into double-stranded random fragments of expected size, and also includes the following The above steps: adding one or more single-stranded or double-stranded oligonucleotides to the obtained double-stranded random fragment population, wherein said oligonucleotides contain the same region and heterologous region as the double-stranded template polynucleotide; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; and combining the resulting population of single-stranded fragments with polymerase incubation, wherein said identical region is sufficient for one member of the pair to initiate replication of the other member, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two more cycles, wherein the mixture produced by the second step in the next cycle comprises the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and this next cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single double-stranded random fragment in the double-stranded random fragment is less than 1% by weight of the total DNA. In a further preferred embodiment, the template double-stranded polynucleotides comprise at least about 100 polynucleotides. In another embodiment, the size of the double-stranded random fragments is about 5 bp-5 kb. In a further embodiment, the fourth step of the method comprises repeating the second and third steps for at least 10 cycles.

The present invention also includes cells comprising a DNA molecule of the present invention, wherein said DNA molecule is not present in its natural cellular environment. In preferred embodiments, said cells are plant cells. In another preferred embodiment, the DNA molecule of the invention is expressible in said cell and is contained in an expression cassette allowing its expression in such cell. In preferred embodiments, the expression cassette is stably integrated into the DNA of such host cells. In another preferred embodiment, the expression cassette is contained within a vector, which is capable of replicating in the cell and is maintained in the cell as an extrachromosomal molecule.

The present invention also includes plants comprising the above-mentioned plant cells. In additional embodiments, the DNA molecules of the invention can be expressed in plants, and expression of any of the DNA molecules of the invention, or portions thereof, in transgenic plants confers resistance to fungal pathogens in the transgenic plants. In a preferred embodiment, the fungal pathogen is preferably capable of infecting living epidermal cells, more preferably the fungal pathogen is from the order Erysiphales (also known as Erysiphales), especially from the genus Erysiphe, most preferably the fungal pathogen is Erysipha graminis . The present invention therefore also encompasses transgenic plants which are resistant to fungal pathogens by expression of any of the DNA molecules of the present invention or parts thereof.

Plants transformed according to the present invention may be monocots or dicots, including but not limited to maize, wheat, barley, rye, sweet potato, kidney bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip , radish, spinach, asparagus, onion, garlic, pepper, celery, pumpkin, zucchini, hemp, courgette, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry , blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugar cane, sugar beet, sunflower, rapeseed (rapeseed), alfalfa, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber , Arabidopsis and woody plants such as conifers and deciduous trees, especially maize, wheat or sugar beets.

Once a desired nucleotide has been transformed into a plant of a particular species, it can be propagated in that species or transferred into other varieties of the same species, including especially commercial varieties, using conventional breeding techniques.

For their expression in transgenic plants, DNA molecules may require modification and optimization. It is known in the art that all organisms have special preferred codons, and the codons in the nucleotide sequence contained in the DNA molecule of the present invention can be changed while maintaining the encoded amino acids to meet the preference of special plants. High expression in plants is best achieved with coding sequences having a GC content of at least 35%, preferably more than 45%. Nucleotide sequences with low GC content are poorly expressed due to the presence of the ATTTA motif, which can destabilize the message, and the AATAAA motif, which can cause inappropriate polyadenylation. Although the preferred gene sequence can be fully expressed in monocot and dicot species, due to their different codon preferences (Murray et al., Nucleic Acids Res. 17:477-498 (1989)), the sequence should be modified from monocotyledonous to satisfying or the special codon bias and GC content bias of dicots. In addition, nucleotide sequences can be screened for the presence of illegitimate splice sites that cause message truncation. All changes required in the nucleotide sequence as described above were made by well known techniques of site-directed mutagenesis, PCR and synthetic gene construction using the methods of published patent applications EP 0385962, EP 0359472 and WO 93/07278.

Modification of sequences adjacent to the initial methionine may be required for efficient initiation of translation. For example, they may be modified by containing sequences known to be effective in plants. A consensus sequence suitable for plants has been proposed by Joshi (NAR 15:6643-6653 (1987)), and a further consensus translation initiation region has been proposed by Clontech (Catalogue 1993/1994, p. 210). These consensus sequences are applicable to the nucleotide sequences of the present invention. These sequences are incorporated into structures containing the nucleotide sequences of the present invention up to and including ATG (while the second amino acid is unmodified), or up to and including GTC after ATG (with the modified transgene second amino acid possibilities).

The DNA molecule in the transgenic plant is driven by a promoter shown to be functional in the plant. The choice of promoter will vary with the temporal and spatial requirements of expression, and may vary with the species of interest. To protect plants against foliar pathogens, preferably expressed in leaves; to protect plants against ear pathogens, preferably expressed in inflorescences (such as spikes, panicles, corn cobs, etc.); to protect plants against root pathogens, preferably expressed in Expression in roots; to protect seedlings against soil-borne pathogens, expression in roots and/or seedlings is preferred. In many cases, however, species will be claimed against more than one type of phytopathogen, and expression in multiple tissues is thus desired. Although many dicot promoters have been shown to be usable in monocots and vice versa, it is desirable to select a dicot promoter for expression in dicots and a monocot promoter for expression in monocots expression. However, there is no restriction on the origin of the selected promoters; it is sufficient that they can drive the expression of the DNA molecule in the intended cell.

Preferred promoters for constitutive expression include those from the Agrobacterium opine synthase gene, such as the nos promoter, or the binary promoter from the Agrobacterium Ti plasmid (Velten et al. (1984) EMBO J.3:2723- 2730), or viral promoters functional in plants, such as the CaMV 35S and 19S promoters, and the promoters of genes encoding actin or ubiquitin. Another preferred promoter is a synthetic promoter, such as the Gelvin Super MAS promoter (Ni et al. (1995) Plant J. 7:661-676). The DNA molecules of the invention may also be expressed under the regulation of chemically regulated promoters. This allows proteins that cause fungal diseases to be synthesized only when crop plants are treated with inducing chemicals. Preferred techniques for chemically inducing gene expression are detailed in published application EP 0332104 and US Patent 5614395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

Preferred promoter species are wound-inducible. A number of promoters have been described for expression at sites of wounding and infection with plant pathogens. Ideally, the promoter should be active only locally at the site of infection, so that the fungal disease-controlling protein accumulates only in the cells that need to synthesize it to kill the invading pest. Such preferred promoters include those described in the following literature: Stanford et al., Mol. Gen. Genet． 215:200-208 (1989); Xu et al., Plant Mol. Biol. 22:573-588 (1993); Logemann et al., Plant Cell 1:151-158 (1989); Rohrmeier and Lehle, Plant Mol. Biol. 22:783 -792 (1993); Firek et al., Plant Mol. Biol. 22: 129-142 (1993) and Warner et al., The Plant Journal 3: 191-201 (1993).

Preferred tissue-specific expression patterns include green tissue-specific, root-specific, stem-specific and flower-specific. Promoters suitable for expression in green tissues include many that regulate genes involved in photosynthesis, many of which have been cloned from both monocots and dicots. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth and Grula, Plant Mol. Biol. 12:579-589 (1989)). A preferred promoter for root-specific expression is the promoter described by de Framond (FEBS290: 103-106 (1991); EP0452269), and a further preferred root-specific promoter is the promoter from the T-1 gene provided by the present invention son. Preferred stalk-specific promoters are those described in US Patent No. 5,625,136 and those driving expression of the maize trpA gene.

A preferred embodiment of the invention is a transgenic plant expressing a DNA molecule in a root-specific manner. A further preferred embodiment is a transgenic plant expressing a DNA molecule in a wound-inducible or pathogen infection-inducible manner.

In addition to the choice of an appropriate promoter, the construct for expressing the protein in plants requires the attachment of an appropriate transcription terminator downstream of the heterologous nucleotide sequence. Several of said terminators are available and known in the art (eg tml from CaMV, E9 from rbcS). Any available terminator known to function in plants may be used in the context of the present invention.

Many other sequences can be incorporated into the expression cassettes of the DNA molecules of the invention. These sequences include those that have been shown to enhance expression, such as intronic sequences (eg, from Adhl and bronzel) and viral leader sequences (eg, from TMV, MCMV, and AMV).

The expression of the DNA molecule is preferably localized to different cellular locations in the plant. In some cases, localization in the cytosol is desirable, while in other cases, localization in certain subcellular organelles is preferred. Subcellular localization of the enzyme encoded by the transgene can be performed using techniques well known in the art. In general, DNA encoding a target peptide from a gene product known to target an organelle is utilized and fused upstream of the nucleotide sequence. Many such target sequences are known for chloroplasts and they have been shown to function in heterologous structures.

Vectors suitable for plant transformation are also described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors with at least one T-DNA border sequence are suitable, while for direct gene transfer any vector is suitable, preferably linear DNA containing only the construct of interest . In the case of direct gene transfer, transformation of individual DNA species or co-transformation can be employed (Schocher et al., Biotechnology 4:1093-1096 (1986)). For direct gene transfer and Agrobacterium-mediated transfer, antibiotics (kanamycin, hygromycin, or methotrexate) or insecticides (glufosinate, glyphosate, or protoporphyrinogen) are usually (but not necessarily) oxidase inhibitors), or a selectable marker that confers a selective advantage on transformed cells (such as the phosphomannose isomerase gene) for transformation. However, the choice of selectable marker is not critical to the invention.

In another preferred embodiment, the DNA molecule of the invention is transformed directly into the plastid genome. Plastid transformation techniques are extensively described in US Patents 5451513, 5545817 and 5545818 and PCT Application WO95/16783 and McBride et al. (1994) Proceedings of the National Academy of Sciences USA 91, 7301-7305. Basic techniques for chloroplast transformation include, for example, introduction of regions of cloned plastid DNA flanking the selectable marker and the DNA molecule of interest using biolistics or protoplast transformation (eg, calcium chloride or PEG-mediated transformation) into the appropriate target tissue. The 1-1.5 kb flanking sequence, called the targeting sequence, promotes homologous recombination with the plastid genome, thus allowing the replacement or modification of specific regions of the plastid system. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin were used as selection markers for transformation (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990 ) Proceedings of the National Academy of Sciences USA 87, 8526-8530; Staub, J.M. and Maliga, P. (1992) The Plant Cell 4, 39-45). This resulted in stable homogeneous transformants with a frequency of approximately 1 transformant per 100 bombardments of the target leaf. The presence of cloning sites between these markers makes it possible to obtain plastid targeting vectors that can be used to introduce foreign genes (Staub, J. M. and Maliga, P. (1993) EMBO J. 12, 601-606, incorporated herein Reference). Replacement of the recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin detoxifying enzyme aminoglycoside-3'-adenylyltransferase, resulted in substantially higher transformation rates (Svab, Z. and Maliga, P. (1993) Proceedings of the National Academy of Sciences USA 90, 913-917). Previously, this marker has been successfully used in the high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucleic Acids Res. 19, 4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present invention. Generally, about 15-20 cell division cycles are required after transformation to reach a homoplastid state. Plastid expression by homologous recombination inserts genes into all thousands of copies of the circular plastid genome in each plant cell has a huge copy number advantage over nuclear expressed genes such that expression levels can easily exceed 100% of total soluble plant protein 10%.

The present invention also encompasses agricultural products including transgenic plants that are resistant to fungal pathogens by expression of any of the DNA molecules of the present invention or produced by any of the methods described below. Since the plants are resistant to fungal pathogens, the growth of pathogens in them is inhibited. Such plants and their produce are therefore less likely to contain mycotoxins that are naturally produced by many fungal pathogens and can be very toxic to humans and animals. Thus, such agricultural products have better phytosanitary properties. In preferred embodiments, such agricultural products are used as feed, silage or food.

Another object of the present invention is to provide a method for producing plants resistant to fungal pathogens. It is a preferred object of the invention to modify the expression of the Mlo protein encoded by the DNA molecule of the invention to confer resistance to fungal pathogens in plants. Another preferred object of the invention is to alter the stability or activity of said protein in its natural environment. Such alterations in the expression, stability or activity of proteins encoded by the DNA molecules of the invention in plants lead to increased resistance of the plants to fungal pathogens. In a preferred embodiment, the protein encoded by the DNA molecule of the invention is a negative regulator of plant resistance to fungal pathogens, since it inhibits a genetic pathway in plants responsible for plant resistance to fungal pathogens. Therefore, a preferred object of the present invention is to reduce the expression of the Mlo protein encoded by the DNA molecule of the present invention in its natural host environment, or to reduce the stability or activity of this protein in its natural host environment.

"Senseful Inhibition"

In a preferred embodiment, the expression of the protein encoded by the DNA molecule of the invention is reduced by "sense suppression" (see eg Jorgensen et al. (1996) Plant Mol. Biol. 31, 957-973). In this case, all or part of the DNA molecule of the invention is contained in an expression cassette to be introduced into a host cell, preferably a plant cell, in which the DNA molecule is expressible. The DNA molecule is inserted into the expression cassette in "sense orientation", that is, in the expression cassette where the 5' end of the DNA molecule is adjacent to the promoter and the coding strand of the DNA molecule can be transcribed. In preferred embodiments, the DNA molecule is fully translatable, and all genetic information contained in the DNA molecule or portion can be translated into protein. In another preferred embodiment, the DNA molecule is partially translatable into short peptides. In a preferred embodiment, this is accomplished by inserting at least one premature stop codon into the DNA molecule, causing translation to stop. In another more preferred embodiment, the DNA molecule is transcribed but no translation product is produced. This is usually done by removing the start codon, such as "ATG", of the protein encoded by the DNA molecule. In a further preferred embodiment, the expression cassette comprising said DNA molecule or part thereof is stably integrated into the host cell genome. In another preferred embodiment, the expression cassette comprising said DNA molecule or a portion thereof is included in an extrachromosomally replicating molecule. In transgenic plants containing one of the aforementioned expression cassettes, the expression of the corresponding gene for the DNA molecule contained within the expression cassette is reduced or eliminated, resulting in reduced or absent levels of the protein in the transgenic plants. The result is that the transgenic plants are resistant to fungal pathogens.

"Antisense" suppression

In another preferred embodiment, expression of the protein encoded by the DNA molecule of the invention is reduced by "antisense" inhibition. All or part of the DNA molecule of the invention is contained in an expression cassette by which the DNA molecule is introduced into a host cell, preferably a plant cell, wherein the DNA molecule is expressible. The DNA molecule is inserted into the expression cassette in an "antisense orientation", that is, in the expression cassette where the 3' end of the DNA molecule is adjacent to the promoter and the non-coding strand of the DNA molecule can be transcribed. In a preferred embodiment, the expression cassette comprising said DNA molecule or a portion thereof is stably integrated into the host cell genome. In another preferred embodiment, the expression cassette comprising said DNA molecule or a portion thereof is included in an extrachromosomally replicating molecule. Several articles describing this method are cited here for further illustration (Green, P.J. et al., Annals of Biochemistry 55:569-597 (1986); van der Krol, A.R. et al., Antisense Nucleic Acids and Proteins, pp.125-141 (1991); Abel, P.P. et al., PNAS USA 86:6949-6952 (1989); Ecker, J.R., et al. August 1986)).

homologous recombination

In another preferred embodiment, at least one genomic copy of the plant genome corresponding to the DNA molecule of the invention is modified by homologous recombination as further described in Paszkowski et al., EMBO Journal 7: 4021-26 (1988) . Said technique utilizes the characteristics of homologous sequences to recognize each other and exchange nucleotide sequences between them using methods known in the art, such as homologous recombination. Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and the introduced copy of the nucleotide sequence transformed into the cell. Specific modifications are thus accurately introduced into the chromosomal copy of the nucleotide sequence. In one embodiment, the regulatory elements of the gene encoding the protein of the invention are modified. An existing regulatory element is replaced by a different regulatory element, thereby reducing protein expression, or the regulatory element is mutated or deleted, thereby eliminating protein expression. In another embodiment, the coding region of the protein is modified by deletion of the entire coding sequence or a portion thereof, or mutation. Expression of mutant proteins can also confer greater resistance to fungal pathogens in plants.

In another preferred embodiment, cells are transformed with a chimeric oligonucleotide comprising a contiguous sequence of RNA and DNA residues terminated in a double helix conformation with double hairpin caps, thereby introducing mutations in the chromosomal copy of the DNA molecule. An additional feature of oligonucleotides is the presence of 2'-O-methylation at RNA residues. The RNA/DNA sequence is designed to be identical to the sequence of the chromosomal copy of the DNA molecule of the invention and to contain expected nucleotide changes. This technique is further described in US Patent 5,501,967.

ribozyme

In another embodiment, the RNA encoding the protein of the invention is cleaved with a catalytic RNA or ribozyme specific for said RNA. The ribozyme is expressed in the transgenic plant, resulting in a reduction in the amount of RNA encoding the protein of the present invention in plant cells, thereby reducing the amount of protein accumulated in the cell and increasing the resistance of the plant to fungal pathogens. This method is further described in US Patent 4,987,071.

dominant negative mutation

In another preferred embodiment, the activity of the protein encoded by the nucleotide sequence of the invention is altered. This is accomplished by expression of a dominant negative mutant of the protein in transgenic plants, resulting in loss of activity of the endogenous protein.

Aptamer

In another embodiment, the activity of the protein encoded by the DNA molecule according to the invention is inhibited by expressing in transgenic plants a nucleic acid ligand which binds specifically to the protein, a so-called aptamer. Preferably obtain aptamer with SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. In the SELEX method, a candidate mixture of single-stranded nucleic acids with randomized sequence regions is contacted with the protein, and those nucleic acids with higher affinity for the target are revealed with the residue of the candidate mixture. To generate a ligand-enriched mixture, the partitioned nucleic acids are amplified. After many iterations, the nucleic acid with the best affinity for the protein was obtained and used for expression in transgenic plants. This method is further described in US Patent 5,270,163.

Method for Separating Nucleotide Sequences Containing Conserved Sequences

The conserved sequence contained in the Mlo protein encoded by the DNA molecule of the present invention was used to isolate other DNA molecules encoding this sequence. In a preferred embodiment, a mixture of degenerate oligonucleotides is produced, including at least one possible oligonucleotide encoding the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The oligonucleotide mixture encoding the sequence described in SEQ ID NO: 1 and the oligonucleotide mixture complementary to the sequence encoding the sequence described in SEQ ID NO: 2 are subjected to a PCR amplification reaction together with the selected template DNA. Mixtures of degenerate oligonucleotides are well known in the art, with the degree of degeneracy varied as desired. In a preferred embodiment, the template DNA is a total DNA sample from a plant, wherein said DNA sample can be obtained by methods well known in the art. The amplified fragments generated by the above PCR reactions were isolated by methods well known in the art and used to isolate the corresponding full-length cDNA by screening cDNA libraries or using the RACE protocol, the latter two techniques also being well known in the art. This approach represents a unique and useful strategy for isolating new genes that confer plant resistance to fungal pathogens.

The present invention will be further described with reference to the following detailed examples. These examples are provided for the purpose of illustration only and are not intended to be limiting unless otherwise stated.

Example

Embodiment 1: Cloning and sequencing of wheat Mlo gene

Mlo gene of wheat was cloned by reverse transcription PCR method. RNA was prepared from leaves of wheat cultivar UC703 and reverse transcribed using the Stratagene RT-PCR kit. The generated cDNA was used in PCR reaction with the following primers:

MLO-26 5'TTC CAG CAC CGG CAC AAG AA 3' (SEQ ID No: 25)

MLO-10 5'AAG AAC TGC CTG AAG AAG GC 3' (SEQ ID No: 23)

MLO-7 5'CAG AAA CTT GTC TCA TCC CTG G 3' (SEQ ID No: 22)

MLO-5 5'ACA GAG ACC ACC TCC TTG GAA 3' (SEQ ID No: 21)

MLO-15 5'CAC CAC CTT CAT GAT GCT CA 3' (SEQ ID No: 24)

PCR with the primers listed below yielded amplified fragments of the indicated sizes:

MLO-26 and MLO-10 503bp

MLO-26 and MLO-7 1481bp

MLO-5 and MLO-15 650bp

The fragment was cloned into pCR2.1 or pCR2.1-TOPO (Invitrogen). Plasmid DNA was prepared from the transformants and DNA sequencing was performed. Sequencing revealed the presence of three different cDNA sequences with high similarity to each other. These wheat Mlo genes are called TrMlo1, TrMlo2 and TrMlo3.

Additional wheat Mlo clones were isolated by screening a cDNA library constructed in the lambda-ZAPII vector. To screen the library, extensive excision was performed to convert the library into a Bluescript-based cDNA clonal population. These clones were grown individually as a pool of 80,000 independent clones and plasmid DNA was prepared. PCR reactions were performed in mixed DNA using oligonucleotide primers MLO-5 and MLO-15 (see above). Three pools yielded a band of the expected size of 650 base pairs. These pools were subsequently fractionated by growing bacterial clones at lower and lower densities, each step followed by plasmid DNA preparation and PCR of each subpool with primers MLO-5 and MLO-15. Single clones containing an insert with the Mlo sequence were obtained after multiple fractionations. Insert sequencing of two of these clones showed that they contained the same insert. The sequence of the third clone showed that the insert was identical to the other two clones except for 40 additional bases at the 5' end.

Cloning of the remainder of wheat Mlo was accomplished by random amplification of cDNA ends (RACE). The RACE reaction of wheat UC703 poly-A+ RNA was performed with Marathon cDNA Amplification Kit (Clontech). Poly-A+ RNA was prepared from wheat total RNA using an oligo-dT cellulose column (Gibco BRL). The oligonucleotides used in the RACE reaction were:

MLO-GSP1 5' TGG ACC TCT TCA TGT TCG ATC CCA TCT G 3' (SEQ ID No: 26)

MLO-GSP2 5'CCT GAC GCT GTT CCA GAA TGC GTT TCA 3' (SEQ ID No: 27)

Amplification with the primer MLO-GSP1 and the 5' adapter primer provided in the kit produces a DNA fragment of about 1300 nucleotides in size. Amplification with primers MLO-GSP2 and a 3' adapter yielded a DNA fragment approximately 600 nucleotides in size. The fragments were cloned into pCR2.1-TOPO, called TrMlo1-5, TrMlo2-5 and TrMlo3-5, which contained the 5' ends of the wheat Mlo genes TrMlo1, TrMlo2 and TrMlo3, respectively. Plasmid DNA was prepared from the clones and the plasmid inserts were sequenced.

Example 2: Cloning of Arabidopsis Mlo cDNA

Using the software TBLASTN to compare the translation products of the Mlo protein sequence and the sequences in the database, it was shown that many of the sequences were similar to Mlo. Among them, the full-length cDNAs corresponding to the three Arabidopsis EST sequences have been cloned, and the accession numbers are H76041, N37544 and T22146. For each EST, oligonucleotides were designed to amplify the sequence corresponding to said EST from an Arabidopsis cDNA library constructed in plasmid pFL61 (Minet et al. (1992) Gene Nov 16;121(2):393-396) . The oligonucleotides used were:

N37544-1 5'AAG ATC AAG ATG AGG ACG TGG AAG TCG TGG 3' (SEQ ID No: 29)

N37544-2 5' AGG CTG AAC CAC TGG GGC GCC TCT CAC CAC 3' (SEQ ID No: 30)

T22146-1 5'CAA GTA TAT GAT GCG CGC TCT AGA GGA TGA 3' (SEQ ID No: 31)

T22146-2 5' AGG TTT CAC CAC TAA GTC TCC TTC AAT GGC 3' (SEQ ID No: 32)

H76041-1 5'GAT CAT TCA AGA CTT AGG CTC ACT CAT GAG 3'(SEQ ID No: 33)

H76041-2 5'AAC AGC AAG GAA GAT TAC AAA TGA TGC CCA 3' (SEQ ID No: 34)

The DNA prepared from the cDNA library was amplified with primers N37544-1 and N37544-2 to obtain a fragment of about 500 base pairs in size, and amplified with primers T22146-1 and T22146-2 to obtain a fragment of about 250 base pairs in size. fragment. Amplification with primers H76041-1 and H76041-2 yielded two fragments of about 350 and about 300 base pair sizes. A fragment of approximately 300 bases was the size expected from the EST sequence and was subsequently used to detect the presence of the cDNA corresponding to the H76041 EST in the cDNA library. DNA from the library was transformed into E. coli and clones were divided into pools of approximately 20,000 clones each. DNA from individual pools was screened by PCR with different primer pairs, and the positive pools were subsequently subdivided so that the number of clones became smaller and smaller. Isolation of single positive clones is accomplished by carrying this process through, or in some cases once the pool size has reached 200 clones or less, by colony hybridization probed with the EST sequence. For ESTs N37544 and T22146, clones corresponding to ESTs were successfully isolated and the inserts were sequenced. The cDNA sequence corresponding to EST N37544 was named CIB10295 in plasmid pCIB10295, and the plasmid containing the cDNA corresponding to EST T22146 (CIB10296) was named pCIB10296. For both ESTs, the corresponding genomic sequences are available as part of the sequence of the Arabidopsis BAC clone recently deposited into GenBank. It should be pointed out that the predicted protein sequence of these genome sequences determined by GenBank does not match the sequence determined by direct cDNA sequencing. Therefore, the amino acid sequences of the corresponding genes of ESTN37544 and T22146 were not evident from GenBank entries, but could only be elucidated by cloning and sequencing of cDNA clones. It has been found that clones isolated with primers for the H76041 EST do not contain the gene for H76041, but contain a novel member of the Mlo gene family as an insert. The insert was completely sequenced and this member of the Mlo gene family was named CIB10259 in plasmid pCIB10259.

Embodiment 3: the construction that is used for the carrier of Mlo gene expression in wheat

Two vectors were constructed for "antisense" expression of the barley Mlo gene in wheat. PCR was performed on MLO-5 and MLO-7 (see (1) above) using barley cDNA and primers, and the reaction generated an amplified fragment of 1124 bp, which was cloned into pGEM-T (Promega). This fragment was excised from pGEM-T with SacII and NotI enzymes. This 1124bp fragment was cloned into pBluescript-SK(+). This time the insert was excised through the BamHI and SacI restriction sites and cloned into BamHI-SacI-digested pCIB9806 (described in patent application 08/838219) with the orientation of the Mlo coding sequence aligned with the maize ubiquitin promoter Son is the opposite. The plasmid was named pCK01.

In order to construct a vector for "antisense" expression of the complete Mlo gene in wheat, the primer pair MLO-1 (5'ATG TCG GAC AAA AAA GGG GT3' (SEQ ID NO: 19)) and MLO-10 (see above (1)) Perform PCR to generate a 635bp amplified fragment, which is cloned into pCR2.1 (Invitrogen). This fragment was excised from pCR2.1 as an EcoRI fragment and inserted into pGEM-9Zf(-) (Promega). A 320 nucleotide fragment spanning the naturally occurring SacI site in Mlo to primer site MLO-10 was excised with SacI and BstXI. pCK01 was digested with SacI and BstXI, and the 320-base fragment was inserted. For the complete construction of the Mlo gene in a monocot expression vector, a SacI fragment of 210 nucleotides was excised from the pGEM-9Zf(-) derivative. This fragment contains the 5' end of the Mlo coding sequence from primer site MLO-1 to the naturally occurring SacI site in the Mlo gene. The pCK01 derivative was digested with SacI and the 210 base fragment was inserted. Clones were analyzed by PCR using primers MLO-1 and MLO-10 to determine the orientation of this 210 base fragment in the newly constructed vector. Only the clone in which the 210 base fragment was inserted in an antisense orientation relative to the ubiquitin promoter produced a product corresponding to the 530 base pair 5' end of the Mlo coding sequence. The resulting plasmid contained the entire Mlo coding sequence in an "antisense" orientation relative to the ubiquitin promoter and was designated pCK02.

To construct a vector for expression of the Mlo gene in "sense" orientation, plasmid pCK02 was digested with BamHI to release the Mlo coding sequence as the insert. The BamHI fragment was ligated back into the pCK02 base vector. Colonies with the Mlo coding sequence reversed relative to pCK02 were identified by SacI digestion, yielding a 1.8 kb fragment in such clones compared to a 210 base fragment in clones of the same structure as pCK02. The clone carrying the Mlo coding sequence in the "sense" orientation relative to the maize ubiquitin promoter was selected and named pCK03.

Embodiment 4: be used for the construction of the vector expressing Mlo gene in Arabidopsis

The Mlo clones in pCIB10259, pCIB10295 and pCIB10296 and pCK02 (barley Mlo gene) were used in PCR reactions and the resulting bands contained the full length gene flanked by BamHI restriction sites. The primer sequences used are as follows: SAS-1: 5' GGA TTA AGA TCT AAT GGC 3' (SEQ ID No: 35, used for pCIB10295) SAS-2: 5' CAA AGA TCT TCA TTT CTT AAA AG 3' (SEQ ID No : 36, for pCIB10295) SAS-3: 5' GCG GAT CCA TGT CGG ACA AAA AAG G 3' (SEQ ID No: 37, for barley Mlo) SAS-4: 5' GCG GAT CCT CAT CCC TGG CTG AAG G 3' (SEQ ID No: 38, for barley Mlo) SAS-5: 5' GGA TCC ACC ATG GCC ACA AGA TG 3' (SEQ ID No: 39, for pCIB10259) SAS-6: 5' GGA TCC TTA GTC AAT ATC ATT AGC 3' (SEQ ID No: 40, for pCIB10259) SAS-7: 5' GCG GAT CCA TGG GTC ACG GAG GAG AAG 3' (SEQ ID No: 41,

For pCIB10269) SAS-8: 5' GCG GAT CCT CAG TTG TTA TGA TCA GGA 3' (SEQ ID No: 42,

for pCIB10296)

These bands were cloned into pCR2.1-TOPO and the insert in the resulting plasmid was sequenced to confirm that no mutations had been introduced by PCR. The plasmid was digested with BamHI, and the insert was purified and cloned into BamHI-digested pPEH28, a gene containing a copy of the Arabidopsis ubiquitin gene promoter UBQ3 located directly downstream of the BamHI site (Norris et al. (1993) Plant Molecular Biology 21: 895-906) shuttle vector. Clones containing the Mlo sequence fused to UBQ3 were identified and restriction enzyme analysis was performed to identify clones containing inserts in the "sense" and "antisense" orientations relative to UBQ3. For each Mlo gene, the clone containing the insert in sense orientation and the clone containing the insert in antisense orientation were digested with XbaI, and the insert was purified and cloned into XbaI digested pCIB200. This approach places the UBQ3-Mlo gene fusion between the T-DNA borders.

Example 5: Transformation of wheat and identification of expressors

Wheat was transformed by particle bombardment of immature embryos as detailed in patent application WO 94/13822. Plantlets will be regenerated in Basta-containing medium and subjected to PCR analysis. To determine the presence of the Mlo transgene by PCR, the primers used were as follows:

MLO-3: 5’ATG CTA CCA CAC GCA GAT CG 3’

ST27: 5’ACT TCT GCA GGT CGA CTC TA 3’

Primer MLO-3 corresponds to the region of the Mlo transgene, while primer ST27 is located within the maize ubiquitin promoter sequence. The simultaneous use of Mlo gene and ubiquitin promoter primers in PCR eliminates false positives that can arise from the use of two Mlo primers, which could be elicited from the chromosomal copy of the Mlo gene present in wheat.

Plants confirmed to contain the Mlo transgene were subjected to RNA gel blot analysis to determine whether they contained altered levels of chromosomally encoded wheat Mlo mRNA. Poly-A ⁺ RNA was prepared from each transgenic line and blotted onto Hybond-N+ filters. The blot was probed with a 530 base fragment corresponding to the 5' end of the Mlo gene. This region was absent in the pCK01 clone; therefore, there was no hybridization with antisense RNA expressed from the transgene in the pCK01 -containing transgenic line. For the pCK02 transgenic line in which the transgene included this 5' end fragment, the probe hybridized to two bands of different sizes. An approximately 2.5 kb mRNA corresponding to the antisense transgene was clearly distinguished from the 2.0 kb mRNA from the wheat chromosome encoding the Mlo gene. The abundance of 2.0 kb mRNA was monitored as a measure of the gene suppression efficiency achieved by the transgene in each line.

Example 6: Disease Detection of Transgenic Wheat Lines

Transgenic and non-transformed UC703 (control) wheat line plants were grown in the greenhouse until they were two weeks old. Plants were transferred into Percival growth chambers (per cycle: 8 hours in the dark, 16°C and 16 hours in the light, 20°C) and thoroughly inoculated with Erysipha graminearum tritici with spores. The extent of fungal sporulation was assessed two weeks after inoculation. Plants were rated as 1 (little to no mycelial growth and no visible sporulation), 2 (some mycelial growth and sporulation, but less than control plants) or 3 (comparable to control plants). silk growth and spore formation). Transgenic wheat lines expressing Mlo-constructs show increased resistance to pathogens. Examples of results obtained with antisense barley Mlo constructs are listed below.

Screening of transgenic lines R1 and R2 sister strains for disease resistance

A sister strain of the Mlo antisense transformant (T2 seeds) was planted, inoculated with Erysiphe graminis, and assessed for disease resistance. R1 R2 UC703 (control) planted seeds twenty four twenty four twenty four germination 14 twenty four twenty one disease score 1 4 2 0 disease score 2 0 0 0 disease score 3 10 twenty two twenty one

The small percentage of R1 and R2 plants exhibiting resistance may be due to the fact that the tested T2 population was still segregated from the transgene.

Example 7: Analysis of Arabidopsis lines expressing the Mlo gene

The pCIB200 derivative containing the Mlo gene was used to transform the Arabidopsis ecotype Ws-O by vacuum infiltration (Bechtold, N., Ellis, J. and Pelletier, G. (1993) C. R. Acad. Sci. Paris 316 , 1194-1199). Progeny were screened by kanamycin selection to identify transformants. For Mlo transgenic lines, plants expressing Mlo were identified by RNA gel blot analysis. For the Mlo gene, transformants were analyzed for changes in the steady-state level of mRNA accumulation by RNA gel blot analysis. Transformants exhibiting sense or antisense suppression of the target gene were detected for altered responses to the phytopathogenic fungi Erysipha bisporus and Peronospora parasitica and the bacterial pathogen Pseudomonas syringae pv tomato. The presence of necrosis was detected by macroscopically and microscopically examining the leaves of the transgenic plants with trypan blue staining.

For powdery mildew inoculation, spores were applied generously to Arabidopsis rosettes and the plants were maintained at 25°C in Percival growth chambers. The extent of fungal sporulation was assessed 10 days after inoculation. Example 8: Isolation of Additional Mlo Gene Family Members Using Similarity Between Mlo Sequences

Alignment of the amino acid sequences predicted to be encoded by the Mlo genes revealed many short regions of high amino acid similarity among all gene products. Design degenerate primers for these regions, and perform PCR reactions with these primers according to the PCR reagent supplier's recommended method. The amplified fragments were used as probes to isolate full-length cDNA or genomic clones of novel Mlo genes. The conserved region of amino acid sequence between the Mlo protein of the invention (bold line) and the degenerate oligonucleotides used for isolation of additional Mlo genes is shown below:

E   L   M   X1  X2  G   X3   I   S   L   L   L   X4WHEATTrMlo1   GAG CTC ATG CTG GTG GGC TTC ATCTrMlo2   GAG CTG ATG CTG GTG GGG TTC ATCTrMlo3   GAG CTG ATG CTG GTG GGA TTC ATCARABIDOPSISCIB10259 GAG CTG ATG ATT CTA GGA TTC ATTCIB10295 GAG CTT ATG CTG TTG GGA TTC ATACIB10296 GAG CTG ATG TTG TTA GGG TTT ATAF19850   GAG CTG ATG GTT CTT GGA TTC ATCU95973   GAG TTG ATG TTG CTG GGA CTT ATA5' GAG CTB ATG MTB BTR GGM TTC AT 3'X5   T   X6   P   L   X7    X8   X9   V   X10   Q   M G   SWHEATTrMlo1                           GCG  CTC  GTC  ACA  CAG ATG GGA TCATrMlo2                           GCG  CTC  GTC  ACA  CAG ATG GGA TCGTrMlo3                           GCG  CTA  GTC  ACA  CAG ATG GGA TCAARABIDOPSISCIB10259                         GCA  CTA  GTT  ACT  CAG ATG GGT TCACIB10295                         GCA  CTT  GTT  ACT  CAG ATG GGT AGTCIB10296                         GCC  ATC  GTC  TCA  CAG ATG GGA AGTF19850 GCA CTC GTA ACT CAG ATG GGT TCTU95973 GTA ATC GTT ACT CAG ATG GGA TCT5' WCC CAT CTG AGT GAC DAG BGC RTA 3

X1=L, V or I, X2=V or L, X3=F or L, X4=T, S or A.

X5=I, V, S or G, X6=F, L or V, X7=Y or N, X8=A or V, X9=L or I, X10=T or S.

R=A, G, Y=C, T, M=A, C, K=G, T, S=C, G, W=A, T, H=A, C, T, B=C, G, T, V=A, C, G, D=A, G, T, N=A, C, G, T.

Example 9: Modification of Coding Sequences and Adjacent Sequences

For expression in transgenic plant hosts, the DNA molecules described in this application can be modified to accomplish and optimize or negatively regulate their expression. The following problems may be encountered and modifications of these DNA molecules can be carried out using techniques well known in the art.

(1) Codon usage. The preferred codon usage in some plants is different from that in other plant species. Plant evolution generally tends to prefer nucleotides C and G at the third base position of monocots, while dicots usually use nucleotides A or T at this position. By modifying the gene to incorporate codons preferred for the particular transgenic species of interest, many of the problems described below with respect to GC/AT content and irrational splicing will be overcome.

(2) GC/AT content. The GC content of plant genes usually exceeds 35%. DNA molecules rich in A and T nucleotides can cause many problems in plants. First, the ATTTA motif is thought to destabilize the messenger and has been found at the 3' end of many short-lived mRNAs. Second, the presence of polyadenylation signals such as AATAAA at inappropriate positions in the messenger is thought to cause premature termination of transcription. In addition, monocots may recognize AT-rich sequences as splice sites (see below).

(3) Sequence adjacent to the starting methionine. It is thought that the ribosome attaches to the 5' end of the message and seeks the first ATG to initiate translation. However, we believe that there is a preference for certain nucleotides adjacent to the ATG and that inclusion of a novel consensus translation initiation codon at the ATG enhances expression of the DNA molecules of the invention. Clontech (1993/1994 catalog, p. 210) has proposed a sequence as a consensus translation initiation region for expression of the E. coli uidA gene in plants. In addition, Joshi (NAR 15:6643-6653 (1987)) has compared many plant sequences adjacent to the ATG and revealed a consensus sequence. Having one of these sequences at the initiation ATG may facilitate translation when expression of the DNA molecule is difficult in plants. In such cases, the last three nucleotides of the consensus region may not be suitable for inclusion in the modified sequence due to the modification of its second amino acid. The preferred sequence adjacent to the starting methionine may vary between different plant species. A survey of 14 maize genes located in the GenBank database yielded the following results:

The position before the start ATG in 14 maize genes

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1

C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0

A 2 3 1 4 3 2 3 7 2 3

G 6 3 6 0 6 5 4 6 1 5

This analysis can be performed on the expected plant species into which the nucleotide sequence will be inserted, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotide.

(4) Removal of unreasonable splicing sites. The DNA molecules of the invention may also contain motifs which in plants would be recognized as 5' or 3' splice sites, and be cleaved, resulting in truncated or deleted messages. These sites can be removed using techniques well known in the art.

(5) Generation of dominant negative mutants

In addition, the DNA molecules of the present invention may also include molecules that are modified such that the activity of the protein encoded by the nucleotide sequence of the present invention is altered. This result is achieved by expression of a dominant negative mutant of the protein in transgenic plants, resulting in the loss of activity of the endogenous protein. The mutation sites leading to the generation of this dominant negative mutant in the Mlo nucleotide sequence are listed below. A single mutation or a combination of different mutations listed below can be introduced.

Techniques for modifying coding sequences and adjacent sequences are well known in the art. If the initial expression level of the DNA molecule of the invention is low and the sequence is amenable to alteration as described above, construction of the synthetic gene is accomplished by methods well known in the art. These methods have been described in published patent content such as EP0385962, EP0359472 and WO93/07278. In most cases, it is preferred to use transient assay methods (well known in the art) to detect expression of the gene construct prior to its transfer into transgenic plants.

Embodiment 10: Construction of plant transformation vector

A number of transformation vectors are available for plant transformation, and the DNA molecules of the invention can be used in conjunction with any such vectors. The choice of vector to be used will depend on the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or insecticide selectable markers may be preferred. Selectable markers routinely used in transformation include the nptII gene which renders plants resistant to kanamycin, paromomycin, geneticin and related antibiotics (Vieira and Messing, 1982, Gene 19:259-268; Bevan et al., 1983, Nature 304:184-187), a bacterial aadA gene encoding aminoglycoside 3'-adenylyltransferase and conferring resistance to streptomycin or spectinomycin (Goldschmidt-Clermont, 1991, Nucleic Acids Res. 19:4083-4089 ), the hph gene conferring resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, 1984, Molecular Cell Biology 4:2929-2931), and the dhfr gene conferring resistance to methotrexate (Bourouis and Jarry, 1983, EMBO J .2:1099-1104). Other useful markers include the phosphinothricin acetyltransferase gene that renders plants resistant to the insecticide phosphinothricin (White et al., 1990, Nucleic Acids Res. 18:1062; Spencer et al. 1990, Theoretical and Applied Genetics 79:625- 631), a mutant EPSP synthase gene encoding glyphosate resistance (Hinchee et al., 1988, Bio/Technology 6:915-922), a mutant acetolactate synthase conferring resistance to imidazolinones or sulfonylureas ( ALS) gene (Lee et al., 1988, EMBO J.7: 1241-1248), a mutant psbA gene conferring resistance to arazine (Smeda et al., 1993, Plant Physiology 103: 911-917) or as in US Patent 5767373 The mutant protoporphyrinogen oxidase gene. Selectable markers that produce positive selection, such as the phosphomannose isomerase gene, can also be used.

Identification of transformed cells can also be accomplished by the expression of selectable marker genes, such as those encoding chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), luciferase, and green fluorescent protein (GFP) Or any other protein that confers a unique phenotypic trait on transformed cells.

(1) Construction of a vector suitable for Agrobacterium transformation

A number of vectors are available for Agrobacterium tumefaciens transformation. These vectors typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucleic Acids Res. (1984)) and pXYZ. The construction of two typical vectors is described below.

Construction of pCIB200 and pCIB2001

Binary vectors pCIB200 and pCIB2001 were used to construct recombinant vectors for Agrobacterium and were constructed in the following manner. The tetracycline resistance gene was excised by digesting pTJS75 with NarI (Schmidhauser & Helinski, J. Bacteriology 164: 446-455 (1985)), and then inserted into the gene from the band NPTII (Vieria & Messing, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983); McBride et al., Plant Mol. Biol. 14:266-276 (1990)) from the AccI fragment of pUC4K, yielding pTJS75kan. The XhoI linker was ligated to the EcoRV fragment of pCIB7 (Rothstein et al., Gene 53:153-161 (1987)) containing the T-DNA left and right border sequences, the plant nos/nptII selectable chimeric gene and the pUC polylinker, and digested with XhoI The fragment was cloned into Sall digested pTJS75kan, resulting in pCIB200 (see also EP 0332104, Example 19). pCIB200 contains the following single polylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI and SalI. pCIB2001 is a derivative of pCIB200 generated by insertion of an additional restriction site polylinker. The single restriction sites in the pCIB2001 polylinker are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BcII, AvrII, ApaI, HpaI and StuI. In addition to containing these unique restriction sites, pCIB2001 also has plant and bacterial kanamycin selection genes, left and right T-DNA border sequences for Agrobacterium-mediated transformation, RK2 for transfer between E. coli and other hosts - Derived trfA function and OriT and OriV functions also from RK2. pCIB2001 is suitable for cloning plant expression cassettes containing self-regulatory signals.

Construction of pCIB200 and its hygromycin selective derivatives

The binary vector pCIB10 contains the kanamycin resistance coding gene that can be used for selection in plants, the left and right border sequences of T-DNA, and incorporates the sequence of the broad-spectrum plasmid pRK252, so that it can be replicated in both Escherichia coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53:153-161 (1987)). Several pCIB10 derivatives have been constructed incorporating the hygromycin B phosphotransferase gene described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable the selection of transgenic plant cells for hygromycin only (pCIB743) or for both hygromycin and kanamycin (pCIB715, pCIB717).

(2) Construction of vectors suitable for non-Agrobacterium transformation

Non-Agrobacterium tumefaciens transformation does not require T-DNA sequences within the chosen transformation vector, so vectors lacking these sequences may also be utilized in addition to the above-described vectors containing T-DNA sequences. Agrobacterium-independent transformation techniques include transformation by particle bombardment, protoplast uptake (eg, PEG and electroporation), and microinjection. The choice of vector depends largely on the chosen transformation species. The construction of some typical vectors is described below.

pCIB3064 build

pCIB3064 is a pUC-derived vector suitable for direct gene transfer in combination with selection for the insecticide Basta (or phosphinothricin). Plasmid pCIB246 contains the CaMV 35S promoter and CaMV 35S transcriptional terminator operably fused to the E. coli GUS gene and is described in PCT Published Application WO 93/07278. The 35S promoter of this vector contains two ATG sequences at the 5' end of the initiation site. These sites were mutated using standard PCR techniques to remove the ATG and generate the restriction sites SspI and PvuII. The new restriction sites were 96 and 37 bp from the unique SalI site and 101 and 42 bp from the exact start site. The resulting pCIB246 derivative was named pCIB3025. The GUS gene was then excised from pCIB3025 by digestion with SalI and SacI, blunt-ended and religated to generate plasmid pCIB3060. Plasmid pJIT82 was obtained from the John Innes Centre, Norwich, and the 400 bp SmaI fragment containing the bar gene from green chromogenic streptomycin was excised and inserted into the HpaI site of pCIB3060 (Thompson et al., EMBO J6:2519-2523 (1987))). This generated pCIB3064, which includes the bar gene under the control of the CaMV 35S promoter and terminator for insecticide selection, the ampicillin resistance gene (for selection in E. Multiple adapters at a single site. This vector is suitable for cloning plant expression cassettes containing self-regulatory signals.

Construction of pSOG19 and pSOG35

pSOG35 is a transformation vector utilizing E. coli dihydrofolate reductase (DHFR) as a selection marker conferring methotrexate resistance. The 35S promoter (about 800 bp), intron 6 from the maize Adhl gene (about 550 bp) and the 18 bp GUS untranslated leader sequence from pSOG10 were amplified by PCR. A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene was also PCR amplified and these two PCR fragments were assembled with the SacI-PstI fragment from pBI221 (Clontech) containing the pUC19 vector backbone and the nopaline synthase terminator together. Assembly of these fragments generated pSOG19, which contains the 35S promoter fused to the intron 6 sequence, the GUS leader sequence, the DHFR gene and the nopaline synthase terminator. The GUS leader sequence in pSOG19 was replaced with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) to generate vector pSOG35. pSOG19 and pSOG35 carry the ampicillin resistance pUC gene and have HindⅢ, SphⅠ, PstⅠ and EcoRI sites which can be used to clone foreign sequences.

Example 11: Requirements for constructing plant expression cassettes

The gene sequence intended to be expressed in the transgenic plant is first assembled into an expression cassette, behind the appropriate promoter and upstream of the appropriate transcription terminator.

promoter selection

The choice of promoter used in the expression cassette will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. The selected promoter will express the transgene in a particular cell type (e.g. leaf epidermal cells, mesophyll cells, root cortex cells) or in a particular tissue or organ (e.g. root, leaf or flower) and the selection will reflect the DNA molecular biology of the present invention. The expected location of the composition. Alternatively, the selected promoter may drive expression of the gene under the control of a light-inducible or other temporally regulated promoter. Another option is that the selected promoter is chemically regulated. This makes it possible to induce expression of nucleotide sequences by chemical inducer treatment only when required.

transcription terminator

A variety of transcription terminators are available for use in expression cassettes. They are responsible for the termination of transcription outside of the transgene and its correct polyadenylation. Suitable transcription terminators known to function in plants can be used, including the CaMV 35S terminator, tml terminator, nopaline synthase terminator, pea rbcS E9 terminator. They are available in both monocots and dicots.

Sequences for enhancing or regulating expression

A number of sequences have been found to enhance gene expression within the transcription unit and these sequences can be used in conjunction with the genes of the invention to increase their expression in transgenic plants.

Various intronic sequences have been shown to enhance expression, especially in monocot cells. For example, it has been found that the intron of the maize Adhl gene, when introduced into maize cells, can significantly enhance the expression of the wild-type gene under the control of its cognate promoter. Intron 1 was found to be particularly effective in enhancing the expression of fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Gene Development 1: 1183-1200 (1987)). In the same experimental system, an intron from the maize bronze1 gene had a similar effect in enhancing expression (Callis et al., supra). Intronic sequences have been routinely introduced into plant transformation vectors, typically within the untranslated leader sequence.

Many untranslated leader sequences from viruses are known to enhance expression, and they are particularly effective in dicot cells. Specifically, leader sequences from tobacco mosaic virus (TMV, 'omega sequence'), maize chlorotic mottle virus (MCMV), and alfalfa mosaic virus (AMV) have been shown to be effective in enhancing expression (eg, Gallie et al., Nucleic Acids Research 15:8693-8711 (1987); Skuzeski et al., Plant Mol. Biol. 15:65-79 (1990)).

Orientation of gene products in cells

Various mechanisms for directing gene products are known to exist in plants, and sequences controlling the function of these mechanisms have been identified to some extent. For example, targeting of gene products to chloroplasts is controlled by signal sequences found at the amino termini of many proteins, which are cleaved during chloroplast import to produce mature proteins (e.g., Comai et al., J. Biol. Chem. 263:15104- 15109 (1988)). These signal sequences can be fused to heterologous gene products to allow import of the heterologous products into chloroplasts (van den Broeck et al., Nature 313:358-363 (1985)). DNA encoding a suitable signal sequence can be isolated from the 5' end of cDNA encoding the following proteins: RUBISCO protein, CAB protein, EPSP synthetase, GS2 protein and many other proteins known to localize to chloroplasts.

Other gene products localize to other organelles such as mitochondria and peroxisomes (eg, Unger et al., Plant Mol. Biol. 13:411-418 (1989)). The cDNAs encoding these products can also be manipulated to direct heterologous gene products to these organelles. Examples of such sequences are nuclear encoded ATPase and mitochondrial specific aspartate aminotransferase isoforms. Work targeting cellular proteosomes has been described by Roger et al. (Proc. National Academy of Sciences USA 82:6512-6516 (1985)).

In addition, sequences were identified that direct gene products to other cellular compartments. The amino-terminal sequence is responsible for extracellular secretion directed to the ER, apoplasts, and aleurone cells (Koehler & Ho, The Plant Cell 2: 769-783 (1990)). Furthermore, the amino-terminal sequence, together with the carboxy-terminal sequence, is responsible for the vacuolar orientation of the gene product (Shinshi et al., Plant Mol. Biol. 14:357-368 (1990)).

By fusing the appropriate targeting sequence described above to the transgene sequence of interest, it is possible to direct the transgene product to any organelle or cell compartment. For example, for chloroplast targeting, the chloroplast signal sequence from the RUBISCO gene, CAB gene, EPSP synthetase gene, or GS2 gene can be fused in frame to the amino-terminal ATG of the transgene. The signal sequence chosen should contain a known cleavage site, and fusion construction should take into account any amino acids required for cleavage after the cleavage site. In some cases, this requirement was achieved by adding a small number of amino acids between the cleavage site and the transgene ATG or by replacing some amino acids in the transgene sequence. In vitro translation of the construct by in vitro transcription and subsequent use of literature (Bartlett et al., In: Edelmann et al. (eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp1081-1091 (1982); Wasmann et al., Molecular and General Genetics 205: 446-453 (1986)) for in vitro chloroplast uptake, allowing the detection of chloroplast uptake efficiency of fusions constructed for import into chloroplasts. These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes. Selective orientation may be required for insecticidal toxins. Usually cytosolic or chloroplast, although in some cases it may also be mitochondrial or peroxisome. Expression of nucleic acid sequences may also require targeting to the ER, apoplast or vacuole.

The above-mentioned cell-directed mechanism can be used not only in combination with its homologous promoter, but also in combination with a heterologous promoter, so as to achieve specific cell-directed activation under the transcriptional regulation of a promoter with a different expression pattern from the promoter from which the directional signal originates. Purpose.

Example 12: Example of expression cassette construction

The present invention encompasses the expression of a DNA molecule under the regulation of a promoter expressible in any plant, regardless of the origin of the promoter. Furthermore, the present invention encompasses the use of any plant expressible promoter in combination with any other sequence required or selected for expression of the DNA molecule. Such sequences include, but are not limited to, transcription terminators, foreign sequences that enhance expression (such as introns [such as Adh intron 1], viral sequences [such as TMV-Ω]) and direct gene products to specific Sequence of organelles and cellular compartments.

Constitutive expression: CaMV 35S promoter

The construction of plasmid pCGN1761 is described in published patent application EP 0392225. pCGN1761 contains a 'double' 35S promoter and a tml transcription terminator with a single EcoRI site between the promoter and terminator, and also includes a pUC-type backbone. A pCGN1761 derivative with a modified polylinker including NotI and XhoI sites in addition to the original EcoRI site was constructed. This derivative was called pCGN1761ENX. For gene expression in transgenic plants under the control of the 35S promoter, pCGN1761ENX can be used to clone cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker. The complete 35S promoter-gene sequence-tml terminator box of this structure can be excised by using the HindIII, SphI, SalI and XbaI sites on the 5' side of the promoter and the XbaI, BamHI and BglI sites on the 3' side of the terminator , which is transferred into a transformation vector such as described in Example 35. In addition, for replacement with another promoter, HindIII, SphI, SalI, XbaI, or PstI can be used to cut at 5', and any of the polylinker restriction sites (EcoRI, NotI, or XhoI) at 3' to remove the double 35S Promoter fragment.

Modification of pCGN1761ENX by optimizing the translation initiation site

As with any of the constructs described in this section, modifications may be made around the cloning site by introducing sequences that enhance translation. This is especially useful when introducing genes from microorganisms into plant expression cassettes, since these genes may not contain, adjacent to their methionine, sequences suitable for translation initiation in plants. If a gene from a microorganism is to be cloned at its ATG into a plant expression cassette, it may be useful to modify its insertion site to optimize its expression. As an example of a way of introducing one of several plant expression optimized sequences, see the modification of pCGN1761ENX (eg Joshi, supra).

Expression under the control of a chemically regulatable promoter

This section describes the replacement of the dual 35S promoter in pCGN1761ENX with any promoter of choice; one example described is replacement with the chemically regulated RP-1a promoter. The selected promoter is preferably excised from its natural source by restriction enzymes, but may alternatively be PCR amplified using primers with appropriate terminal restriction sites. If PCR amplification is performed, after the amplified promoter is cloned into the target vector, the promoter should be re-sequenced to check for amplification errors. The chemically regulatable tobacco PR-1a promoter was excised from plasmid pCIB1004 (see EP 0332104, Example 21 for construction) and transferred into plasmid pCGN1761ENX. pCIB1004 was cut with NcoI, and the 3' overhang of the resulting linear fragment was filled in by T4 DNA polymerase treatment. This fragment was then cleaved with HindIII and the resulting fragment containing the PR-1a promoter was gel purified and cloned into pCGN1761ENX in which the dual 35S promoter had been excised. This was accomplished by cutting with XhoI and filling in with T4 polymerase, followed by cutting with HindIII and isolating the larger fragment containing the vector-terminator cloned into the pCIB1004 promoter fragment. This yielded a pCGN1761ENX derivative containing the PR-1a promoter and tml terminator and an inserted polylinker with unique EcoRI and NotI sites. The DNA molecule of the invention can be inserted into this vector and the fusion product (ie promoter-gene-terminator) can then be transferred into any transformation vector of choice, including the vectors described in this application.

Constitutive Expression: Actin Promoter

Several actin isoforms are known to be expressed in most cell types, so the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Act1 gene has been cloned and characterized (McElroy et al., The Plant Cell 2: 163-171 (1990)). A 1.3 kb fragment of this promoter was found to contain all the regulatory elements required for expression in rice protoplasts. In addition, a number of expression vectors based on the ActI promoter have been constructed specifically for monocotyledonous plants (McElroy et al., Molecular and General Genetics 231: 150-160 (1991)). They incorporated Act1-intron 1, Adh1 5' flanking sequences and Adh1-intron 1 (from the maize alcohol dehydrogenase gene) and sequences from the CaMV 35S promoter. The vectors showing the highest expression were fusions of 35S and the Act1 intron or fusions of the Act1 5' flanking sequence and the Act1 intron. Optimization of the sequence surrounding the initial ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassette described by McElroy et al. (Molecular and General Genetics 231: 150-160 (1991)) can be readily modified for expression of the DNA molecules of the invention, which is particularly suitable for use in monocot hosts. For example, a promoter-containing fragment can be excised from the McElroy construct and used to replace the double 35S promoter in pCGN1761ENX, which can then be used to insert specific gene sequences. The fusion gene thus constructed can then be transferred into a suitable transformation vector. In individual reports, it has also been found that the rice Act1 promoter and its first intron can direct high expression in artificially cultured barley cells (Chibbar et al., Plant Cell Rep. 12: 506-509 (1993)).

Constitutive expression: ubiquitin promoter

Ubiquitin is another gene product known to accumulate in many cell types, and its promoters have been cloned from many species and used in transgenic plants (such as Sunflower-Binet et al., Plant Science 79:87-94 (1991 ), Maize-Christensen et al., Plant Mol. Biol. 12:619-632 (1989)). The maize ubiquitin promoter has been used in transgenic monocotyledonous plant systems, and its sequence and vectors constructed for monocotyledonous plant transformation are disclosed in patent application EP0342926. In addition, Taylor et al. (Plant Cell Rep. 12:491-495 (1993)) described a vector (pAHC25) containing the maize ubiquitin promoter and first intron that, when introduced by particle bombardment, was found in many monocots. Highly active in plant cell suspensions. The ubiquitin promoter is perfectly suitable for the expression of the DNA molecules according to the invention in transgenic plants, especially monocotyledonous plants. Suitable vectors are derivatives of pAHC25 modified by introducing appropriate ubiquitin promoter and/or intron sequences or any of the transformation vectors described in this application.

root-specific expression

A preferred mode of expression of the nucleotide sequences of the invention is root expression. Root expression is especially useful for controlling soil-borne fungal pathogens. A suitable root promoter is that described by de Framond (FEBS 290:103-106 (1991)) and also described in published patent application EP0452269. The promoter is transferred into a suitable vector such as pCGN1761ENX for insertion of the nucleotide sequence and subsequent transfer of the complete promoter-gene-terminator cassette into the transformation vector of interest.

wound inducible promoter

Wound-inducible promoters are particularly suitable for the expression of the DNA molecules according to the invention, since they are generally active not only at the site of wound induction, but also at the site of infection by phytopathogens. Many such promoters have been described (such as Xu et al., Plant Molecular Biology 22: 573-588 (1993), Logemann et al., Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molecular Biology 22: 783-792 (1993), Firek et al., Plant Mol. Biol. 22: 129-142 (1993), Warner et al., Plant Journal 3: 191-201 (1993)) and are applicable to the present invention. Logemann et al. (supra) describe the 5' upstream sequence of the dicotyledonous potato wun1 gene. Xu et al. (supra) showed that a wound-inducible promoter from dicotyledonous potato (pin2) was active in monocotyledonous rice. In addition, Rohrmeier & Lehle (supra) describe the cloning of a maize Wipl cDNA that is inducible by wounding and can be used to isolate a homologous promoter by standard techniques. Likewise, Firek et al. (supra) and Warner et al. (supra) have described a wound-inducible gene from the monocotyledonous plant Asparagus officimalis that is expressed in local wounds and sites of pathogen entry. Using cloning techniques well known in the art, these promoters can be transferred into suitable vectors, fused to the DNA molecules of the invention, and used to express these genes at the site of plant pathogen infection.

Myeloid-preferred expression

Patent application WO93/07278 (Ciba-Geigy) describes the isolation of the maize trpA gene preferentially expressed in myeloid cells and gives the sequence of the promoter gene extending from the transcription start to position -1726. Using standard molecular biology techniques, this promoter, or a portion thereof, can be transferred into a vector such as pCGN1761, where it can replace the 35S promoter and be used to drive expression of the DNA molecules of the invention in a myeloid-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred into any vector and modified for use in transgenic plants.

Pollen-specific expression

Patent application WO93/07278 (Ciba-Geigy) further describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene expressed in pollen cells. The gene sequence and promoter extend up to 1400bp from the start of transcription. Using standard molecular biology techniques, this promoter, or a portion thereof, can be transferred into a vector such as pCGN1761, where it can replace the 35S promoter and be used to drive expression of the DNA molecules of the invention. In fact, fragments containing pollen-specific promoters or parts thereof can be transferred into any vector and modified for use in transgenic plants.

leaf-specific expression

The maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Mol. Biol. 12:579-589 (1989)). The promoter of this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants by standard molecular biology techniques.

Chloroplast-directed expression

Chen & Jagendorf (J. Biol. Chem. 268:2363-2367 (1993)) have described the successful use of chloroplast transit peptides to import heterologous transgenes. The peptide used was the transit peptide from the rbcS gene of Nicotiana plumbaginifolia (Poulsen et al., Molecular and General Genetics 205: 193-200 (1986)). The DNA sequence encoding this transit peptide can be excised from plasmid prbcS-8B (Poulsen et al., supra) using the restriction enzymes DraI and SphI, or Tsp509I and SphI, and used with any of the above constructs. The DraI-SphI fragment extends from position -58 relative to the starting rbcS ATG to and includes the first amino acid (also a methionine) of the mature peptide immediately following the import cleavage site, whereas the TspS09I-SphI The fragment extends from the -8 position relative to the starting rbcS ATG to and includes the first amino acid of the mature peptide. In this way, these fragments can be appropriately inserted into the polylinker of any expression cassette of choice, resulting in transcriptional fusions fused to the untranslated leader sequence of the promoter of choice (e.g., 35S, PR-1a, actin, ubiquitin, etc.) , while allowing the insertion of the DNA molecule of the present invention to be correctly fused downstream of the transit peptide. Such constructions are routine in the art. For example, the DraI end is blunt, while the 5' Tsp509I site can be filled in by treatment with T4 polymerase, or can be ligated with a linker or adapter sequence to facilitate its fusion to the promoter of choice. The 3'SphI site may likewise be maintained, or may be joined to a linker or adapter sequence to facilitate its insertion into the vector of choice, thereby providing a suitable restriction site for subsequent insertion of the DNA molecule of the invention. Ideally, the ATG of SphI is maintained and contains the first ATG of the DNA molecule of the invention. Chen & Jagendorf (supra) provide consensus sequences for ideal cleavage of chloroplast import, in each case methionine is preferred at the first position of the mature protein. Subsequent positions are subject to more variation, perhaps the amino acid is not so critical. In any case Bartlett et al. (in: Edelmann et al. (eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp1081-1091 (1982)) and Wasmann et al. (Molecular and General Genetics 205:446-453 (1986) ) method for assessing the efficiency of import of a fusion construct in vitro. Generally speaking, the best method may be to use the DNA molecule of the present invention with no modification at the amino terminus to create a fusion, and only introduce the modification when the fusion cannot be efficiently imported into the chloroplast. In this case, it can be carried out according to the provided literature. Modifications (Chen & Jagendorf, supra; Wasman et al., supra; Ko & Ko, J. Biol. Chem. 267: 13910-13916 (1992)).

Similar manipulations can be performed to utilize other GS2 chloroplast transit peptide coding sequences from other sources (monocots and dicots) or other genes. Furthermore, similar protocols can be followed for targeting to other subcellular compartments such as mitochondria.

sequence listing <110> Novartis <120> Genes controlling disease <130>S-30431/A <140>CGC1989 <141>1998-03-17 <150> US 09/042763 <151>1998-03-17 <160>42 <170>Patent In Ver. 2.0 <210>1 <211>13 <212>PRT <213> Artificial sequence <220> <223> Artificial sequence description: Conserved amino acid sequence <400>1Glu Leu Met Xaa Xaa Gly Xaa Ile Ser Leu Leu Leu Xaa1 5 10 <210>2 <211>14 <212>PRT <213> Artificial sequence <220> <223> Artificial sequence description: Conserved amino acid sequence <400>2Xaa Thr Xaa Pro Leu Xaa Xaa Xaa Val Xaa Gln Met Gly Ser1 5 10 <210>3 <211>1868 <212>DNA <213>Triticum sp. <220> <221> CDS <222>(176). ． (1777) <220> <221>misc_feature <222>(365). ． (403) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1352). ． (1393) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400>3cacgcccaca cttcgccaac acacaacgta cctgcgtacg tacgctttcc atttcctttc 60ttgctccggc cggccggcca cgtagaatag atacccggcc aggtaggtac ctcgttggct 120cagacgaccg gcggctgggt ctccggacaa ggaaagaggt tgcgctcggg gaccg atg  178Met1gcg gac gac gac gag tac ccc cca gcg agg acg ctg ccg gag acg ccg   226Ala Asp Asp Asp Glu Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr Pro5                  10                  15tcc tgg gcg gtg gcc ctc gtc ttc gcc gtc atg atc atc gtg tcc gtc   274Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser Val20                  25                  30ctc ctg gag cac gcg ctc cat aag ctc ggc cat tgg ttc cac aag cgg   322Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys Arg35                  40                  45cac aag aac gcg ctg gcg gag gcg ctg gag aag atc aag gcg gag ctc   370His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu Leu50                  55                  60                  65atg ctg gtg ggc ttc atc tcg ctg ctg ctc gcc gtg acg cag gac ccc   418Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp Pro70              75                      80atc tcc ggg ata tgc atc tcc gag aag gcc gcc agc atc atg cgg ccc   466Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg Pro85                  90                  95tgc aag ctg ccc cct ggc tcc gtc aag agc aag tac aaa gac tac tac   514Cys Lys Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr Tyr100                 105                 110tgc gcc aaa cag ggc aag gtg tcg ctc atg tcc acg ggc agc ttg cac   562Cys Ala Lys Gln Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu His115                 120                 125cag ctg cac ata ttc atc ttc gtg ctc gcc gtc ttc cat gtc acc tac   610Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr Tyr130                     135             140                 145agc gtc atc atc atg gct cta agc cgt ctc aaa atg aga acc tgg aag   658Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp Lys150                 155                 160aaa tgg gag aca gag acc gcc tcc ctg gaa tac cag ttc gca aat gat   706Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn Asp165                 170                 175cct gcg cgg ttc cgc ttc acg cac cag acg tcg ttc gtg aag cgg cac   754Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg His180                 185                 190ctg ggc ctc tcc agc acc ccc ggc gtc aga tgg gtg gtg gcc ttc ttc   802Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe Phe195                 200                 205agg cag ttc ttc agg tcg gtc acc aag gtg gac tac ctc acc ttg agg   850Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu Arg210                 215                 220                 225gca ggc ttc atc aac gcg cat ttg tcg cat aac agc aag ttc gac ttc   898Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp Phe230                 235                 240cac aag tac atc aag agg tcc atg gag gac gac ttc aaa gtc gtc gtt   946His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val Val245                 250                 255ggc atc agc ctc ccg ctg tgg tgt gtg gcg atc ctc acc ctc ttc ctt   994Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe Leu260                 265                 270gac att gac ggg atc ggc acg ctc acc tgg att tct ttc atc cct ctc   1042Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro Leu275                 280                 285gtc atc ctc ttg tgt gtt gga acc aag ctg gag atg atc atc atg gag   1090Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met Glu290                 295                 300                 305atg gcc ctg gag atc cag gac cgg gcg agc gtc atc aag ggg gcg ccc   1138Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala Pro310                 315                 320gtg gtt gag ccc agc aac aag ttc ttc tgg ttc cac cgc ccc gac tgg   1186Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp Trp325                 330                 335gtc ctc ttc ttc ata cac ctg acg cta ttc cag aac gcg ttt cag atg   1234Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met340                 345                 350gca cat ttc gtg tgg aca gtg gcc acg ccc ggc ttg aag aaa tgc ttc   1282Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys Phe355                 360                 365cat atg cac atc ggg ctg agc atc atg aag gtc gtg ctg ggg ctg gct   1330His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu Ala370                 375                 380                 385ctt cag ttc ctc tgc agc tat atc acc ttc ccg ctc tac gcg ctc gtc   1378Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu Val390                 395                 400aca cag atg gga tca aac atg aag agg tcc atc ttc gac gag cag acg   1426Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln Thr405                 410                  415gcc aag gcg ctg aca aac tgg cgg aac acg gcc aag gag aag aag aag   1474Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys Lys420                 425                 430gtc cga gac acg gac atg ctg atg gcg cag atg atc ggc gac gcg acg   1522Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala Thr435 440                 445ccc agc cga ggg gcg tcg ccc atg cct agc cgg ggc tcg tcg cca gtg   1570Pro Ser Arg Gly Ala Ser Pro Met Pro Ser Arg Gly Ser Ser Pro Val450                 455                 460                 465cac ctg ctt cac aag ggc atg gga cgg tcc gac gat ccc cag agc acg   1618His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser Thr470                 475                 480cca acc tcg cca agg gcc atg gag gag gct agg gac atg tac ccg gtt   1666Pro Thr Ser Pro Arg Ala Met Glu Glu Ala Arg Asp Met Tyr Pro Val485 490                     495gtg gtg gcg cat cca gtg cac aga cta aat cct gct gac agg aga agg   1714Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg Arg500                 505                 510tcg gtc tcg tcg tcg gca ctc gat gtc gac att ccc agc gca gat ttt 1762Ser Val Ser Ser Ser Ala Leu Asp Val Asp Ile Pro Ser Ala Asp Phe515                 520                 525tcc ttc agc cag gga tgagacaagt ttctgtattg atgttagtcc aatgtatagc   1817Ser Phe Ser Gln Gly530caacatagga tgtcatgatt cgtacaataa gaaatacaaa tttttactga g          1868 <210>4 <211>534 <212>PRT <213>Triticum sp. <400> 4MET ALA ALA ASP ASP ASP GLU TYR Pro Pro Ala Ala ARG THR Leu Glu THR1 5 10 15Pro Serp Ala Val Phe Ala Val Met Ile Val SELE Val SELE VAL GLU HIS ALA Leu His Lys Trp Phe His Lys35                 40                   45Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu50                   55                  60Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp65                  70              75                      80Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg85                  90                  95Pro Cys Lys Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr100                 105                 110Tyr Cys Ala Lys Gln Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu115                 120                 125His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr130                     135                 140Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp145                 150                 155                 160Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn165                 170                 175Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg180                 185                 190His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe195                 200                 205Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu210                 215                 220Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp225                 230                 235                 240She His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val245                 250                 255Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe260                 265                 270Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro275                 280                 285Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met290                 295                 300Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala305                 310                 315                 320Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp325                 330                 335Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln340                 345                 350Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys355                 360                 365Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu370                 375                 380Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu385                 390                 395                 400Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln405 410                 415Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys420                  425                 430Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala435                 440                 445Thr Pro Ser Arg Gly Ala Ser Pro Met Pro Ser Arg Gly Ser Ser Pro450                 455                 460Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser465                 470                 475                 480Thr Pro Thr Ser Pro Arg Ala Met Glu Glu Ala Arg Asp Met Tyr Pro485                 490                 495Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala ASP ARG ARG500 505 510ARG Ser Val Ser Ser Ala Leu ASP Val Asp Ile Pro Sering <210>5 <211>1693 <212>DNA <213>Triticum sp. <220> <221> CDS <222> (1). ． (1602) <220> <221>misc_feature <222>(190). ． (228) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1177). ． (1218) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400> 5ATG GCG GAG GAC TAC GAG TAG TAC CCC CCC CCG GCG CGG CTG CTG CCG GAG 48MET ALA Glu ASP Tyr Pro Pro Ala Ala GCCG GCG GCG G GCG G GCG G GCG G GCG G TC -C -C -C -C -TC's trTC -m thatces -m -tr that atc gtg tcc 96Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser20                  25                  30gtc ctc ctg gag cac gcg ctc cac aag ctc ggc cat tgg ttc cac aag 144Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys35                  40                  45cgg cac aag aac gcg ctg gcg gag gcg ctg gag aag atc aaa gcg gag 192Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu50                  55                  60ctg atg ctg gtg ggg ttc atc tcg ctg ctg ctc gcc gtg acg cag gac 240Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp65                  70                  75                  80cca atc tcc ggg ata tgc atc tcc gag aag gcc gcc agc atc atg cgg 288Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg85                  90                  95ccc tgc agc ctg ccc cct ggt tcc gtc aag agc aag tac aaa gac tac 336Pro Cys Ser Leu Pro Pro Gly Ser Val Lys Sar Lys Tyr Lys Asp Tyr100                 105                 110tac tgc gcc aaa aag ggc aag gtg tcg cta atg tcc acg ggc agc ttg 384Tyr Cys Ala Lys Lys Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu115                 120                 125cac cag ctc cac atg ttc atc ttc gtg ctc gcc gtc ttc cat gtc acc 432His Gln Leu His Met Phe Ile Phe Val Leu Ala Val Phe His Val Thr130                 135                 140tac agc gtc atc atc atg gct cta agc cgt ctc aaa atg agg aca tgg 480Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp145                 150                 155                 160aag aaa tgg gag aca gag acc gyc tcc ttg gaa tac cag ttc gca aat 528Lys Lys Trp Glu Thr Glu Thr Xaa Ser Leu Glu Tyr Gln Phe Ala Asn165                 170                 175gat cct gcg cgg ttc cgc ttc acg cac cag acg tcg ttc gtg aag cgt 576Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg180                  185                 190cac ctg ggc ctc tcc agc acc ccc ggc atc aga tgg gtg gtg gcc ttc 624His Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe195                 200                 205ttc agg cag ttc ttc agg tcg gtc acc aag gtg gac tac ctc acc ctg 672Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu210                 215                 220agg gca ggc ttc atc aac gcg cat ttg tcg cat aac agc aag ttc gac 720Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp225 230                 235                 240ttc cac aag tac atc aag agg tcc atg gag gac gac ttc aaa gtc gtc 768Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val245                 250                 255gtt ggc atc agc ctc ccg ctg tgg tgt gtg gcg atc ctc acc ctc ttc 816Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe260                 265                 270ctt gat att gac ggg atc ggc acg ctc acc tgg att tct ttc atc cct 864Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro275 280                 285ctc gtc atc ctc ttg tgt gtt gga acc aag ctg gag atg atc atc atg 912Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met290                 295                 300gag atg gcc ctg gag atc cag gac cgg gcg agc gtc atc aag ggg gcg 960Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala305                 310                 315                 320ccc gtg gtt gag ccc agc aac aag ttc ttc tgg ttc cac cgc ccc gac 1008Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp325 330                 335tgg gtc ctc ttc ttc ata cac ctg acg ctg ttc cag aat gcg ttt cag 1056Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln340                 345                 350atg gca cat ttc gtc tgg aca gtg gcc acg ccc ggc ttg aag aaa tgc 1104Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys355                 360                 365ttc cat atg cac atc ggt ctg agc atc atg aag gtc gtg ctg ggg ctg 1152Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu    370 375                 380gct ctt cag ttc ctc tgc agc tat atc acc ttc ccc ctc tac gcg ctc 1200Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu385                 390                 395                 400gtc aca cag atg gga tcg aac atg aag agg tcc atc ttc gac gag cag 1248Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln405                 410                 415acg gcc aag gcg ctg acc aac tgg cgg aac acg gcc aag gag aag aag 1296Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys420 425                 430aag gtc cga gac acg gac atg ctg atg gcg cag atg atc ggc gac gcg 1344Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala435                 440                 445acg ccc agc cga ggc acg tcg ccg atg cct agc cgg gct tcg tca ccg 1392Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Ala Ser Ser Pro450                 455                 460gtg cac ctg ctt cac aag ggc atg gga cgg tcc gac gat ccc cag agc 1440Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser465                 470 475                 480gcg ccg acc tcg cca agg acc atg gag gag gct agg gac atg tac ccg 1488Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro485                 490                 495gtt gtg gtg gcg cat ccc gtg cac aga cta aat cct gct gac agg cgg 1536Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg500                 505                 510agg tcg gtc tct tcg tcg gca ctc gat gcc gac atc ccc agc gca gat 1584Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp Ile Pro Ser Ala Asp515                 520 525ttt TCC TTC ATC CAG GGA TGAGACAGT TTCTGTAGTAGTCC 1632PHE Ser Gln Gly530AatgtataGA TGTCATAAAAAAAAATTTTTTGA 1692G 169939939993GA <210>6 <211>534 <212>PRT <213>Triticum sp. <400> 6MET ALA Glu ASP Tyr Glu Tyr Pro Pro Ala Ala ARG THR Leu Glu THR1 5 10 15Pro Serp Ala Val Phe Ala Val Met Ile Val SELE Val Glu Glu His Lys Leu His Lys Lys Trp Phe His Lys35                  40                  45Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu50                  55                  60Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp65                  70                  75                  80Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg85                  90                  95Pro Cys Ser Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr100                 105                 110Tyr Cys Ala Lys Lys Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu115             120                     125His Gln Leu His Met Phe Ile Phe Val Leu Ala Val Phe His Val Thr130             135                     140Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp145             150                      155                 160Lys Lys Trp Glu Thr Glu Thr Xaa Ser Leu Glu Tyr Gln Phe Ala Asn165                 170                 175Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg180                 185                 190His Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe195                 200                 205Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu210                 215                 220Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp225                 230                 235                 240Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val245                 250                 255Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe260                 265                 270Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro275                 280                 285Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met290                 295                 300Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala305                 310                 315                 320Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp325                 330                 335Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln340                 345                 350Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys355                 360                 365Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu370                 375                 380Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu385                 390                 395                 400Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln405 410                 415Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys420                 425                 430Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala435                 440                 445Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Ala Ser Ser Pro450                 455                 460Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser465                 470                 475                 480Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro485                 490                 495Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala ASP ARG ARG500 505 510ARG Ser Val Ser Sera Leu ASP ALA Asp Ile Pro Sero Sera Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala ALA ALA ALA ALE515 <210>7 <211>1886 <212>DNA <213>Triticum sp. <220> <221> CDS <222>(198). ． (1799) <220> <221>misc_feature <222>(387). ． (425) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1374). ． (1415) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400>7gcagcaacaa gctagacata cctgcgtgcg tacgtacgtt ttcgttttcc tttcttgctc 60cggccggccg gccggccacg tagaatagat acctgcccag gtacgtacct cgttggctca 120gacgatcggc ggttggactt gggtgcgcgc cctgccctgc tccggccaag gaaagaggtt 180gcgctaaaga cgggcgg atg gca aag gac gac ggg tac ccc ccg gcg cgg    230Met Ala Lys Asp Asp Gly Tyr Pro Pro Ala Arg1               5                  10acg ctg ccg gag acg ccg tcc tgg gcg gtg gcg ctg gtc ttc gcc gtc   278Thr Leu Pro Glu Thr Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val15                      20              25atg atc atc gtc tcc gtc ctc ctg gag cac gcg ctc cac aag ctc ggc   326Met Ile Ile Val Ser Val Leu Leu Glu His Ala Leu His Lys Leu Gly30                      35              40cat tgg ttc cac aag cgg cac aag aac gcg ctg gcg gag gcg ctg gag   374His Trp Phe His Lys Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu45                      50              55aag atg aag gcg gag ctg atg ctg gtg gga ttc atc tcg ctg ctg ctc   422Lys Met Lys Ala Glu Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu60                      65              70                  75gcc gtc acg cag gac cca atc tcc ggg ata tgc atc tcc cag aag gcc   470Ala Val Thr Gln Asp Pro Ile Ser Gly Ile Cys Ile Ser Gln Lys Ala80                  85                  90gcc agc atc atg cgc ccc tgc aag gtg gaa ccc ggt tcc gtc aag agc   518Ala Ser Ile Met Arg Pro Cys Lys Val Glu Pro Gly Ser Val Lys Ser95                 100                 105aag tac aag gac tac tac tgc gcc aaa gag ggc aag gtg gcg ctc atg   566Lys Tyr Lys Asp Tyr Tyr Cys Ala Lys Glu Gly Lys Val Ala Leu Met110                 115                 120tcc acg ggc agc ctg cac cag ctc cac ata ttc atc ttc gtg cta gcc   614Ser Thr Gly Ser Leu His Gln Leu His Ile Phe Ile Phe Val Leu Ala125                 130                 135gtc ttc cat gtc acc tac agc gtc atc a tc atg gct cta agc cgt ctc   662Val Phe His Val Thr Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu140                 145                 150                 155aag atg aga aca tgg aag aaa tgg gag aca gaa acc gcc tcc ttg gaa   710Lys Met Arg Thr Trp Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu160                 165                 170tac cag ttc gca aat gat cct gcg cgg ttc cgc ttc acg cac cag acg   758Tyr Gln Phe Ala Asn Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr175                 180                 185tcg ttc gtg aag cgg cac ctg ggc ctg tcc agc acc ccc ggc gtc aga   806Ser Phe Val Lys Arg His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg190                 195                 200tgg gtg gtg gcc ttc ttc agg cag ttc ttc agg tcg gtc acc aag gtg   854Trp Val Val Ala Phe Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val205                 210                 215gac tac ctc acc ttg agg gca ggc ttc atc aac gcg cac ttg tcg cag   902Asp Tyr Leu Thr Leu Arg Ala Gly Phe Ile Asn Ala His Leu Ser Gln220                 225                 230                 235aac agc aag ttc gac ttc cac aag tac atc aag agg tcc atg gag gac   950Asn Ser Lys Phe Asp Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp240                 245                 250gac ttc aaa gtc gtc gtt ggc atc agc ctc ccg ctg tgg gct gtg gcg   998Asp Phe Lys Val Val Val Gly Ile Ser Leu Pro Leu Trp Ala Val Ala255                 260                 265atc ctc acc ctc ttc ctt gat atc gac ggg atc ggc aca ctc acc tgg   1046Ile Leu Thr Leu Phe Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp270                 275                 280gtt tct ttc atc cct ctc atc atc ctc ttg tgt gtt gga acc aag cta   1094Val Ser Phe Ile Pro Leu Ile Ile Leu Leu Cys Val Gly Thr Lys Leu285                 290                 295gag atg atc atc atg ggg atg gcc ctg gag atc cag gac cgg tcg agc   1142Glu Met Ile Ile Met Gly Met Ala Leu Glu Ile Gln Asp Arg Ser Ser300                 305                 310                 315gtc atc aag ggg gca ccc gtg gtc gag ccc agc aac aag ttc ttc tgg   1190Val Ile Lys Gly Ala Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp320                 325                 330ttc cac cgc ccc gac tgg gtc ctc ttc ttc ata cac ctg acg ctg ttc   1238Phe His Arg Pro Asp Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe335                 340                 345cag aac gcg ttt cag atg gca cat ttc gtg tgg aca gtg gcc acg ccc   1286Gln Asn Ala Phe Gln Met Ala His Phe Val Trp Thr Val Ala Thr Pro350                 355                 360ggc ttg aag gac tgc ttc cat atg aac atc ggg ctg agc atc atg aag   1334Gly Leu Lys Asp Cys Phe His Met Asn Ile Gly Leu Ser Ile Met Lys365                 370                 375gtc gtg ctg ggg ctg gct ctc cag ttc ctg tgc agc tac atc acc ttc   1382Val Val Leu Gly Leu Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe380                 385                 390                 395ccc ctc tac gcg cta gtc aca cag atg gga tca aac atg aag agg tcc   1430Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser400                 405                 410atc ttc gac gag cag aca gcc aag gcg ctg acc aac tgg cgg aac acg   1478Ile Phe Asp Glu Gln Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr415                 420                 425gcc aag gag aag aag aag gtc cga gac acg gac atg ctg atg gcg cag   1526Ala Lys Glu Lys Lys Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln430                 435                 440atg atc ggc gac gca aca ccc agc cga ggc acg tcc ccg atg cct agc   1574Met Ile Gly Asp Ala Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser445                 450                 455cgg ggc tca tcg ccg gtg cac ctg ctt cag aag ggc atg gga cgg tct    1622Arg Gly Ser Ser Pro Val His Leu Leu Gln Lys Gly Met Gly Arg Ser460                 465                 470                 475gac gat ccc cag agc gca ccg acc tcg cca agg acc atg gag gag gct   1670Asp Asp Pro Gln Ser Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala480                 485                 490agg gac atg tac ccg gtt gtg gtg gcg cat cct gta cac aga cta aat   1718Arg Asp Met Tyr Pro Val Val Val Ala His Pro Val His Arg Leu Asn495                 500                 505cct gct gac agg cgg agg tcg gtc tct tca tca gcc ctc gat gcc gac   1766Pro Ala Asp Arg Arg Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp510                 515                 520atc ccc agc gca gat ttt tcc ttc agc cag gga tgagacaagt ttctgtattg 1819Ile Pro Ser Ala Asp Phe Ser Phe Ser Gln Gly525                 530atgttagtcc aatgtatagc caacatagga tgtgatgatt cgtacaataa gaaatacaat 1879tttttac                                                           1886 <210>8 <211>534 <212>PRT <213>Triticum sp. <400> 8MET ALA LY LY ASP ASP GLY TYR Pro Pro Ala Ala ARG THR Leu Glu THR1 5 10 15Pro Serp Ala Val Ala Val Met Ile Ile Val SELE Val SELE VAL GLU HIS ALA Leu His Lys Trp Phe His Lys35                  40                  45Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Met Lys Ala Glu50                  55                  60Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp65                  70                  75                  80Pro Ile Ser Gly Ile Cys Ile Ser Gln Lys Ala Ala Ser Ile Met Arg85                  90                  95Pro Cys Lys Val Glu Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr100                 105                 110Tyr Cys Ala Lys Glu Gly Lys Val Ala Leu Met Ser Thr Gly Ser Leu115                 120                 125His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr130                 135                 140Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp145                 150                 155                 160Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn165                 170                 175Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg180                 185                 190His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe195                 200                 205Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu210                 215                 220Arg Ala Gly Phe Ile Asn Ala His Leu Ser Gln Asn Ser Lys Phe Asp225                 230                 235                 240Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val245                 250                 255Val Gly Ile Ser Leu Pro Leu Trp Ala Val Ala Ile Leu Thr Leu Phe260                 265                 270Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Val Ser Phe Ile Pro275                 280                 285Leu Ile Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met290                 295                 300Gly Met Ala Leu Glu Ile Gln Asp Arg Ser Ser Val Ile Lys Gly Ala305                 310                 315                 320Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp325                 330                 335Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln340                 345                 350Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Asp Cys355                 360                 365Phe His Met Asn Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu370                 375                 380Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu385                 390                 395                 400Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln405 410                 415Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys420                 425                 430Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala435                 440                 445Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Gly Ser Ser Pro450                 455                 460Val His Leu Leu Gln Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser465                 470                 475                 480Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro485                 490                 495Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala ASP ARG ARG500 505 510ARG Ser Val Ser Sera Leu ASP ALA Asp Ile Pro Sero Sera Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala ALA ALA ALA ALE515 <210>9 <211>2197 <212>DNA <213> Arabidopsis thaliana <220> <221> CDS <222>(331). ． (2037) <220> <221>misc_feature <222>(589). ． (627) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1603). ． (1644) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400>9agtaatttag ctgttcttct acccctctga tctctcacag gggatcaaat agttttgata 60catagagcca caacagtgac attagtgtgt tgttgactac tgtaagggtt gggttttgaa 120aagagacatg aaggagtgtt attaggttga ttgtcttcaa gtacctccag tgtcaaacaa 180acattgacga ttgattctct tcccataatt tattgttatg cattacatat cacagtaaac 240ggactttcaa gtcaacaccg catttatttg ccctcttcat tgtttcacgt acgtaatcaa 300ggaccaaggg attttgttct tttggctacc atg gcc aca aga tgc ttt tgg tgt  354Met Ala Thr Arg Cys Phe Trp Cys1               5tgg acc act ttg ctc ttc tgc tct cag ctg ctt acc ggc ttt gcc cga   402Trp Thr Thr Leu Leu Phe Cys Ser Gln Leu Leu Thr Gly Phe Ala Arg10                  15                  20gct tcc tct gca ggc ggc gcc aaa gag aaa gga ctc tcc caa act ccc   450Ala Ser Ser Ala Gly Gly Ala Lys Glu Lys Gly Leu Ser Gln Thr Pro25                  30                  35                  40acc tgg gcc gtt gcc ctc gtc tgt acc ttt ttc att ctt gtc tcc gtc   498Thr Trp Ala Val Ala Leu Val Cys Thr Phe Phe Ile Leu Val Ser Val45                      50                  55ctt ctc gag aag gct ctt cac aga gtt gcc acg tgg ttg tgg gag aaa   546Leu Leu Glu Lys Ala Leu His Arg Val Ala Thr Trp Leu Trp Glu Lys60                  65                      70cat aag aac tct ctg ctt gaa gcc ttg gaa aaa ata aag gcc gag ctg   594His Lys Asn Ser Leu Leu Glu Ala Leu Glu Lys Ile Lys Ala Glu Leu75                  80                      85atg att cta gga ttc att tcc ttg ttg cta acc ttc gga gag cag tac   642Met Ile Leu Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Glu Gln Tyr90 95                     100att ctc aag att tgt att cct gaa aag gct gca gcc tct atg tta cct   690Ile Leu Lys Ile Cys Ile Pro Glu Lys Ala Ala Ala Ser Met Leu Pro105                 110                 115                 120tgt cca gct cct tct act cat gac caa gac aag acc cac cgc aga cgt   738Cys Pro Ala Pro Ser Thr His Asp Gln Asp Lys Thr His Arg Arg Arg125                 130                 135cta gct gct gct acg acc tct tcc cgc tgc gat gag ggt cat gaa cca   786Leu Ala Ala Ala Thr Thr Ser Ser Arg Cys Asp Glu Gly His Glu Pro140 145                 150ctc ata cct gcc acg ggt ttg cac cag cta cac att cta ttg ttc ttc   834Leu Ile Pro Ala Thr Gly Leu His Gln Leu His Ile Leu Leu Phe Phe155                 160                 165atg gct gcc ttt cat atc ctc tac agt ttc atc acc atg atg ctt ggc 882Met Ala Ala Phe His Ile Leu Tyr Ser Phe Ile Thr Met Met Leu Gly170                 175                 180aga ctc aag atc cgt ggc tgg aaa aag tgg gag cag gag aca tgt tct   930Arg Leu Lys Ile Arg Gly Trp Lys Lys Trp Glu Gln Glu Thr Cys Ser185                 190 195                 200cat gat tac gag ttt tca atc gat cca tca aga ttc aga ctc act cat   978His Asp Tyr Glu Phe Ser Ile Asp Pro Ser Arg Phe Arg Leu Thr His205                 210                 215gag acg tcc ttt gtt aga caa cat tcc agt ttc tgg aca aaa atc ccc 1026Glu Thr Ser Phe Val Arg Gln His Ser Ser Phe Trp Thr Lys Ile Pro220                 225                 230ttc ttc ttt tat gct ggg tgc ttc cta cag cag ttt ttc cga tct gtc   1074Phe Phe Phe Tyr Ala Gly Cys Phe Leu Gln Gln Phe Phe Arg Ser Val235                 240 245ggg agg act gac tac tta act ctg cgc cat ggc ttc atc gct gcc cat   1122Gly Arg Thr Asp Tyr Leu Thr Leu Arg His Gly Phe Ile Ala Ala His250                 255                 260tta gct cca gga aga aag ttc gac ttc cag aag tat atc aaa aga tca   1170Leu Ala Pro Gly Arg Lys Phe Asp Phe Gln Lys Tyr Ile Lys Arg Ser265                 270                 275                    280ttg gaa gac gat ttc aag gtg gta gtt gga ata agt cct ctt ttg tgg   1218Leu Glu Asp Asp Phe Lys Val Val Val Gly Ile Ser Pro Leu Leu Trp285                 290 295gca tca ttt gta att ttc cta ctt ctg aat gtt aat ggc tgg gaa gca   1266Ala Ser Phe Val Ile Phe Leu Leu Leu Asn Val Asn Gly Trp Glu Ala300                 305                 310ttg ttt tgg gcg tca atc cta cct gta ctt atc att cta gct gtc agt   1314Leu Phe Trp Ala Ser Ile Leu Pro Val Leu Ile Ile Leu Ala Val Ser315                 320                 325acg aag ctt caa gcg atc cta aca aga atg gct ctg gga atc acg gag   1362Thr Lys Leu Gln Ala Ile Leu Thr Arg Met Ala Leu Gly Ile Thr Glu330                 335                 340aga cac gca gtt gtt caa ggg ata cct ctc gtg cat ggt tca gat aag   1410Arg His Ala Val Val Gln Gly Ile Pro Leu Val His Gly Ser Asp Lys345                 350                 355                 360tac ttt tgg ttt aat cgc cct cag ttg cta ctt cat ctt ctt cac ttc   1458Tyr Phe Trp Phe Asn Arg Pro Gln Leu Leu Leu His Leu Leu His Phe365                 370                 375gcc tta ttt cag aat gct ttc cag cta aca tac ttc ttc tgg gtc tgg   1506Ala Leu Phe Gln Asn Ala Phe Gln Leu Thr Tyr Phe Phe Trp Val Trp380                 385                 390tat tcc ttt ggg cta aaa tct tgc ttt cac acg gat ttc aaa cta gtc   1554Tyr Ser Phe Gly Leu Lys Ser Cys Phe His Thr Asp Phe Lys Leu Val395                 400                 405atc gta aaa ctc tct cta ggc gtt gga gct ttg att ttg tgc agc tac   1602Ile Val Lys Leu Ser Leu Gly Val Gly Ala Leu Ile Leu Cys Ser Tyr410                 415                 420atc aca ctt cct ttg tat gca cta gtt act cag atg ggt tca aac atg   1650Ile Thr Leu Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser Asn Met425                 430                 435                 440aag aaa gct gtg ttt gat gag caa atg gca aaa gcg ttg aag aaa tgg   1698Lys Lys Ala Val Phe Asp Glu Gln Met Ala Lys Ala Leu Lys Lys Trp445                 450                 455cac atg act gtg aag aag aag aaa ggc aaa gcg aga aag cca cca aca   1746His Met Thr Val Lys Lys Lys Lys Gly Lys Ala Arg Lys Pro Pro Thr460                 465                 470gag acc ctt ggt gtt tct gac act gtc agc acc tct acc tca tcc ttt   1794Glu Thr Leu Gly Val Ser Asp Thr Val Ser Thr Ser Thr Ser Ser Phe475                 480                 485cac gcc tct gga gcc act cta ctc cgc tcc aag acc act ggt cac tcg   1842His Ala Ser Gly Ala Thr Leu Leu Arg Ser Lys Thr Thr Gly His Ser490                 495                 500aca gcc tct tat atg agt aat ttc gag gac caa agc atg tct gat ctt   1890Thr Ala Ser Tyr Met Ser Asn Phe Glu Asp Gln Ser Met Ser Asp Leu505                 510                 515                 520gaa gct gag cca tta tcc cct gaa cca ata gag ggg cac act ctc gtc   1938Glu Ala Glu Pro Leu Ser Pro Glu Pro Ile Glu Gly His Thr Leu Val525                 530                 535agg gtt ggt gat cag aac aca gag ata gaa tat act gga gat att agt   1986Arg Val Gly Asp Gln Asn Thr Glu Ile Glu Tyr Thr Gly Asp Ile Ser540                  545                550cct gga aac caa ttc tcc ttt gtg aag aac gtt cct gct aat gat att   2034Pro Gly Asn Gln Phe Ser Phe Val Lys Asn Val Pro Ala Asn Asp Ile555                 560                 565gac taatattcaa aatgaatgca gaacaaatcc atcatccggt ctttattttc        2087Asptattacatgt atgccaacaa ttgcttcgcc aagtgttacc aactaggttt tctgtataag 2147gctgtatttt agagctaaaa aaaaaaaaaa aaaaaaaaaa ctaaattact            2197 <210>10 <211>569 <212>PRT <213> Arabidopsis thaliana <400>10Met Ala Thr Arg Cys Phe Trp Cys Trp Thr Thr Leu Leu Phe Cys Ser1               5                  10                  15Gln Leu Leu Thr Gly Phe Ala Arg Ala Ser Ser Ala Gly Gly Ala Lys20                  25                  30Glu Lys Gly Leu Ser Gln Thr Pro Thr Trp Ala Val Ala Leu Val Cys35                  40                  45Thr Phe Phe Ile Leu Val Ser Val Leu Leu Glu Lys Ala Leu His Arg50                  55                  60Val Ala Thr Trp Leu Trp Glu Lys His Lys Asn Ser Leu Leu Glu Ala65                  70                  75                  80Leu Glu Lys Ile Lys Ala Glu Leu Met Ile Leu Gly Phe Ile Ser Leu85                  90                  95Leu Leu Thr Phe Gly Glu Gln Tyr Ile Leu Lys Ile Cys Ile Pro Glu100                 105                 110Lys Ala Ala Ala Ser Met Leu Pro Cys Pro Ala Pro Ser Thr His Asp115                 120                 125Gln Asp Lys Thr His Arg Arg Arg Leu Ala Ala Ala Thr Thr Ser Ser130                 135                 140Arg Cys Asp Glu Gly His Glu Pro Leu Ile Pro Ala Thr Gly Leu His145                 150                 155                 160Gln Leu His Ile Leu Leu Phe Phe Met Ala Ala Phe His Ile Leu Tyr165                 170                 175Ser Phe Ile Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly Trp Lys180                 185                 190Lys Trp Glu Gln Glu Thr Cys Ser His Asp Tyr Glu Phe Ser Ile Asp195                 200                 205Pro Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg Gln His210                 215                 220Ser Ser Phe Trp Thr Lys Ile Pro Phe Phe Phe Tyr Ala Gly Cys Phe225                 230                 235                 240Leu Gln Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu Thr Leu245                 250                 255Arg His Gly Phe Ile Ala Ala His Leu Ala Pro Gly Arg Lys Phe Asp260                 265                 270Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys Val Val275                 280                 285Val Gly Ile Ser Pro Leu Leu Trp Ala Ser Phe Val Ile Phe Leu Leu290                 295                 300Leu Asn Val Asn Gly Trp Glu Ala Leu Phe Trp Ala Ser Ile Leu Pro305                 310                 315                 320Val Leu Ile Ile Leu Ala Val Ser Thr Lys Leu Gln Ala Ile Leu Thr325                 330                 335Arg Met Ala Leu Gly Ile Thr Glu Arg His Ala Val Val Gln Gly Ile340                 345                 350Pro Leu Val His Gly Ser Asp Lys Tyr Phe Trp Phe Asn Arg Pro Gln355                 360                 365Leu Leu Leu His Leu Leu His Phe Ala Leu Phe Gln Asn Ala Phe Gln370                 375                 380Leu Thr Tyr Phe Phe Trp Val Trp Tyr Ser Phe Gly Leu Lys Ser Cys385                 390                 395                 400Phe His Thr Asp Phe Lys Leu Val Ile Val Lys Leu Ser Leu Gly Val405 410                 415Gly Ala Leu Ile Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr Ala Leu420                 425                 430Val Thr Gln Met Gly Ser Asn Met Lys Lys Ala Val Phe Asp Glu Gln435                 440                 445Met Ala Lys Ala Leu Lys Lys Trp His Met Thr Val Lys Lys Lys Lys450                 455                 460Gly Lys Ala Arg Lys Pro Pro Thr Glu Thr Leu Gly Val Ser Asp Thr465                 470                 475                 480Val Ser Thr Ser Thr Ser Ser Phe His Ala Ser Gly Ala Thr Leu Leu                485                 490                 495Arg Ser Lys Thr Thr Gly His Ser Thr Ala Ser Tyr Met Ser Asn Phe500                 505                 510Glu Asp Gln Ser Met Ser Asp Leu Glu Ala Glu Pro Leu Ser Pro Glu515                 520                 525Pro Ile Glu Gly His Thr Leu Val Arg Val Gly Asp Gln Asn Thr Glu530                 535                 540Ile Glu Tyr Thr Gly Asp Ile Ser Pro Gly Asn Gln Phe Ser Phe Val545 550 555 560Lys Asn Val Pro Ala Asn Asp Ile Asp565 <210>11 <211>1935 <212>DNA <213> Arabidopsis thaliana <220> <221> CDS <222>(89). ． (1807) <220> <221>misc_feature <222>(269). ． (307) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1370). ． (1411) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400>11cagtgtgagt aatttagtaa aaagacaaga tctctggtct ggaattagaa gaatcttatt 60tgggtttttt tcttaggatt aagctcta atg gca gat caa gta aaa gag cgg    112Met Ala Asp Gln Val Lys Glu Arg1              5act tta gag gag acc tct acg tgg gca gtt gct gtt gtt tgc ttt gtc   160Thr Leu Glu Glu Thr Ser THR TRP ALA Val Ala Val Val Cys PHE VAL10 15 20TTA CTC TTT ATT TCG ATT GTC GAA CAA CAA CAA CAC AAA ATT GGA 208leu PHE Ile Val Leu His LYS Ile Gly25 35 40 40 40 40 40 40 40Acly 25 40 40 40 40 40 40 40 40Acly 25 40 40 40 40 40 40 40 40 40 40Acly 25 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40Acly 25 aag cac aag cag gct ctt ttt gaa gct ctt gaa   256Thr Trp Phe Lys Lys Lys His Lys Gln Ala Leu Phe Glu Ala Leu Glu45                  50                  55aag gtc aaa gca gag ctt atg ctg ttg gga ttc ata tca cta cta ctc   304Lys Val Lys Ala Glu Leu Met Leu Leu Gly Phe Ile Ser Leu Leu Leu60                  65                  70aca att gga caa aca cca atc tca aat atc tgc atc tcc cag aaa gtt   352Thr Ile Gly Gln Thr Pro Ile Ser Asn Ile Cys Ile Ser Gln Lys Val75                  80                  85gcg tca aca atg cac cct tgc agt gct gct gaa gaa gct aaa aaa tac   400Ala Ser Thr Met His Pro Cys Ser Ala Ala Glu Glu Ala Lys Lys Tyr90                  95                 100ggc aag aaa gac gcc gga aag aaa gat gat gga gat gga gat aaa ccc   448Gly Lys Lys Asp Ala Gly Lys Lys Asp Asp Gly Asp Gly Asp Lys Pro105                 110                 115                 120ggt cga aga ctt ctt ctt gag tta gct gaa tct tat atc cat aga aga   496Gly Arg Arg Leu Leu Leu Glu Leu Ala Glu Ser Tyr Ile His Arg Arg125                 130                 135agt tta gcc acc aaa ggc tat gac aaa tgt gca gag aag ggg aaa gtg   544Ser Leu Ala Thr Lys Gly Tyr Asp Lys Cys Ala Glu Lys Gly Lys Val140                 145                 150gct ttt gta tct gct tat gga atc cac cag ctg cat ata ttc atc ttc   592Ala Phe Val Ser Ala Tyr Gly Ile His Gln Leu His Ile Phe Ile Phe155                 160                 165gtg ctc gcg gtt gtt cat gtt gtt tac tgc att gtt act tat gct ttc   640Val Leu Ala Val Val His Val Val Tyr Cys Ile Val Thr Tyr Ala Phe170                 175                 180gga aag atc aag atg agg acg tgg aag tcg tgg gag gaa gag aca aag   688Gly Lys Ile Lys Met Arg Thr Trp Lys Ser Trp Glu Glu Glu Thr Lys185                 190                 195                 200aca ata gag tat cag tat tcc aac gat cct gag agg ttc agg ttt gcg   736Thr Ile Glu Tyr Gln Tyr Ser Asn Asp Pro Glu Arg Phe Arg Phe Ala205                 210                 215agg gac aca tct ttt ggg aga aga cat ctc aat ttc tgg agc aag acg   784Arg Asp Thr Ser Phe Gly Arg Arg His Leu Asn Phe Trp Ser Lys Thr220                 225                 230aga gtc aca cta tgg att gtt tgt ttt ttt aga cag ttc ttt gga tct   832Arg Val Thr Leu Trp Ile Val Cys Phe Phe Arg Gln Phe Phe Gly Ser235                 240                 245gtc acc aaa gtt gat tac tta gca cta aga cat ggt ttc atc atg gcg   880Val Thr Lys Val Asp Tyr Leu Ala Leu Arg His Gly Phe Ile Met Ala250                 255                 260cat ttt gct ccc ggt aac gaa tca agattc gat ttc cgc aag tat att   928His Phe Ala Pro Gly Asn Glu Ser Arg Phe Asp Phe Arg Lys Tyr Ile265                 270                 275                 280cag aga tca tta gag aaa gac ttc aaa acc gtt gtt gaa atc agt ccg   976Gln Arg Ser Leu Glu Lys Asp Phe Lys Thr Val Val Glu Ile Ser Pro285                 290                 295gtt atc tgg ttt gtc gct gtg cta ttc ctc ttg acc aat tca tat gga   1024Val Ile Trp Phe Val Ala Val Leu Phe Leu Leu Thr Asn Ser Tyr Gly300                 305                 310tta cgt tct tac ctc tgg tta cca ttc att cca cta gtc gta att cta   1072Leu Arg Ser Tyr Leu Trp Leu Pro Phe Ile Pro Leu Val Val Ile Leu315                 320                 325ata gtt gga aca aag ctt gaa gtc ata ata aca aaa ttg ggt cta aga   1120Ile Val Gly Thr Lys Leu Glu Val Ile Ile Thr Lys Leu Gly Leu Arg330                 335                 340atc caa gag aaa ggt gat gtg gtg aga ggc gcc cca gtg gtt cag cct   1168Ile Gln Glu Lys Gly Asp Val Val Arg Gly Ala Pro Val Val Gln Pro345                 350                 355                 360ggt gat gac ctc ttc tgg ttt ggc aag cca cgc ttc att ctt ttc ctt   1216Gly Asp Asp Leu Phe Trp Phe Gly Lys Pro Arg Phe Ile Leu Phe Leu365                 370                 375att cac ttg gtc ctt ttt acg aat gca ttt caa ctt gca ttc ttt gcc   1264Ile His Leu Val Leu Phe Thr Asn Ala Phe Gln Leu Ala Phe Phe Ala380                 385                 390tgg agt acg tat gaa ttc aat ctc aat aat tgt ttc cat gaa agc act   1312Trp Ser Thr Tyr Glu Phe Asn Leu Asn Asn Cys Phe His Glu Ser Thr395                 400                 405gca gat gtg gtc att aga ctt gta gtt gga gct gtt gtg cag ata ctt   1360Ala Asp Val Val Ile Arg Leu Val Val Gly Ala Val Val Gln Ile Leu410                 415                 420tgc agc tat gtg act ctt cca ctc tat gca ctt gtt act cag atg ggt   1408Cys Ser Tyr Val Thr Leu Pro Leu Tyr Ala Leu Val Thr Gln Met Gly425                 430                 435                 440agt aaa atg aag cca aca gta ttc aac gat aga gta gcc acg gca tta   1456Ser Lys Met Lys Pro Thr Val Phe Asn Asp Arg Val Ala Thr Ala Leu445                 450                 455aag aag tgg cat cac act gca aag aac gag acg aaa cac gga aga cac   1504Lys Lys Trp His His Thr Ala Lys Asn Glu Thr Lys His Gly Arg His460                 465                 470tcg gga tcc aat aca cct ttc tct agc cgt cca act aca cca aca cat   1552Ser Gly Ser Asn Thr Pro Phe Ser Ser Arg Pro Thr Thr Pro Thr His475                 480                 485ggc tca tct cca atc cat ctc ctt cac aat ttc aat aac cgg agc gtt   1600Gly Ser Ser Pro Ile His Leu Leu His Asn Phe Asn Asn Arg Ser Val490                 495                 500gaa aat tac cca agt tct cct tct cct aga tac tct ggt cat ggt cat   1648Glu Asn Tyr Pro Ser Ser Pro Ser Pro Arg Tyr Ser Gly His Gly His505                 510                 515                 520cat gaa cac caa ttt tgg gat cct gag tct caa cac caa gaa gct gaa   1696His Glu His Gln Phe Trp Asp Pro Glu Ser Gln His Gln Glu Ala Glu525                 530                 535act tcc aca cat cat tct ctt gcg cat gaa agc tca gaa cct gtt ctt   1744Thr Ser Thr His His Ser Leu Ala His Glu Ser Ser Glu Pro Val Leu540                 545                 550gca tct gtg gaa ctt cct cct ata agg act agc aaa agc tta aga gat   1792Ala Ser Val Glu Leu Pro Pro Ile Arg Thr Ser Lys Ser Leu Arg Asp555                 560                 565ttt tct ttt aag aaa tgatgattct tgtttgctat atttgatttc gtacagtggg   1847Phe Ser Phe Lys Lys570aattttgtca tatgaaaata atttcttgta cattactagt ttgataagaa ataaccatat 1907ctatatggat acaaaaaaaa aaaaaaaa <210>12 <211>573 <212>PRT <213> Arabidopsis thaliana <400> 12MET ALA ASLN VAL LYS GLU ARG THR Leu Glu THR Serp1 5 10 15ALA Val Ala Val Val Leu Phe Ile Val Leu20 25 30glu see pHRS LYS LYS ILE GLLT THR THR TRT LYS LYS HIS LYS30 45GLN ALA Leu Phe Glu Ala Leu Lys Val Lys Ala Glu Leu Met Leu 50 60leu Gly PHE ILEU Leu Thr Ile Gln THRLN THRLLLLLSN Ile Cys Ile Cys Ile Cys Thr Met His Pro Cys Ser85                  90                  95Ala Ala Glu Glu Ala Lys Lys Tyr Gly Lys Lys Asp Ala Gly Lys Lys            100                 105                 110Asp Asp Gly Asp Gly Asp Lys Pro Gly Arg Arg Leu Leu Leu Glu Leu115                 120                 125Ala Glu Ser Tyr Ile His Arg Arg Ser Leu Ala Thr Lys Gly Tyr Asp130                 135                 140Lys Cys Ala Glu Lys Gly Lys Val Ala Phe Val Ser Ala Tyr Gly Ile145                 150                 155                 160His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Val His Val Val165                 170                 175Tyr Cys Ile Val Thr Tyr Ala Phe Gly Lys Ile Lys Met Arg Thr Trp180                 185                 190Lys Ser Trp Glu Glu Glu Thr Lys Thr Ile Glu Tyr Gln Tyr Ser Asn195                 200                 205Asp Pro Glu Arg Phe Arg Phe Ala Arg Asp Thr Ser Phe Gly Arg Arg210                 215                 220His Leu Asn Phe Trp Ser Lys Thr Arg Val Thr Leu Trp Ile Val Cys225                 230                 235                 240Phe Phe Arg Gln Phe Phe Gly Ser Val Thr Lys Val Asp Tyr Leu Ala245                 250                 255Leu Arg His Gly Phe Ile Met Ala His Phe Ala Pro Gly Asn Glu Ser260                 265                 270Arg Phe Asp Phe Arg Lys Tyr Ile Gln Arg Ser Leu Glu Lys Asp Phe275                 280                 285Lys Thr Val Val Glu Ile Ser Pro Val Ile Trp Phe Val Ala Val Leu290                 295                 300Phe Leu Leu Thr Asn Ser Tyr Gly Leu Arg Ser Tyr Leu Trp Leu Pro305                 310                 315                 320Phe Ile Pro Leu Val Val Ile Leu Ile Val Gly Thr Lys Leu Glu Val325                 330                 335Ile Ile Thr Lys Leu Gly Leu Arg Ile Gln Glu Lys Gly Asp Val Val340                 345                 350Arg Gly Ala Pro Val Val Gln Pro Gly Asp Asp Leu Phe Trp Phe Gly355                 360 365Lys Pro Arg Phe Ile Leu Phe Leu Ile His Leu Val Leu Phe Thr Asn370                 375                 380Ala Phe Gln Leu Ala Phe Phe Ala Trp Ser Thr Tyr Glu Phe Asn Leu385                 390                 395                 400Asn Asn Cys Phe His Glu Ser Thr Ala Asp Val Val Ile Arg Leu Val405                 410                 415Val Gly Ala Val Val Gln Ile Leu Cys Ser Tyr Val Thr Leu Pro Leu420                 425                 430Tyr Ala Leu Val Thr Gln Met Gly Ser Lys Met Lys Pro Thr Val Phe435                 440                 445Asn Asp Arg Val Ala Thr Ala Leu Lys Lys Trp His His Thr Ala Lys450                 455                 460Asn Glu Thr Lys His Gly Arg His Ser Gly Ser Asn Thr Pro Phe Ser465                 470                 475                 480Ser Arg Pro Thr Thr Pro Thr His Gly Ser Ser Pro Ile His Leu Leu485                 490                 495His Asn Phe Asn Asn Arg Ser Val Glu Asn Tyr Pro Ser Ser Pro Ser500                 505                 510Pro Arg Tyr Ser Gly His Gly His His Glu His Gln Phe Trp Asp Pro515                 520                 525Glu Ser Gln His Gln Glu Ala Glu Thr Ser Thr His His Ser Leu Ala530                 535                 540His Glu Ser Ser Glu Pro Val Leu Ala Ser Val Glu Leu Pro Pro Ile545 550 555 560Arg Thr Ser Lys Ser Leu Arg Asp Phe Ser Phe Lys Lys565 5 7   <210>13 <211>1811 <212>DNA <213> Arabidopsis thaliana <220> <221> CDS <222>(56). ． (1633) <220> <221>misc_feature <222>(236). ． (274) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1328). ． (1369) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400>13gttcccagat tcatctttac ttattgtcta aattctctct ggtgtgagaa gtaaa atg  58Met1ggt cac gga gga gaa ggg atg tcg ctt gaa ttc act ccg acg tgg gtc   106Gly His Gly Gly Glu Gly Met Ser Leu Glu Phe Thr Pro Thr Trp Val5                  10                  15gtc gcc gga gtt tgt acg gtc atc gtc gcg att tca ctg gcg gtg gag   154Val Ala Gly Val Cys Thr Val Ile Val Ala Ile Ser Leu Ala Val Glu20                  25                  30cgt ttg ctt cac tat ttc ggt act gtt ctt aag aag aag aag caa aaa   202Arg Leu Leu His Tyr Phe Gly Thr Val Leu Lys Lys Lys Lys Gln Lys35                  40                  45ccc ctt tac gaa gcc ctt caa aag gtt aaa gaa gag ctg atg ttg tta   250Pro Leu Tyr Glu Ala Leu Gln Lys Val Lys Glu Glu Leu Met Leu Leu50                  55                  60                  65ggg ttt ata tcg ctg tta ctg acg gta ttc caa ggg ctc att tcc aaa   298Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Leu Ile Ser Lys70                  75                  80ttc tgt gtg aaa gaa aat gtg ctt atg cat atg ctt cca tgt tct ctc   346Phe Cys Val Lys Glu Asn Val Leu Met His Met Leu Pro Cys Ser Leu85                  90                  95gat tca aga cga gaa gct ggg gca agt gaa cat aaa aac gtt aca gca   394Asp Ser Arg Arg Glu Ala Gly Ala Ser Glu His Lys Asn Val Thr Ala100                 105                 110aaa gaa cat ttt cag act ttt tta cct att gtt gga acc act agg cgt   442Lys Glu His Phe Gln Thr Phe Leu Pro Ile Val Gly Thr Thr Arg Arg115                 120                 125cta ctt gct gaa cat gct gct gtg caa gtt ggt tac tgt agc gaa aag   490Leu Leu Ala Glu His Ala Ala Val Gln Val Gly Tyr Cys Ser Glu Lys130                 135                 140                 145ggt aaa gta cca ttg ctt tcg ctt gag gca ttg cac cat cta cat att   538Gly Lys Val Pro Leu Leu Ser Leu Glu Ala Leu His His Leu His Ile150                 155                 160ttc atc ttc gtc ctc gcc ata tcc cat gtg aca ttc tgt gtc ctt acc   586Phe Ile Phe Val Leu Ala Ile Ser His Val Thr Phe Cys Val Leu Thr165                 170                 175gtg att ttt gga agc aca agg att cac caa tgg aag aaa tgg gag gat   634Val Ile Phe Gly Ser Thr Arg Ile His Gln Trp Lys Lys Trp Glu Asp180                 185                 190tcg atc gca gat gag aag ttt gac ccc gaa aca gct ctc agg aaa aga   682Ser Ile Ala Asp Glu Lys Phe Asp Pro Glu Thr Ala Leu Arg Lys Arg195                 200                 205agg gtc act cat gta cac aac cat gct ttt att aaa gag cat ttt ctt   730Arg Val Thr His Val His Asn His Ala Phe Ile Lys Glu His Phe Leu210                 215                 220                 225ggt att ggc aaa gat tca gtc atc ctc gga tgg acg caa tcc ttt ctc   778Gly Ile Gly Lys Asp Ser Val Ile Leu Gly Trp Thr Gln Ser Phe Leu230                 235                 240aag caa ttc tat gat tct gtg acg aaa tca gat tac gtg act tta cgt   826Lys Gln Phe Tyr Asp Ser Val Thr Lys Ser Asp Tyr Val Thr Leu Arg245                 250                 255ctt ggt ttc att atg aca cat tgt aag gga aac ccc aag ctt aat ttc   874Leu Gly Phe Ile Met Thr His Cys Lys Gly Asn Pro Lys Leu Asn Phe260                 265                 270cac aag tat atg atg cgc gct yta gag gat gat ttc aaa caa gtt gtt   922His Lys Tyr Met Met Arg Ala Xaa Glu Asp Asp Phe Lys Gln Val Val275                 280                 285ggt att agt tgg tat ctt tgg atc ttt gtc gtc atc ttt ttg ctg cta   970Gly Ile Ser Trp Tyr Leu Trp Ile Phe Val Val Ile Phe Leu Leu Leu290                 295                 300                 305aat gtt aac gga tgg cac aca tat ttc tgg ata gca ttt att ccc ttt   1018Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe Ile Pro Phe310                 315                 320gct ttg ctt ctt gct gtg gga aca aag ttg gag cat gtg att gca cag   1066Ala Leu Leu Leu Ala Val Gly Thr Lys Leu Glu His Val Ile Ala Gln325                 330                 335tta gct cat gaa gtt gca gag aaa cat gta gcc att gaa gga gac tta   1114Leu Ala His Glu Val Ala Glu Lys His Val Ala Ile Glu Gly Asp Leu340                 345                 350gtg gtg aaa ccc tca gat gag cat ttc tgg ttc agc aaa cct caa att   1162Val Val Lys Pro Ser Asp Glu His Phe Trp Phe Ser Lys Pro Gln Ile355                 360                 365gtt ctc tac ttg atc cat ttt atc ctc ttc cag aat gct ttt gag att   1210Val Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala Phe Glu Ile370                 375                 380                 385gcg ttt ttc ttt tgg att tgg gtt aca tac ggc ttc gac tcg tgc att   1258Ala Phe Phe Phe Trp Ile Trp Val Thr Tyr Gly Phe Asp Ser Cys Ile390                 395                 400atg gga cag gtg aga tac att gtt cca aga ttg gtt atc ggg gtc ttc   1306Met Gly Gln Val Arg Tyr Ile Val Pro Arg Leu Val Ile Gly Val Phe405                 410                 415att caa gtg ctt tgc agt tac agt aca ctg cct ctt tac gcc atc gtc   1354Ile Gln Val Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala Ile Val420                 425                 430tca cag atg gga agt agc ttc aag aaa gct ata ttc gag gag aat gtg   1402Ser Gln Met Gly Ser Ser Phe Lys Lys Ala Ile Phe Glu Glu Asn Val435                 440                 445cag gtt ggt ctt gtt ggt tgg gca cag aaa gtg aaa caa aag aga gac   1450Gln Val Gly Leu Val Gly Trp Ala Gln Lys Val Lys Gln Lys Arg Asp450                 455                 460                 465cta aaa gct gca gct agt aat gga aac gaa gga agc tct cag gct ggt 1498Leu Lys Ala Ala Ala Ser Asn Gly Asn Glu Gly Ser Ser Gln Ala Gly470                 475                 480cct ggt cct gat tct ggt tct ggt tct gct cct gct gct ggt cct ggt   1546Pro Gly Pro Asp Ser Gly Ser Gly Ser Ala Pro Ala Ala Gly Pro Gly485                 490                 495gca ggt ttt gca gga att cag ctc agc aga gta aca aga aac aac gca   1594Ala Gly Phe Ala Gly Ile Gln Leu Ser Arg Val Thr Arg Asn Asn Ala500                 505                 510ggg gac aca aac aat gag att aca cct gat cat aac aac tgagcagaga    1643Gly Asp Thr Asn Asn Glu Ile Thr Pro Asp His Asn Asn515                 520 525tattatcttt tccatttaga ggatcatcat cagattttag cttcaaggtc cggttttgtg 1703gtttatacat aagttatagt gacttgattt ttttgttttg ttacaaagtt accatctttg 1763gattagaatt gggaaattga atctgtttgt aaaaaaaaaa aaaaaaaa              1811 <210>14 <211>526 <212>PRT <213> Arabidopsis thaliana <400>14Met Gly His Gly Gly Glu Gly Met Ser Leu Glu Phe Thr Pro Thr Trp1               5                  10                  15Val Val Ala Gly Val Cys Thr Val Ile Val Ala Ile Ser Leu Ala Val20                  25                  30Glu Arg Leu Leu His Tyr Phe Gly Thr Val Leu Lys Lys Lys Lys Gln35                  40                  45Lys Pro Leu Tyr Glu Ala Leu Gln Lys Val Lys Glu Glu Leu Met Leu50                  55                  60Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Leu Ile Ser65                  70                  75                  80Lys Phe Cys Val Lys Glu Asn Val Leu Met His Met Leu Pro Cys Ser85                  90                  95Leu Asp Ser Arg Arg Glu Ala Gly Ala Ser Glu His Lys Asn Val Thr100                 105                 110Ala Lys Glu His Phe Gln Thr Phe Leu Pro Ile Val Gly Thr Thr Arg115                 120                 125Arg Leu Leu Ala Glu His Ala Ala Val Gln Val Gly Tyr Cys Ser Glu130                 135                 140Lys Gly Lys Val Pro Leu Leu Ser Leu Glu Ala Leu His His Leu His145                 150                 155                 160Ile Phe Ile Phe Val Leu Ala Ile Ser His Val Thr Phe Cys Val Leu165                 170                 175Thr Val Ile Phe Gly Ser Thr Arg Ile His Gln Trp Lys Lys Trp Glu180                 185                 190Asp Ser Ile Ala Asp Glu Lys Phe Asp Pro Glu Thr Ala Leu Arg Lys195                 200                 205Arg Arg Val Thr His Val His Asn His Ala Phe Ile Lys Glu His Phe210                 215                 220Leu Gly Ile Gly Lys Asp Ser Val Ile Leu Gly Trp Thr Gln Ser Phe225                 230                 235                 240Leu Lys Gln Phe Tyr Asp Ser Val Thr Lys Ser Asp Tyr Val Thr Leu245                 250                 255Arg Leu Gly Phe Ile Met Thr His Cys Lys Gly Asn Pro Lys Leu Asn260                 265                 270Phe His Lys Tyr Met Met Arg Ala Xaa Glu Asp Asp Phe Lys Gln Val275                 280                 285Val Gly Ile Ser Trp Tyr Leu Trp Ile Phe Val Val Ile Phe Leu Leu290                 295                 300Leu Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe Ile Pro305                 310                 315                 320Phe Ala Leu Leu Leu Ala Val Gly Thr Lys Leu Glu His Val Ile Ala325                 330                 335Gln Leu Ala His Glu Val Ala Glu Lys His Val Ala Ile Glu Gly Asp340                 345                 350Leu Val Val Lys Pro Ser Asp Glu His Phe Trp Phe Ser Lys Pro Gln355                 360                 365Ile Val Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala Phe Glu370                 375                 380Ile Ala Phe Phe Phe Trp Ile Trp Val Thr Tyr Gly Phe Asp Ser Cys385                 390                 395                 400Ile Met Gly Gln Val Arg Tyr Ile Val Pro Arg Leu Val Ile Gly Val405 410                 415Phe Ile Gln Val Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala Ile420                 425                 430Val Ser Gln Met Gly Ser Ser Phe Lys Lys Ala Ile Phe Glu Glu Asn435                 440                 445Val Gln Val Gly Leu Val Gly Trp Ala Gln Lys Val Lys Gln Lys Arg450                 455                 460Asp Leu Lys Ala Ala Ala Ser Asn Gly Asn Glu Gly Ser Ser Gln Ala465                 470                 475                 480Gly Pro Gly Pro Asp Ser Gly Ser Gly Ser Ala Pro Ala Ala Gly Pro485                 490                 495Gly Ala Gly Phe Ala Gly Ile Gln Leu Ser Arg Val Thr Arg Asn Asn500 505 510Ala Gly Asp Thr Asn Asn Glu Ile Thr Pro Asp His Asn Asn515 520 5 5 <210>15 <211>1782 <212>DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1). ． (1779) <220> <221>misc_feature <222>(274). ． (313) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1327). ． (1370) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400> 15ATG GGA ATC ATC GAC GAC GGT TCT TTG CTT CGG CGG TTG ATT TGT CTC TGT 48MET GLY Ile ASP GLY Serg acg gcg gag gat   96Leu Trp Cys Leu Leu Gly Gly Gly Val Thr Val Val Thr Ala Glu Asp20                  25                  30gag aag aaa gtg gta cat aaa cag ctt aat caa act ccg act tgg gct   144Glu Lys Lys Val Val His Lys Gln Leu Asn Gln Thr Pro Thr Trp Ala35                  40                  45gtt gct gct gtt tgt act ttc ttc atc gtt gtt tct gtt ctt ctt gaa   192Val Ala Ala Val Cys Thr Phe Phe Ile Val Val Ser Val Leu Leu Glu50                  55                  60aaa ctt ctt cac aaa gtt gga aag gtt cta tgg gat cgg cac aag aca   240Lys Leu Leu His Lys Val Gly Lys Val Leu Trp Asp Arg His Lys Thr65                  70                  75                  80gct ctt ctt gac gct ttg gag aag atc aaa gca gag ctg atg gtt ctt   288Ala Leu Leu Asp Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Val Leu85                  90                  95gga ttc atc tct ttg ctt ctg aca ttt gga caa acc tac att ttg gat   336Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Gln Thr Tyr Ile Leu Asp100                 105                 110att tgt atc cct tca cat gtt gct cgt acg atg ctc ccg tgt cct gct   384Ile Cys Ile Pro Ser His Val Ala Arg Thr Met Leu Pro Cys Pro Ala115                 120                 125cct aac ttg aaa aag gag gat gat gac aat ggt gaa agt cac agg aga   432Pro Asn Leu Lys Lys Glu Asp Asp Asp Asn Gly Glu Ser His Arg Arg130                 135                 140ctc ttg tcg ttt gag cac aga ttt tta tct gga ggt gaa gca tct ccc   480Leu Leu Ser Phe Glu His Arg Phe Leu Ser Gly Gly Glu Ala Ser Pro145                 150                 155                 160act aaa tgc acg aag gag ggt tat gta gag ctt atc tct gcc gag gca   528Thr Lys Cys Thr Lys Glu Gly Tyr Val Glu Leu Ile Ser Ala Glu Ala165                 170                 175ctc cat cag ttg cac atc ctt ata ttc ttc tta gcc att ttc cac gtt   576Leu His Gln Leu His Ile Leu Ile Phe Phe Leu Ala Ile Phe His Val180                 185                 190ctt tac agc ttc tta act atg atg ctt gga agg ttg aag att cgc gga   624Leu Tyr Ser Phe Leu Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly195                 200                 205tgg aag cat tgg gag aat gag aca tca tcc cat aat tac gag ttt tca   672Trp Lys His Trp Glu Asn Glu Thr Ser Ser His Asn Tyr Glu Phe Ser210                 215                 220aca gac act tcc agattc agg cta act cat gaa aca tct ttt gtg aga   720Thr Asp Thr Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg225 230                 235                 240gcg cac acc agt ttc tgg acc cgg att cca ttc ttt ttc tat gtt gga   768Ala His Thr Ser Phe Trp Thr Arg Ile Pro Phe Phe Phe Tyr Val Gly245                 250                  255tgc ttt ttc aga cag ttt ttc aga tcc gtt ggg aga act gac tat ttg   816Cys Phe Phe Arg Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu260                 265                 270aca ttg aga aat ggt ttc atc gct gtt cat tta gct cca gga agt caa   864Thr Leu Arg Asn Gly Phe Ile Ala Val His Leu Ala Pro Gly Ser Gln275 280                 285ttt aac ttc caa aaa tac att aaa aga tcg ttg gag gat gat ttc aag   912Phe Asn Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys290                 295                 300gta gtc gtt gga gtc agc cct gtc ttg tgg gga tct ttt gtg cta ttc 960Val Val Val Gly Val Ser Pro Val Leu Trp Gly Ser Phe Val Leu Phe305                 310                 315                 320ctc ctc cta aat att gac ggt gag tat atg atg ttc atc ggc act gca   1008Leu Leu Leu Asn Ile Asp Gly Glu Tyr Met Met Phe Ile Gly Thr Ala325 330                 335ata ccg gtt att atc att tta gct gta ggg aca aag ctt caa gcg att   1056Ile Pro Val Ile Ile Ile Leu Ala Val Gly Thr Lys Leu Gln Ala Ile340                 345                 350atg aca agg atg gct ctt ggt atc aca gat aga cat gcg gta gtt caa 1104Met Thr Arg Met Ala Leu Gly Ile Thr Asp Arg His Ala Val Val Gln355                 360                 365gga atg ccg ctt gta caa ggc aac gat gag tat ttc tgg ttc ggt cgt   1152Gly Met Pro Leu Val Gln Gly Asn Asp Glu Tyr Phe Trp Phe Gly Arg370                 375 380ccc cat ttg att ctc cat ctc atg cat ttc gcc ttg ttt cag aac gca   1200Pro His Leu Ile Leu His Leu Met His Phe Ala Leu Phe Gln Asn Ala385                 390                 395                 400ttt cag atc act tat ttc ttc tgg ata tgg tat tcc ttt gga tca gat 1248Phe Gln Ile Thr Tyr Phe Phe Trp Ile Trp Tyr Ser Phe Gly Ser Asp405                 410                 415tct tgc tac cat cct aat ttc aag att gca ctt gta aaa gta gcg att   1296Ser Cys Tyr His Pro Asn Phe Lys Ile Ala Leu Val Lys Val Ala Ile420                 425 430gct tta gga gta ttg tgt ctt tgc agc tac atc aca ctt cct ctt tac   1344Ala Leu Gly Val Leu Cys Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr435                 440                 445gca ctc gta act cag atg ggt tct cgg atg aaa aaa tcg gta ttc gat    1392Ala Leu Val Thr Gln Met Gly Ser Arg Met Lys Lys Ser Val Phe Asp450                 455                 460gaa caa acg tca aaa gca ctc aag aaa tgg aga atg gca gtg aag aag   1440Glu Gln Thr Ser Lys Ala Leu Lys Lys Trp Arg Met Ala Val Lys Lys465                 470                 475 480aag aaa ggt gtg aaa gcc act act aag aga cta ggt gga gat gga agt   1488Lys Lys Gly Val Lys Ala Thr Thr Lys Arg Leu Gly Gly Asp Gly Ser485                 490                 495gcg agc cct acg gca tcg aca gtt agg tct act tcg tct gta cgt tca   1536Ala Ser Pro Thr Ala Ser Thr Val Arg Ser Thr Ser Ser Val Arg Ser500                 505                 510ttg cag cgt tac aaa acc aca cca cat tcg atg aga tac gaa gga ctt   1584Leu Gln Arg Tyr Lys Thr Thr Pro His Ser Met Arg Tyr Glu Gly Leu515                 520                 525gac cct gaa aca tcg gat ctc gac aca gat aat gaa gct ttg act cct   1632Asp Pro Glu Thr Ser Asp Leu Asp Thr Asp Asn Glu Ala Leu Thr Pro530                 535                 540ccc aaa tct cct cca agc ttc gag ctt gtt gtg aaa gtt gaa cca aat   1680Pro Lys Ser Pro Pro Ser Phe Glu Leu Val Val Lys Val Glu Pro Asn545                 550                 555                 560aag acc aat acc ggt gag act agc cgt gac act gaa act gat tct aaa   1728Lys Thr Asn Thr Gly Glu Thr Ser Arg Asp Thr Glu Thr Asp Ser Lys565                 570                 575gag TTC TCT TTC GTC Aag CCT GCT CCG AGT AAT GAA TCA TCT CAA GAC 1776GLU PHE VAL LYS PRO Ala Pro Ser Asn Gln ASP585 590CGG TGA 1782arg <210>16 <211>593 <212>PRT <213> Arabidopsis thaliana <400> 16MET GLY Ile ASP GLY Serg ARG Leu Ile Cys1 5 10 15LEU Leu Leu Leu Gly Gly Val Val THR Ala Glu Alas LYS VAl His LEU ALN Leu Alas LYS LEU Pro Thr Trp Ala35                  40                  45Val Ala Ala Val Cys Thr Phe Phe Ile Val Val Ser Val Leu Leu Glu50                  55                  60Lys Leu Leu His Lys Val Gly Lys Val Leu Trp Asp Arg His Lys Thr65                  70                  75                  80Ala Leu Leu Asp Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Val Leu85                  90                  95Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Gln Thr Tyr Ile Leu Asp100                 105                 110Ile Cys Ile Pro Ser His Val Ala Arg Thr Met Leu Pro Cys Pro Ala115                 120                 125Pro Asn Leu Lys Lys Glu Asp Asp Asp Asn Gly Glu Ser His Arg Arg130                 135                 140Leu Leu Ser Phe Glu His Arg Phe Leu Ser Gly Gly Glu Ala Ser Pro145                 150                 155                 160Thr Lys Cys Thr Lys Glu Gly Tyr Val Glu Leu Ile Ser Ala Glu Ala165                 170                 175Leu His Gln Leu His Ile Leu Ile Phe Phe Leu Ala Ile Phe His Val180                 185                 190Leu Tyr Ser Phe Leu Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly195                 200                 205Trp Lys His Trp Glu Asn Glu Thr Ser Ser His Asn Tyr Glu Phe Ser210                 215                 220Thr Asp Thr Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg225                 230                 235                 240Ala His Thr Ser Phe Trp Thr Arg Ile Pro Phe Phe Phe Tyr Val Gly245                 250                 255Cys Phe Phe Arg Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu260                 265                 270Thr Leu Arg Asn Gly Phe Ile Ala Val His Leu Ala Pro Gly Ser Gln275                 280                 285Phe Asn Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys290                 295                 300Val Val Val Gly Val Ser Pro Val Leu Trp Gly Ser Phe Val Leu Phe305                 310                 315                 320Leu Leu Leu Asn Ile Asp Gly Glu Tyr Met Met Phe Ile Gly Thr Ala325                 330                 335Ile Pro Val Ile Ile Ile Leu Ala Val Gly Thr Lys Leu Gln Ala Ile            340                 345                 350Met Thr Arg Met Ala Leu Gly Ile Thr Asp Arg His Ala Val Val Gln355                 360 365Gly Met Pro Leu Val Gln Gly Asn Asp Glu Tyr Phe Trp Phe Gly Arg370                 375                 380Pro His Leu Ile Leu His Leu Met His Phe Ala Leu Phe Gln Asn Ala385                 390                 395                 400Phe Gln Ile Thr Tyr Phe Phe Trp Ile Trp Tyr Ser Phe Gly Ser Asp405                 410                 415Ser Cys Tyr His Pro Asn Phe Lys Ile Ala Leu Val Lys Val Ala Ile420                 425                 430Ala Leu Gly Val Leu Cys Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr435                 440                 445Ala Leu Val Thr Gln Met Gly Ser Arg Met Lys Lys Ser Val Phe Asp450                 455                 460Glu Gln Thr Ser Lys Ala Leu Lys Lys Trp Arg Met Ala Val Lys Lys465                 470                 475                 480Lys Lys Gly Val Lys Ala Thr Thr Lys Arg Leu Gly Gly Asp Gly Ser485                 490                 495Ala Ser Pro Thr Ala Ser Thr Val Arg Ser Thr Ser Ser Val Arg Ser500                 505                 510Leu Gln Arg Tyr Lys Thr Thr Pro His Ser Met Arg Tyr Glu Gly Leu515                 520                 525Asp Pro Glu Thr Ser Asp Leu Asp Thr Asp Asn Glu Ala Leu Thr Pro530                 535                 540Pro Lys Ser Pro Pro Ser Phe Glu Leu Val Val LYS VAL GLU Pro Asn545 550 560LYS THR Asn Thr GLY GLU THR Serg ARG ARG ARG ASP THR ASP Ser Lys565 575Glu Phe Val Lysn Gln Gln ASP585 595590 595 595585585585 590 <210>17 <211>1629 <212>DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1). ． (1626) <220> <221>misc_feature <222>(184). ． (223) <223>The position of the amino acid sequence shown in SEQ ID NO: 1 <220> <221>misc_feature <222>(1141). ． (1183) <223> The position of the amino acid sequence shown in SEQ ID NO: 2 <400> 17atg Cat atg AAA GAA GGA GGA AGG TCT CTT GCA GAG ACG ACG Act 48MET GLU HIS MET MET MET MET LYS GLU GLY Serg Ser Leu THR PRO THR1 5 10 15TAC TCG GTT GTT GTT GTT TGT GTT TGT GTT TGTGTA tgc ttt ctc   96Tyr Ser Val Ala Ser Val Val Thr Val Leu Val Phe Val Cys Phe Leu20                  25                  30gtt gaa cgc gcc att tac aga ttt gga aag tgg tta aag aag act aga   144Val Glu Arg Ala Ile Tyr Arg Phe Gly Lys Trp Leu Lys Lys Thr Arg35                  40                  45aga aag gca ctt ttt act tca ctt gag aaa atg aaa gag gag ttg atg   192Arg Lys Ala Leu Phe Thr Ser Leu Glu Lys Met Lys Glu Glu Leu Met50                  55                  60ttg ctg gga ctt ata tca ctt ctg ttg tca caa agc gcg aga TGG Att 240leu Leu Ile Seru Leu LEU Leu Serg Trg TRP ILE65 80TCA GAA ATC TGT GTT AAC CTT AGT AAAA TAES SET 288ser Glu Ile Cys Val aser Cys Val Tyr Ile85                  90                  95tgc tct gaa gag gac tat gga atc cat aag aaa gtt ctt ctg gaa cac   336Cys Ser Glu Glu Asp Tyr Gly Ile His Lys Lys Val Leu Leu Glu His100                 105                 110acc tct tct aca aac cag agc tcc tta cct cat cat gga ata Cat Gaa 384thr Serte THR ASN GLN SER Leu PRO HIS HIS GLY Ile His Glu115 125GCC TCT CAA TGT Cat GGC CGT GAA CCA TAT GAG TAG TAG TAT Hisl SERN CYS GLY HIS GLL ARG GLY ARG GLY ARG GLY ARG GLY ARG GLY ARG GLY ARG GLY ARG GLY ARG GLY GLY ARG GLY GLY GLY GLY GLY GLY GLY ARG Glu130                 135                 140gga ctc gag caa ctc cta agattc tta ttc gtc ctg ggt atc act cat   480Gly Leu Glu Gln Leu Leu Arg Phe Leu Phe Val Leu Gly Ile Thr His145                 150                 155                160gtt cta tac agt ggc att gcc att ggt tta gcc atg agc aag att tac   528Val Leu Tyr Ser Gly Ile Ala Ile Gly Leu Ala Met Ser Lys Ile Tyr165                 170                 175agt tgg aga aaa tgg gaa gcc caa gcg atc ata atg gct gaa tca gat   576Ser Trp Arg Lys Trp Glu Ala Gln Ala Ile Ile Met Ala Glu Ser Asp180 185                 190atc cac ctt tgt ttc ctg cgg caa ttt aga ggc tcc ata cga aag tct    624Ile His Leu Cys Phe Leu Arg Gln Phe Arg Gly Ser Ile Arg Lys Ser195                 200                 205gac tac ttc gca ctt cgg tta ggt ttc ctc act aaa cat aat ttg cca 672Asp Tyr Phe Ala Leu Arg Leu Gly Phe Leu Thr Lys His Asn Leu Pro210                 215                 220ttt aca tac aac ttc cat atg tat atg gta cgg acg atg gaa gat gag   720Phe Thr Tyr Asn Phe His Met Tyr Met Val Arg Thr Met Glu Asp Glu225                 230 235                 240ttt cat ggc att gtt gga att agc tgg cca ctt tgg gtt tac gct ata   768Phe His Gly Ile Val Gly Ile Ser Trp Pro Leu Trp Val Tyr Ala Ile245                 250                 255gta tgc atc tgc ata aat gtt cat ggc ctg aat atg tac ttt tgg ata 816Val Cys Ile Cys Ile Asn Val His Gly Leu Asn Met Tyr Phe Trp Ile260                 265                 270tca ttc gtt cct gcc att ctt gtc atg ttg gtt gga acc aaa ctt gag   864Ser Phe Val Pro Ala Ile Leu Val Met Leu Val Gly Thr Lys Leu Glu275                 280 285cat gtt gtc tcc aag ctt gct ctc gag gtt aag gag cag cag aca ggc   912His Val Val Ser Lys Leu Ala Leu Glu Val Lys Glu Gln Gln Thr Gly290                 295                 300aca tct aat ggg gct caa gtc aaa cca cgt gat ggg ctc ttc tgg ttt   960Thr Ser Asn Gly Ala Gln Val Lys Pro Arg Asp Gly Leu Phe Trp Phe305                 310                 315                 320ggg aaa cca gaa att ctg cta cgg ttg ata caa ttt atc att ttt cag   1008Gly Lys Pro Glu Ile Leu Leu Arg Leu Ile Gln Phe Ile Ile Phe Gln325                 330 335aat gca ttt gaa atg gca aca ttc atc tgg ttc ttg tgg gga atc aag   1056Asn Ala Phe Glu Met Ala Thr Phe Ile Trp Phe Leu Trp Gly Ile Lys340                 345                 350gaa aga tct tgc ttc atg aag aac cat gtg atg ata tca agc cgg cta   1104Glu Arg Ser Cys Phe Met Lys Asn His Val Met Ile Ser Ser Arg Leu355                 360                 365att tct ggg gtt ctc gtt cag ttc tgg tgt agt tat ggc act gtg cct   1152Ile Ser Gly Val Leu Val Gln Phe Trp Cys Ser Tyr Gly Thr Val Pro370                 375                  380ctc aat gta atc gtt act cag atg gga tct cgg cat aag aaa gct gtg   1200Leu Asn Val Ile Val Thr Gln Met Gly Ser Arg His Lys Lys Ala Val385                 390                 395                 400ata gca gag agc gta aga gac tca ctt cac agt tgg tgc aag aga gtg   1248Ile Ala Glu Ser Val Arg Asp Ser Leu His Ser Trp Cys Lys Arg Val405                 410                 415aaa gag agg tct aag cac acg aga tca gtg tgt tcc ctt gac aca gca   1296Lys Glu Arg Ser Lys His Thr Arg Ser Val Cys Ser Leu Asp Thr Ala420                 425                 430aca ata gac gag aga gac gag atg aca gtg ggg aca ttg tct agg agc   1344Thr Ile Asp Glu Arg Asp Glu Met Thr Val Gly Thr Leu Ser Arg Ser435                 440                 445tca tcg atg act tca ctg aat cag att acc ata aac tcc ata gac caa   1392Ser Ser Met Thr Ser Leu Asn Gln Ile Thr Ile Asn Ser Ile Asp Gln450                 455                 460gca gag tct ata ttc gga gca gca gct tca tcc agc agt cct caa gat    1440Ala Glu Ser Ile Phe Gly Ala Ala Ala Ser Ser Ser Ser Pro Gln Asp465                 470                 475                 480gga tac acg tcg agg gtg gaa gaa tat ctg tct gaa aca tac aat aac   1488Gly Tyr Thr Ser Arg Val Glu Glu Tyr Leu Ser Glu Thr Tyr Asn Asn485                 490                 495atc ggt tcg ata ccg cct tta aac gat gag att gag att gag att gaa   1536Ile Gly Ser Ile Pro Pro Leu Asn Asp Glu Ile Glu Ile Glu Ile Glu500                 505                 510ggt gaa gaa gat aat gga ggg aga gga agt ggg agt gat gag aat aac   1584Gly Glu Glu Asp Asn Gly Gly Arg Gly Ser Gly Ser Asp Glu Asn Asn515                 520                 525ggt gat gct gga gaa aca ctt ctt gag ttg ttt agg agg act tga 1629Gly Asp Ala Gly Glu Thr Leu Leu Glu Leu Phe Arg Arg Thr530 535 0 4 5 <210>18 <211>542 <212>PRT <213> Arabidopsis thaliana <400> 18MET GLU HIS MET MET LYS GLU GLY ARG Serite Leu lys lys thr arg35 45arg LYS ALA Leu Phe Thr Seru Lys Met Lys Glu Leu Met50 55 60leu Leu Leu Leu Serg Trle65 75 80SER SALS VALS VALS VALS VALS VALS VALS VALS VALS VALS Vals Val Phe Asn Ser Lys Phe Tyr Ile85                  90                  95Cys Ser Glu Glu Asp Tyr Gly Ile His Lys Lys Val Leu Leu Glu His100                 105                 110Thr Ser Ser Thr Asn Gln Ser Ser Leu Pro His His Gly Ile His Glu115                 120                 125Ala Ser His Gln Cys Gly His Gly Arg Glu Pro Phe Val Ser Tyr Glu130                 135                 140Gly Leu Glu Gln Leu Leu Arg Phe Leu Phe Val Leu Gly Ile Thr His145                 150                 155                 160Val Leu Tyr Ser Gly Ile Ala Ile Gly Leu Ala Met Ser Lys Ile Tyr165                 170                 175Ser Trp Arg Lys Trp Glu Ala Gln Ala Ile Ile Met Ala Glu Ser Asp180                 185                 190Ile His Leu Cys Phe Leu Arg Gln Phe Arg Gly Ser Ile Arg Lys Ser195                 200                 205Asp Tyr Phe Ala Leu Arg Leu Gly Phe Leu Thr Lys His Asn Leu Pro210                 215                 220Phe Thr Tyr Asn Phe His Met Tyr Met Val Arg Thr Met Glu Asp Glu225                 230                 235                 240Phe His Gly Ile Val Gly Ile Ser Trp Pro Leu Trp Val Tyr Ala Ile245                 250                 255Val Cys Ile Cys Ile Asn Val His Gly Leu Asn Met Tyr Phe Trp Ile260                 265                 270Ser Phe Val Pro Ala Ile Leu Val Met Leu Val Gly Thr Lys Leu Glu275                 280                 285His Val Val Ser Lys Leu Ala Leu Glu Val Lys Glu Gln Gln Thr Gly290                 295                 300Thr Ser Asn Gly Ala Gln Val Lys Pro Arg Asp Gly Leu Phe Trp Phe305                 310                 315                 320Gly Lys Pro Glu Ile Leu Leu Arg Leu Ile Gln Phe Ile Ile Phe Gln325                 330                 335Asn Ala Phe Glu Met Ala Thr Phe Ile Trp Phe Leu Trp Gly Ile Lys340                 345                 350Glu Arg Ser Cys Phe Met Lys Asn His Val Met Ile Ser Ser Arg Leu355                 360 365Ile Ser Gly Val Leu Val Gln Phe Trp Cys Ser Tyr Gly Thr Val Pro370                 375                 380Leu Asn Val Ile Val Thr Gln Met Gly Ser Arg His Lys Lys Ala Val385                 390                 395                 400Ile Ala Glu Ser Val Arg Asp Ser Leu His Ser Trp Cys Lys Arg Val405                 410                 415Lys Glu Arg Ser Lys His Thr Arg Ser Val Cys Ser Leu Asp Thr Ala420                 425                 430Thr Ile Asp Glu Arg Asp Glu Met Thr Val Gly Thr Leu Ser Arg Ser435                 440                 445Ser Ser Met Thr Ser Leu Asn Gln Ile Thr Ile Asn Ser Ile Asp Gln450                 455                 460Ala Glu Ser Ile Phe Gly Ala Ala Ala Ser Ser Ser Ser Pro Gln Asp465                 470                 475                 480Gly Tyr Thr Ser Arg Val Glu Glu Tyr Leu Ser Glu Thr Tyr Asn Asn485                 490                 495Ile Gly Ser Ile Pro Pro Leu Asn Asp Glu Ile Glu Ile Glu Ile Glu500 505 510GLY GLU GLU ASN GLY GLY GLY ARG GLY Serg GLY Ser ASP GLU ASN515 520 525GLY ALA GLY GLU Leu Leu Leu PHE ARG THR530 535 540 540 <210>19 <211>20 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>19atgtcggacaaaaaaggggt <210>0 <211>20 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>20atgctaccac acgcagatcg 20 <210>21 <211>21 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>21acagagacca cctccttgga a 21 <210>22 <211>22 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>22cagaaacttg tctcatccct gg 22 <210>23 <211>20 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>23aagaactgcc tgaagaaggc 20 <210>24 <211>20 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>24caccaccttc atgatgctca 20 <210>25 <211>20 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>25ttccagcacc ggcacaagaa 20 <210>26 <211>28 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>26tggacctctt catgttcgat cccatctg 28 <210>27 <211>27 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>27cctgacgctg ttccagaatg cgtttca 27 <210>28 <211>20 <212>DNA <213> Artificial sequence <223> Artificial sequence description: oligonucleotides <400>28acttctgcag gtcgactcta 20 <210>29 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>29aagatcaaga tgaggacgtg gaagtcgtgg 30 <210>30 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>30aggctgaacc actggggcgc ctctcaccac 30 <210>31 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>31caagtatatg atgcgcgctc tagaggatga 30 <210>32 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotide aggtttcacc actaagtctc cttcaatggc 30 <210>33 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>33gatcattcaa gacttaggct cactcatgag 30 <210>34 <211>30 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>34aacagcaagg aagattacaa atgatgccca 30 <210>35 <211>18 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>35ggattaagat ctaatggc 18 <210>36 <211>23 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>36caaagatctt catttcttaa aag 23 <210>37 <211>25 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>37gcggatccat gtcggacaaa aaagg 25 <210>38 <211>25 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>38gcggatcctc atccctggct gaagg 25 <210>39 <211>23 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>39ggatccacca tggccacaag atg 23 <210>40 <211>24 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>40ggatccttag tcaatatcat tagc 24 <210>41 <211>27 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>41gcggatccat gggtcacgga ggagaag 27 <210>42 <211>27 <212>DNA <213> Artificial sequence <220> <223> Artificial sequence description: oligonucleotides <400>42gcggatcctc agttgttatg atcagga 27

Claims (28)

1. coding Mlo protein DNA molecule, wherein said Mlo protein comprises listed at least a sequence among SEQ ID NO:1 or the SEQ ID NO:2, and said Mlo protein is given the resistance of plant to fungal pathogens.

2. the dna molecular of claim 1, any listed among wherein said dna molecular and SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7 nucleotide sequence is identical or similar substantially, perhaps identical the or similar substantially Mlo protein of listed Mlo protein among coding and SEQ ID NO:4, SEQ ID NO:6 or the SEQ IDNO:8.

3. the dna molecular of claim 1, any listed among wherein said dna molecular and SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ IDNO:17 nucleotide sequence is identical or similar substantially, perhaps identical with listed Mlo protein among SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ IDNO:18 or the basic similar Mlo protein of coding.

4. each dna molecular among the claim 1-3, wherein said dna molecular is not to derive from barley.

5. each dna molecular among the claim 1-4, wherein said DNA are modified so that the loss of activity of endogenous protein.

6. the dna molecular of claim 5, wherein said dna modification causes that one of following change, whole or various combination are arranged in the respective egg white matter aminoacid sequence:

-tryptophane (163) is changed into arginine

-proline(Pro) (396) back frameshit

-tryptophane (160) back frameshit

-methionine(Met) (1) is changed into Isoleucine

-glycine (227) is changed into aspartic acid

-methionine(Met) (1) is changed into Xie Ansuan

-arginine (11) is changed into tryptophane

-lose phenylalanine (183), Threonine (184)

-Xie Ansuan (31) is changed into L-glutamic acid

-Serine (32) is changed into phenylalanine

-leucine (271) is changed into Histidine.

With claim 1-6 in the dna molecular of each dna molecular antisense.

8. the protein that comprises one of listed sequence among SEQ ID NO:1 or the SEQ ID NO:2 at least, wherein said protein are Mlo protein and give the resistance of plant to fungal pathogens.

9. the protein of claim 8, wherein said protein is by identical or similar substantially with listed any sequence among SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7 nucleotide sequence coded, and is perhaps identical or similar substantially with listed any protein among SEQ ID NO:4, SEQ ID NO:6 or the SEQ IDNO:8.

10. the protein of claim 8, wherein said protein is by identical or similar substantially with listed any sequence among SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or the SEQ ID NO:17 nucleotide sequence coded, and is perhaps identical or similar substantially with listed any protein among SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ IDNO:18.

11. each protein among the claim 8-10, wherein said protein is not to derive from barley.

12. contain the expression cassette that right requires each dna molecular among the 1-7.

13. contain the carrier of the expression cassette of the dna molecular that comprises claim 12.

14. comprise that containing right requires the expression cassette of each dna molecular among the 1-7 or the cell of its part, wherein the said dna molecular in this expression cassette is effable in said cell.

15. the cell of claim 14, wherein said dna molecular is stabilized in the genome that is integrated into said cell.

16. each cell in claim 14 or 15, wherein said cell is a vegetable cell.

17. comprise that containing right requires the expression cassette of each dna molecular among the 1-7 or the plant of its part, wherein the said dna molecular in this expression cassette is effable in said plant.

18. the plant of claim 17, wherein said dna molecular is stabilized in the genome that is integrated into this plant.

19. comprise containing the agricultural-food that right requires the plant of each DNA isolation molecule among the 1-7, wherein said agricultural-food have the Plant Quarantine characteristic of improvement.

20. the method for the plant of preparation resistant to fungal pathogens may further comprise the steps:

A) in said plant, express among the claim 1-6 each dna molecular with " justice is arranged " direction; Or

B) in said plant, express among the claim 1-6 each dna molecular with " antisense " direction; Or

C) in said plant, express the special cutting of energy by ribozyme corresponding to the coded messenger RNA(mRNA) transcript of the native gene of each dna molecular among the claim 1-6; Or

D) expression is specific to coded protein or its a part of aptamer of each dna molecular among the claim 1-6 in plant; Or

E) in plant, express the sudden change or the clipped form of each dna molecular among the claim 1-6; Or

F) in plant, modify at least one chromosome copies corresponding to the gene of each dna molecular among the claim 1-6 by homologous recombination.

21. plant with the resistant to fungal pathogens of the method for claim 20 preparation.

22. the plant of claim 21, wherein said fungal pathogens can infect epidermic cell alive.

23. the plant of claim 21, wherein said fungal pathogens is from Erysiphales.

24. the plant of claim 21, wherein said fungal pathogens is from Erysiphe.

25. the plant of claim 21, wherein said fungal pathogens is the standing grain powdery mildew.

26. the tool that obtains with the method for claim 20 improves the agricultural-food of Plant Quarantine characteristic.

27. a method of separating the dna molecular of coding Mlo protein may further comprise the steps:

A) will encode at least 6 amino acid whose degenerate oligonucleotides among the SEQ ID NO:1 and be complementary to the degenerate oligonucleotide and the DNA that extracts from plant of at least 6 amino acid whose sequences among the coding SEQ ID NO:2 mix under said degenerate oligonucleotide and the condition that described DNA is hybridized allowing;

B) amplification said DNA of plants dna fragmentation, wherein said dna fragmentation its a left side and right end contain can with said degenerate oligonucleotide annealed nucleotide sequence in the step a); With

C) acquisition contains the full length cDNA clone of the dna fragmentation of step b).

28. the method for the dna molecular of mutagenesis claim 1, wherein said dna molecular have been cut into the double-stranded random fragment of expection size, this method may further comprise the steps:

A) add one or more strand or double chain oligonucleotide in the double-stranded random fragment group of gained, wherein said oligonucleotide comprises zone identical with the double-stranded template polynucleotide and allogenic zone;

B) the mixture sex change with double-stranded random fragment of gained and oligonucleotide becomes single-chain fragment;

C) causing said single-chain fragment to form under the annealing fragment paired condition in said same area annealing, with gained single-chain fragment group and polysaccharase insulation, wherein said same area is enough to make the member in the pairing to cause duplicating of another member, thereby forms the double-stranded polynucleotide through mutagenesis; With

D) repeat second and at least two circulations of third step again, wherein the mixture that produces of second step in next circulation comprise from last circulation third step through the double-stranded polynucleotide of mutagenesis, and this next circulation has formed the double-stranded polynucleotide of further mutagenesis.