JPH05199877A - Controlling region for induction type plant protecting gene of potato and rice plant, its use and method for assay - Google Patents

Abstract

(57) [Summary] [Construction] Transgenic plant cells, tissues and plants, and transgenic plants into which the promoter of the potato or rice PAL gene, an expression cassette containing the promoter and a structural gene, and the expression cassette are introduced. It relates to a screening method for agricultural substances and the like characterized by being used. [Effect] The PAL promoter of the present invention responds to a stimulus to various plants and initiates transcription of a gene bound thereto, and a plant into which an expression cassette containing the promoter is introduced is used. This makes it possible to screen for agricultural substances that induce plant defense genes.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to plant biotechnology. More specifically, the promoters (control sequences) of inducible plant defense genes from potato and rice, and methods for identifying and isolating these related sequences, as well as their uses, especially for the construction of transgenic plants and plant compositions. Regarding the use of. Furthermore, the present invention relates to a method for identifying pesticides and organisms that induce plant defense genes.

[0002]

2. Description of the Related Art Mechanisms of resistance to plant diseases include physical and chemical barriers in plants and reactions induced or activated by damage or attack of pathogens. One of the important induced defense reactions is the production of phytoalexin, which is defined as a low molecular weight antibacterial compound synthesized and accumulated at the site of infection of plants. Phytoalexins are primarily phenylpropanoids, isoprenoids, and acetylenes. The first step in the biosynthesis of phenylpropenoids in higher plants is the formation of ammonia and trans-cinnamate from L-phenylalanine. This reaction is catalyzed by oxygen L-phenylalanine ammonia lyase (PAL). The PAL gene is composed of legumes, especially common bean (Phaseolus vulgaris).
s vulgaris)). Cram
er et al., 1989, Plant Molecular Biology (Pl
ant Mol. Biol.) 12: 367-383. In the kidney bean genome, three different PAL genes are
There are gPAL-1, gPAL-2 and gPAL-3.

[0003]

Transcription of the PAL gene is rapidly activated by a wide range of stimuli, including damage, glycan elicitor compositions from the fungal cell wall and reduced forms of the cellular metabolite glutathione. Not all PAL genes respond similarly to these stimuli. Therefore, there is a need to identify new PAL promoters that respond to fungal or bacterial elicitors or damage and the like. Such novel PAL promoters can be used for transgenic plant material and transgenic plant creation and novel pesticide screening.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel PAL promoter which responds to fungal or bacterial elicitor, damage and the like. further,
It is an object of the present invention to create transgenic plant material and transgenic plants using these novel promoters. Another object is to provide new methods of identifying and isolating new PAL promoters. A further object of the present invention is to use in a method for screening a novel pesticide capable of inducing a normal PAL-related defense reaction in a plant and inducing expression of a structural gene associated with a corresponding PAL promoter in a plant. To provide a transgenic plant and a screening method or technology for a new pesticide that can be used. Another object of the present invention is also an improvement for identifying pesticides that induce plant defense gene promoters or regulatory regions, particularly pesticides capable of inducing these plant gene regulatory regions immediately after or very early in exposure. To provide a method. Finally, another object of the invention is to provide a method for identifying organisms which induce plant defense gene regulatory regions.

The definitions of the terms used in the present specification are as follows. As used herein, "PAL" refers to phenylalanine ammonia lyase. PAL catalyzes the conversion of the amino acid L-phenylalanine to trans-cinnamic acid and NH ₄ ⁺ . This is the first reaction in the synthesis of a wide range of natural plant materials based on the phenylpropane skeleton including lignins, flavonoids, isoflavonoids, coumarins and hydroxycinnamonates. As used herein, "exogenous regulatory plant gene regulatory regions or elements" refers to nucleic acid sequences that affect the transcription of structural genes that are operably linked or linked in response to foreign stimuli. . Examples of exogenous regulatory plant gene regulatory sequences include inducible plant defense gene regulatory regions or promoters (eg PAL promoters), elicitor regulatory activator domains, upstream silencer domains and the like. "Promoter" used in the text
Is an untranslated region DN involved in RNA polymerase binding.
A and factors that initiate or regulate transcription to produce RNA transcripts. The promoter may be natural or synthetic. Promoters are constitutive or regulated, and constitutive promoters are always on. Controllable promoters require specific signals to activate or deactivate. A developmentally controlled promoter is one that operates or stops as a function of development. The promoters of the invention respond directly or indirectly to exogenous elicitors and / or damage, as described above. Stated another way, exogenous elicitors and / or lesions induce the promoters of the invention to induce transcription of related structural genes. In the present specification and claims, the terms "promoter" and "gene control region" are used interchangeably.

As used herein, "elicitor" refers to the action of exogenous regulatory plant gene regulatory elements, such as the inducible stress regulatory promoter of the plant gene encoding the phenylpropanoid biosynthetic enzyme, phenylalanine ammonia lyase (PAL). A compound or organism that directly or indirectly regulates (eg, starts, stops, increases or decreases). As elicitor, glutathione reduced type, homoglutathione reduced type, reduced form of glutathione and other peptide analogs; hexa (β-D-glucopyranosyl) -D
-Glycan elicitors such as glucitols, lipid elicitors such as arachidonic acid and eicosapentaenoic acid, glycoprotein elicitors, fungal elicitors, abiotic elicitors such as mercury chloride HgCl ₂ , molds (eg. Examples thereof include, but are not limited to, elicitors released from whole mold), bacteria (eg elicitors released from whole bacteria), and the like. As used herein , "fungal elicitor" refers to a high molecular weight substance that is heat released from the mycelial cell wall of the pathogenic legume fungus Collectotrichim lindemuthianum or from the pathogenic rice fungus Pyricularia oryzae .

As used herein, a "probe" or "primer" is a single strand that has sufficient complementarity with a target sequence to hybridize or bind to the appropriate (target) sequence under appropriate hybridization conditions. A nucleic acid sequence (naturally occurring, synthetically produced, or product of restriction enzyme digestion). The probe may contain any number of nucleotides, may contain only about 10 or may contain hundreds of thousands of nucleotides. In a preferred form, the probe or primer has the same or very high complementarity with the appropriate (target) sequence. Conditions and protocols for such hybridization reactions are well known to those of skill in the art, as are the effects of probe size, temperature, degree of mismatch, salt concentration and other parameters in the hybridization reaction. For example, the lower the temperature at which the hybridization reaction takes place and the higher the salt concentration, the greater the degree of mismatch present in the hybrid molecule.
For use as a hybridization probe, the DNA sequence must be detectable by being labeled with ³² P, ³ H or ¹⁴ C, or by other means, including non-radioactive labels and chemical labels. Non-radioactive labels known in the art can be used as well, as they will serve as a source of DNA within the available time (for cell culture, DNA extraction and hybridization assays) as a source of DNA. The probe must be sufficiently sensitive to be detected by. Once sequences with sufficiently high homology to the probe have been identified, such sequences are described, for example, in Maniatis et al. ((1982) Molecular Cloning: Laboratory Manual (Molecula).
r Cloning: A Laboratory Manual), ColdSpring Har
bor Laboratoyr Press, Cold Spring Harbor, NY) and easily isolated by standard techniques. All assays and procedures used herein are carried out under conditions recognized by those of skill in the art as standard conditions, unless otherwise specified.

The "DNA coding sequence" used in the text is
By a DNA sequence that results in the formation of a polypeptide when transcribed and translated. As used herein, "transcription" means RNA synthesis from a DNA template. As used herein, the term “terminator” or “3 ′ terminator” means a DNA sequence which is located at the 3 ′ end of a gene or transcript and which causes RNA polymerase to terminate transcription. Examples of terminators include the 3'flanking region of the nopaline synthase (NOS) gene and the 3'flanking region of the octapin synthase (OCS) gene. As used herein, "RNA transcript" means a ribonucleic acid sequence produced after initiation of transcription following RNA polymerase recognition of the promoter sequence or control region. As used herein, the "transcription initiation site" means the position on DNA corresponding to the first base incorporated into RNA. For convenience, the transcription start site is indicated as +1 in the DNA sequence. "Expression cassette" used in the text means
By a DNA construct containing sequences that function for expression in the chosen host and, if desired, processing and secretion of the mature protein. Since such fragments are designed to move from vector to vector into host cells for replication and expression, they are often referred to by those of skill in the art as "expression cassettes." Thus, the expression cassette includes a promoter region, a transcription terminator region, and sequences sufficient for translation, as well as DNA encoding all other regulatory signals that affect the proper processing of the expressed protein or peptide.

As used herein, "DNA construct" includes an expression cassette and also includes a DNA fragment containing one or more expression cassettes. As used herein, the terms "operatively linked or operatively linked or associated" are equivalent terms that can be used interchangeably. is there. In particular, these terms refer to the binding of a promoter or non-coding gene regulatory sequence to a DNA sequence encoding RNA, the RNA transcript corresponding to the DNA coding sequence when the promoter or regulatory sequence is recognized by the appropriate polymerase. By the ability of a control sequence or promoter to induce production. All three terms refer to the binding DNA sequences (eg promoters, structural genes (eg reporter genes, terminator sequences, etc.) being operably or functionally linked, ie, for their intended purpose. Means to act on.
In other words, operably linked (op
By eratively linked or operably linked or associated is meant that the structural gene is expressed upon appropriate activation of the promoter after the respective DNA segments have been linked. "Marker" or "reporter gene" used in the text means
By gene that encodes protein production that is easily assayed. Examples of markers or reporter genes are chloramphenicol acetyl transferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glutaronidase (GUS). , Acetohydroxy acid synthase (A
There are genes encoding HAS), β-galactosidase (β-GAL), and luciferase (LUX). In the method of the invention, the marker or reporter gene is the gene (CA) operably linked thereto.
T, GUS, β-galactosidase, or luciferase reporter gene) by regulating the transcription of the plant gene regulatory site in response to an external stimulus. As used herein, "naturally occurring or naturally occurring gene" means a naturally occurring gene.

As used herein, "chimeric gene" or "chimeric DNA sequence" means an artificially constructed or engineered gene or DNA sequence. A chimeric gene or DNA sequence has at least two different parts. For example, a portion isolated from a naturally-occurring DNA sequence that is not naturally linked or at least one DNA that is not naturally found in synthetic origin.
Having a DNA portion containing the portion. As used herein, "appropriate plant material" means and includes plant protoplasts, plant cells, plant callus, plant tissues, seedlings, immature plants and mature plants. As used herein, "plant protoplast" refers to a single plant cell that does not have a plant cell wall. "Wild type plant" used in the text
Refers to a plant that is the same species or identical to the transgenic plants, but does not contain DNA or RNA encoding a heterologous gene expressed by the transgenic plant. As used herein, "transgenic plant" or "plant composition" means a plant or plant composition in which heterologous or foreign DNA is expressed or the expression of genes naturally present in the plant is altered. To do. Such DNA is operably linked to plant biochemical control signals and sequences. Expression can be constitutive or controllable. This DNA can be integrated into the chromosome or integrated into episomal elements such as chloroplasts, or it can remain episomal elements. In making transgenic plants or plant compositions, any method of introducing DNA known to those of skill in the art can be used.

As used herein and in the claims, "substantial sequence homology" is used to mean that DNA or RNA sequences are sufficiently similar to each other to form stable hybrids. . Such conditions are preferably such that DNA molecules having at least 60% complementarity form stable hybrids. As is known to those skilled in the art, nucleic acid fragments having different sequences are
It can be detected by hybridizing to the same "target" nucleic acid under the same conditions. The two nucleic acid fragments hybridize and are detectable under stringent conditions over a sufficiently long hybridization period because they are complementary (or complementary to the sequence of one segment in another nucleic acid fragment). One fragment which is (mostly complementary) contains a segment of at least 10 nucleotides. If the time during which hybridization can occur is kept constant, and two nucleic acid fragments having completely complementary base pairing sites hybridize under the stringency conditions selected in advance, and the detection is possible, then Introducing a sequence away from perfect complementarity at the base pairing site still causes sufficient base pairing to detect hybridization. The greater the deviation from complementarity between the base pairing segments of two nucleic acids, and the more stringent the hybridization conditions, the more hybridisable the two segments will be to detect each other. The probability of doing it is low. The two single-stranded nucleic acid segments are
If both (a) both form a base pair duplex and (b) the melting points of the two duplexes in a 0.5 × SSPE solution differ by less than 10 ° C., It has “substantially identical sequence” within the meaning. When the segments to be compared have the same number of bases, their sequence differences are usually less than 1 per 10 bases because they have "substantially identical sequences". . The method for measuring the melting point of a double-stranded nucleic acid is well known. For example, Meinkoth and Wahl, Analytical Biochemistry (Anal. Bi
ochem.) 138: 267-284 (1984) and the references cited therein.

DN in the specification and claims
"Isolated" used as a modifier of A or RNA
Means that DNA or RNA has been separated from their in vivo cellular environment by human effort; the result of this separation is that the isolated DNA or RNA differs from the unseparated, impure DNA or RNA. Means useful in. The present invention provides a promoter (regulatory sequence) of a plant gene encoding a potato or rice phenylalanine ammonia lyase (PAL). This promoter is inducible by an exogenous elicitor or damage and is capable of controlling transcription of related DNA sequences in transgenic plant cells, tissues and plants containing the promoter as part of the chimeric construct. The present invention includes methods for obtaining these novel PAL promoters. The present invention provides a PAL promoter, a probe sequence, a chimeric potato and a rice PAL promoter construct, in which the PAL promoter is operably linked to a structural gene such as a reporter gene, and these Transgenic potato and rice plant cells, plant tissues and transgenic plants containing the chimeric constructs of. This chimeric construct and the transgenic plant material containing them are useful in pesticide assays. The present invention also provides methods of inducing transcription of chimeric genes in transgenic plants. The present invention further provides a method for identifying pesticides capable of inducing plant defense gene elements, in particular pesticides capable of inducing particularly plant gene regulation shortly after or very early in exposure. Finally, the invention provides methods for identifying organisms capable of inducing plant defense genetic elements.

The present invention comprises an isolated or isolatable promoter for a plant defense gene encoding a potato or rice phenylalanine ammonia lyase (PAL), the promoter comprising a ligation DNA sequence in a suitable host. Of the present invention is characterized by that the promoter is inducible by an exogenous elicitor and / or injury, or is otherwise responsive to them either directly or indirectly. To do. As an example of the promoter of the present invention, clone λpPA
L-1, λpPAL-2, λpPAL-3, λpPAL
-4, λpPAL-6, λpPAL-7 (λpPAL-
6 and λpPAL-7 are identical to those described in Example IIC, referred to herein as λpPAL-6 / 7),
And the potato PAL promoter linked to the PAL structural gene sequence contained in λpPAL-8 and clone λ
Rice PA included in pPAL-2 and λpPAL-4
The L promoter can be mentioned.

In another aspect, the potato and rice PAL promoters of the present invention are operably linked to at least one related DNA sequence encoding a protein that directly or indirectly produces a phenotypic trait. There is. The binding can be at the transcriptional level (ie, "transcriptional fusion") such that the protein that produces this phenotypic trait is expressed as a non-fusion peptide. Also, particularly in the case of a marker gene, this binding can be at a translation level (ie, a "translational fusion") such that the marker protein is expressed as a fusion peptide with the amino-terminal portion of the native PAL protein. In the present invention, a phenotypic trait is a herbicide, fungus, virus, bacterium, insect, nematode or arachnid resistance or resistance, secondary metabolite production, male or female infertility, or an enzyme or reporter. Such as the production of compounds. Where this related structural gene encodes a reporter enzyme or compound, it is preferably chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS). ), Β-
1,3-glucuronidase (GUS), acetohydroxy acid (AHAS), β-galactosidase (β-GA
L) or luciferase (LUX). According to the present invention, the potato and rice PAL promoter-related DNA sequence constructs are transformed into suitable hosts, transgenic plant compositions such as transgenic plant protoplasts, plant cells, plant callus, plant tissue,
Outbreak plantlet, immature whole plant, mature whole plant,
Alternatively, it is used to produce seeds. The present invention includes these transformed host and transgenic plant compositions, particularly transgenic potato and rice plant and seed PAL promoters containing the potato and rice PAL promoters and related DNA sequences.

The present invention provides the DNA sequences identified as SEQ ID NOS: 1-9. SEQ ID NOs: 1 to 4 are potato PAL clones λpPAL-1 to λp, respectively.
PAL-4, SEQ ID NOS: 5 and 6 are λpPAL-6 (two sequences), and SEQ ID NO: 7 are DNs derived from λpPAL-8.
Represents the A sequence. Sequence Nos. 8 and 9 are DNs derived from rice PAL clones λrPAL-2 and λrPAL-4.
The A array. More specifically, λ shown in FIGS.
The B → S sequence of pPAL-1 is shown as SEQ ID NO: 1. The (E) → P sequence of λpPAL-2 is SEQ ID NO: 2. The (E) → P sequence of λpPAL-3 is SEQ ID NO: 3. The E → P array of λpPAL-4 is array code 4.
The H → S sequence of λpPAL-6 is λpPAL-6 (a) and is SEQ ID NO: 5. The S → H sequence of λpPAL-6 (shown as the sense sequence and converted from the antisense sequence) is λpPAL-6 (b), which is shown as SEQ ID NO: 6. The (E) → P fragment of λpPAL-8 is SEQ ID NO: 7. λrPAL-2 is SEQ ID NO: 8 and λrPAL-4 is SEQ ID NO: 9. The DNA sequences of the present invention can be used as probes to identify identical or similar PAL sequences. Preferably,
The probe is at least 10 nucleotides in length, most preferably from about 100 to about 500 nucleotides in length.
Individual nucleotides. For example, potato PAL clones, λpPAL-1, λpPAL-2, λpPAL
-3, λpPAL-4, λpPAL-6 / 7 or λp
The coding sequence from PAL-8, or λrPAL-2 or λrPAL-4, was cloned to search for identical sequences.
It can be used to probe NA or genomic libraries. These same sequences are linked to an inducible promoter within the scope of the invention.

Promoters may not be functionally equivalent, even though the relevant PAL coding regions have sufficient homology to hybridize to each other under mild hybridization conditions. For this reason, the invention includes a method of finding sequences that are not only homologous to the sequences of the invention, but are also regulated by the inducible PAL promoter. According to this method, the plant is subjected to an elicitor, and RNA is then isolated from said plant. Poly A ⁺ RNA is selected by using, for example, an oligo (dT) column. A cDNA library is prepared from this RNA and cloned into a suitable vector. This library is used for the nucleotide sequence of the coding region of the elicitor-inducible gene, for example, SEQ ID NO: 2-7 (λpPAL-2, λpPAL-3, λpP.
AL-4, λpPAL-6, λpPAL-8) or SEQ ID NOs: 8 and 9 (λrPAL-2 and λrPAL-
Search with a probe consisting of the coding sequence of 4). The cDNA that hybridizes to the probe (ie, the positive clone) is subcloned and sequenced. Search the genomic library from the target plant with cDNA,
A segment of genomic DNA that hybridizes to the probe is identified and identified. Antisense RNA transcripts are made from this genomic DNA and labeled (eg, by radioactivity)
To do. This is then used as a probe to hybridize to mRNA from elicitor-treated and untreated plants.
The hybridizing mixture is then subjected to RNase degradation of total single stranded RNA. If your transcript is total RNA
RNA from elicitor-treated plants, if present in
A double-stranded RNA-RNA hybrid is formed which contains no mismatches protected by RNA but not RNA from a non-elicitor, and said RN
not affected by the ase process. The size of this product can be identified by gel electrophoresis. Promoters from genomic clones that produce antisense RNA transcripts protected by RNA from elicitor-treated plants but not RNA by non-elicitor-treated plants are inducible by elicitors. The promoter is isolated to produce a chimeric construct for use in pesticide assays to produce transformed cells and transgenic plants and transgenic plant compositions.

The present invention also includes a method of identifying and identifying an exogenous elicitor capable of directly or indirectly inducing a potato or rice PAL promoter. According to this method, a suitable host (eg, plant composition) is transformed with a potato or rice PAL promoter operably linked to a structural gene whose expression is detectable (eg, a marker gene). The putative exogenous elicitor is then applied to the transformed host. It can be concluded that the exogenous elicitor capable of inducing the expression of the marker gene is an elicitor capable of inducing the potato or rice PAL promoter. In a related method, plants are used in this assay. According to this method, transgenic plants containing at least one chimeric DNA sequence consisting of a potato or rice PAL promoter operably linked to a reporter structural gene are produced. A putative exotic elicitor is then applied to this plant. It can be concluded that the elicitor that induces the expression of the reporter gene is an elicitor that can induce the expression of the potato or rice PAL promoter. The invention also provides a method of identifying an elicitor-inducible promoter. According to this method, the plant is subjected to an elicitor, and then RNA is isolated from this plant. A cDNA library is prepared from this isolated RNA, which is then probed with a probe consisting of nucleotide sequences from the transcribed coding and / or non-coding regions of the gene of interest. Next, cD hybridized to the probe
Search for genomic libraries from the plant in question using NA. Genome D hybridized to the probe
The segment of NA is identified and identified, from which a labeled antisense RNA transcript is made. This labeled antisense RNA transcript is used as a probe to hybridize to mRNA from elicitor-treated and non-elicitor-treated plants. The hybridized mixture was treated with RNa
Antisense RNA transcripts that are protected against degradation by elicitor-treated RNA but not protected by non-elicitor-treated plants are identified.
Promoters from genomic clones producing antisense RNA transcripts protected only by RNA from elicitor-treated plants are inducible by elicitors.

The present invention also discloses related methods for identifying and identifying elicitor inducible promoters. According to this method, a plant genomic library is searched with probes consisting of nucleotide sequences derived from the transcriptional coding and / or non-coding regions of the gene of interest. The genomic DNA site that hybridizes to the probe is identified. A labeled antisense RNA transcript is prepared from a segment of genomic DNA hybridized to the probe. Using this labeled antisense RNA transcript as a probe,
M from elicitor-treated and non-elicitor-treated plants
Hybridizes to RNA. The hybridized mixture is subjected to RNase to identify antisense RNA transcripts protected against degradation by elicitor-treated RNA but not degraded by non-elicitor-treated plants. Promoters from genomic clones producing antisense RNA transcripts protected only by RNA from elicitor-treated plants are inducible by elicitors. In another aspect, the present invention discloses an amplification method for identifying and identifying a pesticide capable of inducing the expression of plant genes. According to the method of the present invention,
RNA is isolated from plant material that has not been exposed to the putative inducer, and from this isolated RNA the RNA encoded by the gene of interest is identified by hybridization with a probe. R from this identified gene
NA was cloned into self-retained replication or 3SR ^™ (SIBIA La, Jolla, CA 92037-464 for amplification of specific RNA sequences.
1) Amplified using primers specific for the technique and inducible gene transcript of interest. Guatelli et al., 1990.
Proc. Natl. Acad. Sci USA 8
7: 1874-1878. The unexposed plant material from which the first RNA was obtained is then exposed to the putative chemical inducer. The RNA encoded by the gene of interest is then differentially identified and amplified with 3SR. Compare amplification products from unexposed and exposed plant material. If the predicted product level in RNA amplification from exposed plant material is higher than in RNA amplification from unexposed plant material, it can be concluded that this pesticide is able to induce expression of the gene of interest.

Regarding the amplification method, its sensitivity has two important advantages. First of all, very small samples can be analyzed and thus plant and tissue culture cells such as Arabidopsis can be used for pesticide screening. Second, some pesticides can have very small but very important effects on plant genes, such as plant defense genes, which response is more difficult to detect than 3SR in a less sensitive way. . The 3SR amplification method of the present invention made it possible for the first time to identify these chemical elicitors. In yet another aspect, the present invention discloses a novel method for identifying and identifying organisms capable of inducing plant defense genes. According to the method, at least 1
Transgenic plants containing at least one chimeric DNA sequence consisting of an inducible plant defense gene promoter operably linked to a single reporter structural gene are exposed to a potentially inducible organism. The organism may be living, dead or otherwise impaired. By monitoring the expression of the reporter gene, which organism induces the transcription of the reporter gene and is therefore a candidate for use as an "inducer" or plant vaccines or potentially pathogenic to plants. It is possible to conclude that it is sex.

[0020]

The present invention provides a PAL promoter, a plant defense gene promoter that is induced in response to various exogenous stimuli, and the promoter of the present invention comprises:
Transgenic plant cells, which can control transcription of plant genes and are produced by introducing an expression cassette incorporating this promoter into plant cells, tissues, plants, etc., tissues and plants, agricultural chemicals, etc. Can be used for screening.

[0021]

The invention is described in greater detail in the following examples, which are given for illustrative purposes only and are not limiting. Example I: Isolation of potato-derived arachidonic acid-derived cDNAs A. Construction of cDNA library Potato tubers ( Solanum tuberosum cv. Desiree)
Obtained from the US Department of Agriculture. The tubers were washed with deionized water to remove soil, then soaked in 95% ethanol for 1 minute,
It was washed 3 times with sterile purified water. Tuber is peeled and the surface contains 2 drops of TWEEN 20 ^™ per 100 ml of solution 1
It was sterilized in 0% Purex ^™ (commercial bleach) for 15 minutes and washed 3 times with sterile distilled water. A cork borer (diameter: about 8 mm) is inserted in the major axis direction of the marrow tissue of the tuber, the tuber is cut out into a disc shape, and sliced on a disc with a thickness of about 3 mm. Tuberdiscs were transferred to potato callus induction medium. Potato callus induction medium Murashige and Skoog salt-based thiamine · HCl 0.1 mg / l pyridoxine · HCl 0.5 mg / l nicotinic acid 0.5 mg / l glycine 2.0 mg / l 2,4-dichlorophenoxyacetic acid 10.0 mg / l myo-inositol 100.0 mg / l sucrose 30.0 g / l tissue culture agar 8.0 g / l pH 5.7 Induced callus was maintained in growth on LS2T medium. LS2T medium composition mg / l NH ₄ NO ₃ 1650.0 KNO ₃ 1900.0 CaCl ₂ .2H ₂ O 440.0 MgSO ₄ .7H ₂ O 370.0 KH ₂ PO ₄ 170.0 Na ₂ -EDTA 37.3 FeSO ₄ .7H ₂ O 27.8 H ₃ BO ₃ 6.2 MnSO ₄ .H ₂ O 16.9 ZnSO ₄ .7H ₂ O 8.6 KI 0.83 Na ₂ MoO ₄ .2H ₂ O 0.25 CuSO _{4 · 5H 2 O 0.025 CoCl 2} · 6H 2 O 0.025 thiamine-HCl 0.5 pyridoxine-HCl 0.5 nicotinic acid 5.0 glycine 2.0 biotin 0.05 folic acid 0.5 trans-zeatin 0 .1, 2,4-dichlorophenoxyacetic acid 2.0 myo-inositol 100.0 sucrose 300.0 pH 5.8 potato suspension culture Was al induction. Cultures were maintained and grown in LS2T medium and incubated in the orbital incubator at a speed of 125 rpm at 27 ° C in the dark. Desiee suspension cultures were subcultured weekly. In each subculture, 5 g fresh weight of cells was added to 100 ml of fresh L
Transferred to S2T medium. It was observed to grow 5-fold during the 7-day culture period. Three hours before RNA isolation, this culture was treated with arachidonic acid (Sigma, St.
(Louis, MO) at a final concentration of 0.1 mM. Arachidonic acid, a fatty acid normally found in fungal cell walls, induces expression of at least some PAL genes (Fritze
meier et al., 1987, Plant Physology
iol.) 85: 34-41). After induction, Haffner et al., 19
78, Canadian Journal of Biochem. (Can. J. Biochem.) 56: 729-733. Total RNA was isolated according to a modified procedure.

RNA was isolated by a modification procedure as follows: Weigh 3-5 g of tissue. Grinding to a fine powder using a mortar and pestle (tissue keep pre Ji because cooled with liquid N ₂ and). 2. Tissue, 10 ml saturated phenol + 0.1 M Tris
Add to 5 ml of Cl (pH 9). Homogenize using vortex. All of the following operations must be performed at 4 ° C. 3. Centrifuge at 3,000 xg for 10 minutes. 50 ml of water layer
Transfer to a test tube. 4. Back-extract the phenol. 0.1M Tris-C
l (pH 9) 5 ml is added, mixed and re-centrifuged. 5. Combine the aqueous layers, add 1 equivalent of chloroform, mix and centrifuge again. Remove the lower chloroform layer. repeat. 6. The aqueous layer is transferred to another test tube, and 0.1 volume of 3M sodium acetate (pH 5.2) +2.5 volume of ethanol is added. Let stand at -20 ° C for 2 hours (minimum). 10,000x in 7.30 ml Corex test tube
Precipitate RNA for 30 min at 30 g. 8. Resuspend the pellet in 6 ml H ₂ O. After dissolution, 2.0 ml of 8M LiCl is added. Mix well. Let stand overnight at + 4 ° C. 9. The LiCl precipitated RNA is centrifuged at 10,000 xg for 30 minutes. The pellet is washed with 10 ml 70% ethanol and recentrifuged. Decant and remove liquid. 10. Resuspend the pellet in 5 ml H ₂ O. 11. 0.5 ml of 3M sodium acetate (pH 5.2) and 13
Add ml ethanol. 2 hours at -20 ° C (minimum)
Let stand. 12. Centrifuge RNA at 10,000 xg for 30 minutes. 5
Wash with ml 70% ethanol, re-centrifuge and dry. 13. Resuspend the pellet in 1 ml H ₂ O. -70 ° C
Save with. Poly A ⁺ RNA from 400 μg of total RNA from mRNA purification kit (Pharmacia, Piscataway, N.
J; catalog number # 27-9258) and according to the manufacturer's instructions. About 3.1 μg of poly A ⁺ was recovered, and the BRL cDNA synthesis system protocol (BRL, Be
thesda, MD; catalog number # 8269SA)
Used for A synthesis. The cDNA was treated with 9 units of T4 DNA polymerase (BRL) for 30 minutes, then a 50-fold molar excess of EcoRI adapter (Pharmacia (Phar) was used.
macia)) was bound to this cDNA (15 ° C. overnight).
Maniatis et al., Molecular Cloning: A Laboratory Manua
l), Cold Spring Harbor Laboratory Press, Cold Spri
The cDNA was isolated from excess adapters according to ng Harbor, NY, 1982, and the cDNA purified according to the manufacturer's instructions was ligated into an EcoRI digested λgt10 arm (Promege, Madison, Wis.). Gigapack ^™ 11 Gold according to the manufacturer's instructions
Packaging Extract (Stratagene)
, La Jolla, CA). cd
E. NA library. coli strain C600 (Promega (Prom
titers were tested using ega), Madison, WI)). The titer of the library was determined to be 2.6 x 10 ⁷ plaque forming units / ml.

B. Library Screening This library was screened with a 0.9 kb fragment of pUC19-based plasmid pCP63.15, which has a cDNA encoding a portion of the potato PAL exon II gene sequence (Fritzemeier et al., 1987, Plant Phys.
iol. 85: 34-41). The screening conditions were as follows. Hybridization: 42 ° C, 35% formamide, 5 x Denhardts, 5 x SSC, 0.2%
SDS, 200 μg / ml salmon sperm DNA. Washing: 2 × SSC, 0.1% SDS, 4 times in total, repeated at 50 ° C. for 1 hour each. Three PAL-encoding cDNAs were identified by screening and 5 plaque washes and were identified as λpPAL-3, λpPAL-2.
1 and λpPAL-25. C. Subcloning and characterization Ligation was performed with an insert to vector ratio of 5: 1. Three λ clones, λpPAL-
3, λpPAL-21 and λpPAL-25 were digested with EcoRI to give 450 and 5, respectively.
50 and 250 bp inserts were released. After gel purification, each insert was EcoRI-digested pUC.
119 (Vieira, J. and Messing, J., 1987, Methods in Enzymolog
y)) (edited by R. Wu and L. Grossman), 153, 3-
(Page 11, Academic Press, New York), ligation and ligation were separately transformed into DH5α cells. A
mp ^R colonies were selected. Colonies with the correct plasmid released 450,000 or 250 bp fragments, respectively, upon digestion of the plasmid DNA with EcoRI. EcoRI insert for Sequencing for double-stranded DNA
The nucleotide sequence of each was sequenced using the enase (UB Biochemical, Cleveland, OH) protocol, and the nucleotide sequence of each was prepared using the bean gPAL-2 gene (Ceamer et al., 1989, Plant Mol. Biol.) 12: 3.
67-383). The similarities were as follows: homology to potato clone # gPAL-2 3 55% 21 62% 25 No significant homology.

Example II: Isolation of genomic clone corresponding to arachidonic acid-derived cDNA from potato A. Construction of genomic library A genomic library was constructed from total genomic DNA isolated from Solanum tuberosum cv. Desiree shoots . DNA
Isolation by Bendich, 1988, Plant Molecular Biology Manual.
, Kleuwer Academic Publishers, Section A6, 1
According to the operation on page-10. Genomic DNA is Sau3A
Partial digestion with I generated a fragment 9-23 kb in size. The Sau3AI ends are dATP and dGT.
Partially filled with P. X about 0.43 μg fragment
hoI-digested plasmid λFIXII ^™ (Stratagene) 1
bound with μg. It had an overhang partially filled with dTTP and dCTP.
Gigapack ^TM 11 Gold Packgin according to manufacturer's instructions
Binding reactions were packaged using g mix (Stratagene, La Jolla, CA). Four libraries were constructed. Three contained approximately 3 × 10 ⁵ clones, the fourth had 7 × 10 ⁵ clones. B. Library screening Solanum tuberosum cv. Desi
ree cDNA clone pPAL-3 (450 bp, Ec
oRI fragment) and pPAL-21 (500 bp, Ec)
oRI fragments) and screened under high stringency conditions using an equal mixture of fragments isolated as a probe (see potato run IB). The screening conditions were as follows: Hybridization: 42 ° C, 50% formamide, 5x Denhardts, 5x SSC, 0.2%.
SDS, 200 μg × ml salmon sperm DNA. Washing: 0.2 x SSC, 0.1% SDS, total 3 times, 45 ° C
For 15 minutes each; and 0.2 x SSC, 0.1%
SDS, washed once at 65 ° C for 15 minutes. A total of 43 putative PAL-encoding genomic DNA clones were identified from this screen. Twelve clones were selected and subjected to 5 plaque hybridizations. Nine clones were selected for further characterization. This selected clone described in the text was λpPAL-1.
Was designated as λpPAL-8.

C. Clone Analysis By restriction enzyme digestion analysis of these 9 clones, λpPAL-6 and λpPAL-7 are overlapping clones having several restriction enzyme fragments in common,
Also, it was revealed that the remaining 7 clones were unique (Valley Fig. 1). (A map of λpPAL-2 is shown in Figure 2). A map of λpPAL-5 is not shown as no sequence similarity to PAL is found in this insert. By restriction enzyme analysis and hybridization analysis (pPAL-3 and p
Using PAL-21 as a probe), the open boxed segment of the genomic clone shown in FIG. 1 was determined to be part of the probe (hybridized insert. The appropriately digested plasmid pGEM- which was cleaved by digestion with the appropriate restriction enzymes (see Figure 1).
Ligation into 7Z (-) (Promega, Madison, WI) gave the following subclone plasmid: Genome clone # insert (5'-3 ') subclone plasmid λpPAL-1 BamHI / SstI pPAL-1 λpPAL. -2 EcoRI / EcoRI pPAL-2 [lambda] pPAL-3 EcoRI / EcoRI pPAL-3 [lambda] pPAL-4 EcoRI / BglII pPAL-4 [lambda] pPAL-6 HindIII / EcoRI pPAL-5 [lambda] pPAL-7 BamHI / EcoRIpELPEL-EcoRIpPAL-L7pPAL-4. EcoRI pPAL-8 insert DNA is Se for double-stranded (dideoxynucleotide)
quenase protocol (US Biochemical Corporatio
n) and was sequenced and analyzed using the University of Wisconsin Genetics (UWG) comparison program, FASTA. The partial sequence of the genomic clone excluding λpPAL-5 is Solanum tuberosum cv. Datula (So
PAL c isolated from lanum tuberosum cv. Datura)
It showed identity with the DNA clone. Furthermore, λpPAL
-2, λpPAL-3, λpPAL-4 and λpPA
Subsequences of L-8 showed greater than 90% sequence similarity with each other; subsequences of λpPAL-6 and λpPAL-7 were identical; and subsequences of λpPAL-1 were λpPAL-6 and λpPAL. It showed 62% sequence similarity with -7. These results show that λpPAL-2,
λpPAL-3, λpPAL-4, and λpPAL-
The gene corresponding to 8 is a member of the same PAL gene subfamily, λpPAL-6 and λpPA
It was suggested that L-7 is a genomic clone of the same PAL gene, and that λpPAL-1 has a PAL gene different from the PAL gene contained in λpPAL-6.

Example III: Potato RNase Protection Assay RNase Protection Assay (Winter et al., 1985, Proc. Nat
L. Acad. Sci. 82: 7575-7579) is the single-stranded RN for degradation by the single-stranded RNA-specific enzyme RNase.
It is based on the difference in resistance between A and RNA-RNA hybrids. In these assays, total RNA was hybridized to a radiolabeled antisense probe complementary to the transcript of interest, followed by RNase total single-stranded RN.
Decompose A. If the transcript in question is present in total RNA, then it contains no mismatches 2
The double-stranded RNA-RNA hybrid is formed to form the RN.
It will not be affected by the ase process. This product can be identified by size-discriminating gel electrophoresis. Potato Example II. C. In order to determine whether the gene that induced the genomic subclone described in (1) was induced by arachidonic acid, labeled antisense transcripts generated from each subclone were treated with arachidonic acid-treated and untreated potato (Solanum tuberosum cv. Desiree). ) Hybridization to total RNA derived from suspension-cultured cells,
A protection assay was performed. The hybridization mixture was then treated with RNase and analyzed by electrophoresis. The protection of labeled antisense probes from degradation is shown to be found in RNA from arachidonic acid-treated cells and not in untreated cell-derived RNA, where expression of the corresponding gene is induced by arachidonic acid. Therefore, this assay method allowed the determination of whether the gene corresponding to the genomic clones was regulated by the arachidonic acid inducible promoter. A. In the RNase protection assay described in the protocol, the transcript in question was defined by the genomic subclone as follows: Genomic subclone used insert RNase expressing subclone λpPAL-1 1250bp BamHI / SstI pPAL-1 * λpPAL-2 271 bp EcoRI / EcoRI pPAL-2EP ** λpPAL-3 276 bp EcoRI / EcoRI pPAL-3EP *** λpPAL-4 276 bp EcoRI / BglII pPAL-4EP * λpPAL-8 278 bp EcoRI / PstI ** PPAL-PstIpPAL-PstI * PPAL-PstL ** pPAL 6 500 bp HindIII / SstI pPAL-6HS *** This antisense transcript is transcribed using the SP6 promoter and the sense transcript is transcribed using the T7 promoter. ** This antisense transcript is transcribed using the T7 promoter and the sense transcript is transcribed using the SP6 promoter. The insert described excludes the 600 bp insert of λpPAL-6 bound in pGEM-11Z (+) (Promega), with the appropriately digested pGEM-7z (−).
Was combined with. The polylinker region in both of these plasmids is flanked by SP6 and T7 RNA polymerase promoters, and SP6 or T7
RNA polymerase can be used to generate the ³² P-labeled antisense RNA transcript of the insert. The choice of which polymerase to use depends on the orientation of the gene fragment in the polylinker. ^{The 32} P-labeled antisense RNA transcript is a riboprobe Gemini system (Ribo
Subclone pPAL with probe Gemini System) II (Promega) and according to the manufacturer's instructions
-1, pPAL-2EP, pPAL-3EP, pPAL
-4EP, pPAL-8EP, and pPAL-6HS
In vitro. This RNA is then called Solanum
tuberosum cv. Desiree R of total RNA derived from suspension culture cells
Used as a probe in Nase protection assay.
The suspended cells were prepared according to the procedure of Example I. Total RNA was isolated from the cells prepared as described in A. and treated with freshly prepared 0.1 mM arachidonic acid (induced) or water (non-induced) for 3 hours. Was isolated as described above). The isolated RNAs were hybridized separately at 45 ° C. overnight to prepare 6 different antisense probes. Hybridization conditions were as follows: 40 mM Pipes, pH 6.
4, 1 mM EDTA, 400 mM NaCl, 50%
Formamide. The hybridization mixture was then subjected to RNase (40 μg / ml; Sigma (Si
gma), St. Louis, MO) and RNaseT1 (2U
/ Ml; BRL, Bethesda, MD) digested with a mixture of 6
% Polyacrylamide, 8M urea gel analysis. To provide a positive control in each of these experiments, sense R
NA in vitro with subclones pPAL-1, pPA
L-2EP, pPAL-3EP, pPAL-4EP, p
Transcribed from PAL-8EP and pPAL-6HS,
Hybridized with corresponding labeled antisense RNA. These hybrids must be a perfect match. This antisense RNA transcript was protected from degradation by RNase as expected when hybridized with sense RNA synthesized from the same subclone, but was completely degraded in the absence of sense RNA. pPAL-2EP, pPAL-3EP, p
PAL-4EP, pPAL-8EP, and pPAL-
RNA antisense transcripts from each of the 6HS were isolated from freshly prepared arachidonic acid Solanum tube
Protected from degradation by RNA prepared from rosum cv. Desiree cells. From these results, it was confirmed that the promoter bound to the gene (s) to which these clones correspond could be induced by arachidonic acid. Also, some protection was obtained from cells in which RN was not induced.
A, except that it was derived from cell-derived R
It was much lower than that observed in NA. PAL
This result was not surprising, as a significant background level of gene expression was consistently observed in these cells. The pPAL-1 derived probe was equally well protected from degradation by RNA from both induced and uninduced cells. This result suggested that the gene corresponding to λpPAL-1 was not the arachidonic acid-inducible PAL gene.

Example IV: Isolation of the potato PAL promoter A. Cloning Approximately 6 kb SstI-BamHI fragment derived from λpPAL-2 was gel-purified and subjected to standard DNA cloning procedures (Mani
subcloned into pGEM11Z (+) (Promega) using atis et al., supra). The binding mixture is DH5α
Transformed into, and AmP ^R colonies were selected. Correct construct was constructed with SstI-BamHI digested DNA
Confirmed by the presence of 6 and 3.2 kb bands of
It was named D2.2. The 1.4 kb Kpn-1SstI fragment and the 3.3 kb SstI-EcoRI fragment were combined with λpPAL.
Purified from -7. These fragments were mixed in an equimolar ratio and pUC1 previously digested with KpnI and EcoRI.
Subcloned into The resulting reaction is DH5
Transformed into α cells, Amp ^R colonies were selected. The correct construct was confirmed by the presence of the 4.7 and 3.2 kb bands by two digests with EcoRI-KpnI and was named 7P. B. Characterization The complete promoter-containing region and part of the coding region of λpPAL-7 and the majority of the complete promoter-containing region and part of the coding region of λpPAL-2 were sequenced. The two PAL coding regions share 80% sequence similarity at the nucleotide level.

Example V: Potato Arachidon Induced Gene Expression A. pUC-GUS. 1, pUC-GUS. 2, pUC
-GUS. Construction of 3 GUS lacking a promoter (β-glucuronidase)
Gene cassettes are plasmids pBI101, pBI
Hing I in 101.2 and pBI101.3
Available as II-EcoRI insert. (Clontech, Palo Alto, CA). GUS in this plasmid pBI101.2 and pBI101.3
The cassettes are identical to those in pBI101, except that their open reading frames are shifted by 1 and 2 nucleotides respectively with respect to the polylinker. As a result, the promoter fragment and part of the coding region can be inserted in all three open reading frames upstream of the GUS gene, producing translational and transcriptional fusions. The GUS coding insert was removed from each plasmid and each insert was separately ligated with HindIII-EcoRI digested pUC119. This ligation product was transformed into DH5α cells and Amp ^R colonies were selected. The correct construct shows 2.2 and 3.2 kb bands upon digestion with EcoRI and HindIII, respectively p
UC-GUS. 1, pUC-GUS. 2 and pUC-
GUS. It was named 3. B. Construction of PAL-GUS-fusion vector 1. The λpPAL-2 promoter plasmid pD2.2 (Valley Example IX.A) was added to Ha.
digested with eIII and the inserts separately Sma
I digested pUC-GUS. 1, pUC-GUS. 2 and pUC-GUS. Bound to 3. After transformation into DH5α cells and selection of Amp ^R colonies, Hind I
The correct constructs, confirmed by the 1.8 kb release by II digestion, were identified as pPAL2.1, pPAL2.2 and pPAL2.2.
It was named 3. Plasmids pPAL2.1, 2.2 and 2.
3 was digested with XbaI and SstI and each insert was ligated to XbaI- and SstI-digested pBI101. Plasmid pBI101 (Clontech, Palo A
lto, CA) is a binary vector, Agrobacter
It is a wide range of host plasmids used in the plant transformation system using ium tumefaciens . The ligation was transformed into DH5α cells and Kan ^R colonies were selected. Correct construct was 6k by digestion with XbaI and SstI
Confirmed by release of the b fragment, pBIN-2.
1, pBIN-2.2, and pBIN-2.3. 2. The promoter λpPAL-7 plasmid 7P was digested with PstI and EcoRV and the insert was digested with PstI and SmaI.
UC-GUS. 1, pUC-GUS. 2, and pUC
-GUS. Combined with 3. After transformation into DH5α cells and selection of Amp ^R colonies, the correct construct was
Confirmed by release of the 4.7 kb band upon digestion with coRI and BamHI. The correct constructs were designated pGUS7.1, pGUS7.2, and pGUS7.3, respectively.
I named it. Plasmids pGUS7.1, 7.2 and 7.3
Was digested with HindIII and EcoRI and each insert was ligated to HindIII- and EcoRI digested pBIN19. Plasmid pBIN19
Is a binary vector, Agrobacterium tumefa
A wide range of host plasmids used in ciens- based transformation systems, available from Clontech. This ligation product was transformed into TB-1 cells and Kan ^R colonies were selected. Correct constructs are designated Hind III and E
Confirmed by the release of a 6 kb fragment upon digestion with coRI, designated pBIN-7.1, pBIN-7.2, and pBIN-7.3, respectively. Prior to transformation of potato, pRK2073 was used as a helper strain to
Plasmids pBIN2.1, 2.2, 2.
3, 7.1, 7.2, and 7.3 in E. coli host to Ag
Transferred to robacteriuum tumefaciens strain LBA4404 (Co
rbin, D.I. Et al., 1982, J. Bacteriol. 149: 221-2.
28).

C. Potato transformation The potato tubers used in this experiment were Solanum tuberosu.
Obtained from potato plant of m cv. Desiree . Tubers stored for 1 week in the dark at 4 ° C. were washed, washed with deionized water to remove the soil, immersed in 95% ethanol for 1 minute and washed with sterile distilled water 3 times to sterilize the surface. Peel the tuber,
Surface in 15% 10% BUREX ^™ (commercial bleach) containing 2 drops of TWEEN 20 ^™ per 100 ml of solution.
It was sterilized for minutes and washed 5 times with sterile distilled water. One quarter of the ends of the tuber was discarded. Sterilized potato tubers were treated with hormone-free MS liquid medium (Murashige and Skoog, 196).
2, Phyiol. Plant 15: 473-496) and immersed for 20 minutes, and then the disc was collected. This explant was transformed into plasmids pBIN2.1, 2.2, 2.3; and pBIN7.1, 7.
Separate Ag with 2,7.3 (Valley Example IV.B)
robacterium tumefaciens LBA4404 (Clontech, Palo
Alto, CA) was suspended in 20 ml of hormone-free MS medium containing an overnight culture. This Agrobacterium
Strains were pre-induced with 50 μM acetosyringone. The tissue and Agrobacterium were incubated at room temperature on a vortex incubator with gentle shaking (about 60 rpm).
After 20 minutes, remove the explants with sterile Whatman filter paper.
The cells were blotted on No. 1 and transferred to an incubation medium, that is, a tobacco feeder plate prepared in advance for 2 days. The feeder plates are Horsch and Jones (In Vitro, 1980, 1
6: 103-108) with the following modifications: Cells from day 6 of suspension culture were sterile 30
Filter through a mesh sieve and collect on two layers of sterile Kimwipe to remove excess medium by wax. The cells were then placed in MS basal medium at 0.5 mg / l 2,4-D and 0.5 mg / l BA.
Resuspended in fresh MM medium supplemented with and to give a final concentration of 0.3 g fresh weight density per ml. Stir this suspension, 1.
Pipette 2 ml of sprout regeneration medium 3C52R with a weight of 5 ml
Transferred onto plates containing medium (Valley Table I) and medium as defined in Potato Table II. Potato Table I Potato Sprout Regeneration Medium 3C52R Medium Murashige and Skoog Salt Medium (4.3 g / L; Gibco, Grand Is land, NY) R3 Vitamins Thiamine / HCl 1.0 mg / l Nicotinic Acid 0.5 mg / l Pyridoxine / HCl 0.5 mg / l trans-zeatin riboside 5.0 μM indole acetic acid / aspartate 3.0 μM myo-inositol 100.0 mg / l sucrose 30.0 g / l tissue culture agar 8.0 g / l pH 5.7 potato table II Sprout induction medium Murashige and Skoog salt base Nitsch vitamins: thiamine / HCl 0.1 mg / l nicotinic acid 0.5 mg / l pyridoxine / HCl 0.5 mg / l glycine 2.0 mg / l D-biotin 0.5. 05 mg / l folic acid 0.5 mg / l diveric acid 0.5 mg / l 6-benzylaminopurine 3.0 mg / l α-naphthanacetic acid 0.1 3 mg / l casein hydrolyzate 1.0 g / l myo-inositol 100.0 mg / l sucrose 30.0 g / l tissue culture agar 8.0 g / l pH 5.7 2 days after the infection tuber disks cefotaxime (500 microns
(g / ml) and kanamycin (100 μg / ml). The composition of the medium was the same as the incubation medium, but without any feeder layer.
Tissues were subcultured in fresh selection medium at 2-week intervals and the cefotaxime concentration was reduced to 250 μg / ml after 4 weeks of culture.
The potato shoots were regenerated after 3 weeks of culture. When the regenerated potato shoots reached 3-5 mm in size, they were cut from tuber discs and grown on selective medium containing 250 μg / ml cefataxime and 100 μg / ml kanamycin. When the putative transgenic potato shoots reached 2 cm in size, they were transferred onto rooting medium containing 250 μg / ml cefataxime and 100 μg / ml kanamycin. This rooting medium was the same as the rooting medium except that no plant hormone was added.
When the shoots rooted, the resulting plantlets were transplanted from medium into soil and grown in a plant growth chamber. At desired time points, transgenic potato plants were assayed for GUS activity. The culture was exposed to 4000 lux light intensity for 16 hours throughout the experiment.
Incubated at 27 ° C.

Example V: Potato Use of Transgenic Potato Plants as a Pesticides Screen Tubers derived from transgenic plants shown in this example to contain stably incorporated PAL-GUS constructs induced by arachidonic acid. To clonally promote a transgenic potato plant containing a PAL-GUS fusion construct. These transgenic plants are maintained under standard greenhouse conditions. For use in pesticide screening assays,
The plants are grown under greenhouse conditions to a size and / or developmental stage that is manageable and resistant to the pathogen in question, eg Phythophthora infestans . The plants are treated with either a leaf spray or soil infiltration with a range of concentrations of an unknown chemical (elicitor). In parallel, additional plants are exposed to water as a negative control or the known elicitor as a positive control. After 6 to 24 hours of treatment, the leaf tissue was removed, and Jefferson et al. (Plant M
ol. Biol. Rep., 1987, 5: 387-405) was used to assay GUS activity. A positive result is indicated by a significantly higher level of GUS activity than that observed with the negative control. Positive results indicate, ie, identify, chemicals that are inducible to the potato PAL promoters.

Example VI: Isolation of PAL promoter from rice genomic DNA Rice genomic library screening Rice ( Oryza sativa ) genomic library (Clontech, Pa
lo Alto, CA; the average insert size in λEMBL3 is 15 kb) and elicitor-induced beans PAL1 exon IIc
Screening with DNA identified clones encoding the rice PAL gene. The cDNAs encoding the PAL1 exon II sequences are pPAL1-B6 and pPAL1-B6.
SPP1 (C. Lamb, The Salk Institute, La Jolla, CA
Obtained from; no published literature). This probe
800 bp insert of pPAL1-B6 and pSP
It was an equal mixture of 500 bp inserts of P1. The screening conditions were as follows: Hybridization: 42 ° C., 35% formamide, 5 × SSC, 5 × Denhardts, 5 × S.
SC, 0.2% SDS, 200 μg / ml salmon sperm DNA. Washing: 2 × SSC, 0.1% SDS, 4 times in total, repeated at 50 ° C. for 1 hour each. Five clones consisting of the putative rice PAL sequence were identified by six rounds of plaque purification. Clones λrPAL-2, λrPAL when hybridized to the probe
-4 and λrPAL-10 showed a strong signal, while λrPAL-8 and λrPAL-12 had weak hybridization. B. Clone characterization 1. Creation of restriction enzyme maps These five clones were characterized by restriction enzyme analysis. Clones λrPAL-8 and λrPAL-12 were identical; clones λrPAL-4 and λrPA.
L-10 had several common fragments; and clone λrPAL-2 was unique. Clone λr
Partial restriction enzyme maps of inserts derived from PAL-2, -4 and -10 are shown in FIG. 2. DNA Sequencing Restriction Enzyme Mapping and Bean PAL1 Exon II DN
By hybridization analysis using the A fragment as a probe, clones λrPAL-2, λrPAL-4, λrPAL- having a PAL hybridizing region were obtained.
The 8 and λrPAL-10 parts were determined. These parts are shown in FIG. 3 of the rice, and in the case of λrPAL-2, in a frame with a dotted line, λrPAL-4 and λrPAL-10.
The case is shown in the shaded area. The in-frame fragment was cut from the λrPAL clone and digested appropriately with pUC119 (Viei
ra and Messing, 1987, Meth. Enzym. 153: 3-1.
Bound to 1). After this ligation reaction was transformed into DH5α cells and Amp ^R colonies were selected, the correct plasmid was identified by digestion with the appropriate enzyme to release a fragment of the appropriate size (ie, insert size). The dotted frame region of λrPAL-2 is in p6 and
The shaded regions of PAL-4 and -10 are p and p, respectively.
296 and -4410. The insert DNA is a Sequenase protocol for double-stranded dideoxynucleotides (U.
S. Biochemical Corporation)
The partial sequence of the PAL region of rPAL-2 is the rice (Oryza
sativa cv. Nipponbare) Genome PAL gene (Minami
Et al., 1989, European Journal of Biochem. 185: 19-25, found to have 84.3% identity to the published sequence; λrPAL-4 and λrPAL. The partial sequence of the −10 derived fragment was identical and had 79.6% identity to the rice PAL genomic sequence; and λr
The partial sequence of the PAL-8 fragment had 53.8% identity with the rice PAL genomic sequence. These data suggest that clones λrPAL-2, λrPAL-4, and λrPAL-10 contain the rice PAL gene, and λrPAL-8 may or may not contain the rice PAL gene. Was. The orientation of the PAL gene within clones λrPAL-2, -4 and -10 was determined from the sequence data. Based on the size and orientation of the insert compared to the published sequence of the rice PAL gene (Minami et al., 1989, supra), all three clones appeared to contain the rice PAL promoter. To determine the promoter region of these clones, the fragments were further subcloned and sequenced. Clone λrPAL-2, Hind III-E
For both the coRI and EcoRI-HindIII fragments (Figure 3 in rice), the shaded boxes were subcloned separately into pUC119. λrPAL-4 and λrP
Since the region of similarity at the 5'end of the AL-10 clone and the published rice PAL genomic sequence is at the right end of the SalI fragment (shaded box in FIG. 3 of rice), λrPAL-10.
The derived SalI fragment (black box) was subcloned into pUC119 and sequenced. The λrPAL-4-derived promoter fragment was AccI-SstI-digested pGEM5Z (Promega (Prom) as a 3 kb ClaI-SstI fragment.
ega), Madison, WI). This 3 kb fragment is the right arm of EMBL3 of about 300 bp, 18
00 bp SalI fragment (black box in rice 3) and 18
It has a 50 bp SalI fragment (hatched frame).
The structural sequence of λrPAL-4 and -10 is shown as SEQ ID NO: 9 (λrPAL-4). The sequence of the promoter region and the coding region portion of λrPAL-2 is set to the sequence 8 (λr
It is shown as PAL-2). From this sequencing data,
λrPAL-4 has an additional 8 compared to λrPAL-10.
ΛrP except that it contains a 50 bp upstream sequence
It was shown that parts of the promoter regions of AL-4 and λrPAL-10 and their coding regions are identical. From a comparison of the published rice ( Oryzae sativa) PAL genomic sequences (Minami et al., 1989, supra) and the sequences of λrPAL-4 and λrPAL-10 subclones,
Translation initiation (ATG) of the published sequence is λrPAL-4
And was located 5 base pairs upstream of the SalI restriction site of λrPAL-10 (SEQ ID NO: 9). Therefore, the translation initiation site of the PAL gene encoded by λrPAL-4 and λrPAL-10 is located at this S
It may be near the alI site. The plant translation initiation consensus sequence (GNNATGG) is located at position 1704 of the structural sequence in (SEQ ID NO: 9) in λrPAL-4 and λrPAL-10. Computational translation of a nucleotide sequence starting at ATG at position 1702 and extending to position 1828 shows 78% similarity to the amino acid sequence predicted from the published rice PAL gene sequence (Minami et al., 1989, ibid.). And 51% of the amino acid sequence deduced from λrPAL-4 and λrPAL-10
(Cramer et al., 1989, Plant Mol. Biol).

Example VII: Construction of rice PAL-GUS construct A. GUS marker-hygromycin selective expression vector GUS lacking promoter (β-glucuronidase)
The gene cassette consists of plasmids pBI101, pBI1.
2 and HindIII-EcoRI in pBI101.3
Available as insert (Clontech
tech), Palo Alto, CA). Plasmid pBI101,
The GUS cassettes in bBI1.2 and pBI101.3 are identical to those in pBI101, except that their open reading frames are shifted by one and two nucleotides relative to the polylinker, respectively. As a result, by inserting a promoter fragment and a portion of the gene coding region into each vector, the G
Both translational and transcriptional fusions are made in all three open reading frames upstream of the US gene. All ligations were performed with a 5: 1 molar ratio of insert to vector. Therefore, the three plasmids pB
I101, pBI1.2 and pBI101.3, respectively, were digested with HindIII and EcoRI to give the GU
The S-code insert was isolated and purified on a 1% TBE gel. Hind III-EcoRI for each insert separately
Ligated into digested pUC119. The ligation product was transformed into DH5α cells and Amp ^R colonies were selected. The correct plasmids are EcoRI and Hin
Upon digestion with dIII, bands of 2.2 and 3.2 kb in size were shown, and pUC-GUS. 1, pUC-GUS. 2 and pUC-GUS. We named them 3 respectively. E. coli hygromycin B gene-containing plasmid pSV2hyg (J. Kwoh, Baxter Healthcare, San Die
go, CA) was digested with Hind III and Bgl II, and the entire digest was digested with T4 DNA polymerase (BRL, Be).
thesda, MD). The fragment consisting of the hygromycin gene was isolated on a 1% TBE gel and PstI digested T4 D using the established cloning procedure.
It was ligated to NA polymerase treated pCANVCN DNA (Pharmacia, Piscathaway, NJ). The ligation reaction was transformed into DH5α cells and Amp ^R colonies were selected. The correct plasmid released a 2 kb fragment upon digestion with HindIII,
It was named p35S-hyg. Plasmid p35S-h
Yg was digested with HindIII and this 2 kb fragment was digested with 1%
Purified on TBE gel. Next, this fragment is called Hind I
II digested pUC-GUS vector (pUC-GU
S. 1, pUC-GUS. 2 and pUC-GUS.
Separately bound to 3). The ligation reaction was transformed into DH5α cells and Amp ^R colonies were selected. The correct plasmid is 2k after digestion with Hind III
b fragment is released and pHyg-GUS. 1, pHyg-G
US. 2, and pHyg-GUS. We named them 3 respectively. In all three plasmids, the direction of transcription of the hygromycin resistance gene was opposite to that of the GUS gene; therefore, any GUS activity detected in plants transformed with these vectors would result in CaMV35S. It would not be due to read-through transcription from the promoter. B. Construction of expression vector having rice PAL promoter 1. The clone λrPAL-4 λrPAL-4-derived promoter retaining fragment was ClaI-S.
From the stI subclone (rice example VI.B.2), BssHII digestion of the subclone, Klenow treatment, Pst
Isolated after I digestion. Separately generated fragments of about 1850 bp,
Furthermore, pHy digested with SmaI and PstI
s-GUS. 1, pHys-GUS. 2 and pHys
-GUS. Ligated into 3 plasmids. The combined product is D
Amp ^R colonies were selected by transformation into H5alpha cells. The correct construct was made 70 by EcoRI digestion.
Determined by linearization of the 00 bp plasmid, rPAL4.
1, rPAL4.2 and rPAL4.3, respectively. 2. rPAL-10-derived promoter A λrPAL-10-derived promoter fragment was isolated by the following method. Plasmid pSa11000 (800 bp Sal derived from the black frame of λrPAL-10 in FIG. 3 of rice.
The I fragment, ligated to pUC119) was digested with SalI and PstI. Plasmid p4410 (see rice Example VIB.2) was digested with BssHII and Klen
ow treatment, digestion with SalI and SstII (to remove other BssHII-SalI fragments that interfere with binding). The 800 bp fragment and the entire SalI
-SstII digested product with SmaI- and PstI digested pH
yg-GUS. 1, p-Hyg-GUS. 2 and pH
yg-GUS. It was attached to 3 in three different ways. The ligation product was transformed into DH5alpha cells and transformed into Am
p ^R colonies were selected. Correct construction with EcoRI
RPAL10.1, rPAL10.2, and rPAL determined by linearization of the 6200 bp plasmid by digestion
They were named 10.3. 3. λrPAL-2-derived promoter A λrPAL-2-derived promoter fragment was digested with EcoRI,
After Klenow treatment, phosphorylated BamHI linkers (New England Biolabs, Inc., Beverly, MA) were added. Generated by established cloning operations
The 2100 bp fragment was separately separated from each other by BamHI-Sma.
I digested pHyg-GUS. 1, p-Hyg-GUS. Two
And pHyg-GUS. Bound to 3. The ligation was transformed into DH5alpha cells and Amp ^R colonies were selected. The correct construct is rPAL2.1, rPA
They were named L2.2 and rPAL2.3, respectively.

Example VIII: Rice A. Isolation and transformation of protoplasts Rice suspension culture ( Oryza sativa IR54) was transformed into C. Lam.
b, The Salt Institute, La Jolla, CA). This suspension culture was maintained and grown on 2.3 g / l proline-added N6 medium (rice table III). Rice Table III: N6 medium KNO ₃ 283.0 (HN ₄ ) ₂ SO ₄ 463.0 KH ₂ PO ₄ 400.0 MgSO ₄ .7H ₂ O 185.0 CaCl ₂ .2H ₂ O 166.0 Na ₂ -EDTA _{37.25 FeSO 4 · 7H 2 O 27.85} KI 0.8 H 3 BO 3 1.6 ZnSO 4 · H 2 O 1.5 MnSO 4 · 7H 2 O 3.3 thiamine · HCl 0.1 pyridoxine · HCl 0.5 Nicotinic acid 0.5 Glycine 2.0 2,4-Dichlorophenoxyacetic acid 2.0 L-Tryptophan 50.0 Myo-inositol 100.0 Sucrose 30.000.0 pH 5.8 Weekly passage of the main culture Cultured. In each subculture, 2.5 g fresh weight of rice suspension cells was transferred to 50 ml fresh medium. Rotate the main culture at 27 ° C in a dark place at a speed of about 125 rp.
Incubated at m. Suspended cells on day 5 ( Oryza sa
tiva IR54) 10 g fresh weight in a dark place in a greenhouse, 1%
The cells were incubated in 100 ml of protoplast wash solution (pH 6.0) containing cellulase RS, 0.1% pectolyase Y-23 at a speed of 50 rpm for 4 hours in an orbital incubator. Rice Table IV: Protoplast Wash Solution Composition Amount KH ₂ PO ₄ 27.2 mg / l KNO ₃ 101.0 mg / l CaCl ₂ .2H ₂ O 1.4 mg / l MgSO ₄ .7H ₂ O 246.0 mg / l KI 0. _{16mg / l CuSO 4 · 7H 2} O 0.025mg / l MES 5.0mM mannitol 72.9 g / l pH 5.8

The enzyme protoplast mixture was passed through a 300 mesh tissue sieve to remove debris, then about 1
Centrifuge at 47 xg for 10 minutes at room temperature. The pelleted protoplasts were resuspended in 35 ml of the protoplast washing solution and centrifuged at about 147 × g for 10 minutes to repeat the washing twice. Protoplasts were purified by centrifugation with a Percoll solution step gradient. Protoplasts were resuspended in 6 ml of 70% Percoll solution (Rice Table V), 50% Percoll solution was overlaid on this resuspended protoplast, and 6 ml of 25% Percoll solution was overlaid with 50% Percoll solution. did. Rice Table V: Percoll Solution (1) 70% Percoll Solution Percoll Solution 70.0 ml / 100 ml Commercial Murashige & Skoog Salt Base 0.43 g / 100 ml Sucrose 3.0 g / 100 ml Manitol 0.3M (2) 50% Percoll Solution Percoll solution 50.0 ml / 100 ml Commercial Murashige & Skoog Salt base 0.43 g / 100 ml Sucrose 3.0 g / 100 ml Manitol 0.3M (3) 25% Percoll solution Percoll solution 25.0 ml / 100 ml Commercial Murashige & Skoog Salt base 0.43 g / 100 ml sucrose 3.0 g / 100 ml Manitol 0.3M Percoll protoplast gradient for about 15 minutes at room temperature, about 2 min.
Centrifuge at 97 × g. Protoplasts were collected at the interface of 25% and 50% Percoll solutions using a sterile Pasteur pipette and transferred to 25 ml of protoplast wash solution. Add protoplasts to the protoplast wash solution 25
The cells were resuspended in ml and centrifuged twice at about 147 × g for 10 minutes. The protoplasts were resuspended in protoplast wash solution at a density of approximately 1 × 10 ⁷ protoplasts per ml. Transformation with PEG was carried out as follows: 1 ml volume of each protoplast,
It was mixed with 100 μg / ml of plasmid DNA derived from either rPAL2.2 or rPAL4.3 (Rice Examples VII.B.1, and 3). Medium (Krens's F solution; Krens et al., 1982, Nature, 296: 72-74) equivalent amount of polyethylene glycol (PEG8000, 40% w)
/ V) was added to the mixture of protoplasts and plasmid DNA. 45 ° C to the protoplast-PEG mixture
Heat shock was applied for 5 minutes and cooled with ice for 20 seconds. The solution was then brought to room temperature and incubated at 30 ° C for 30 minutes. The protoplast-PEG mixture was then diluted with Krens' F solution until the PEG concentration was below 2% with the timetable below as a standard. 0-2 minutes 2 drops every 30 seconds 2-5 minutes 5 drops every 30 seconds 5-10 minutes 0.5 ml every 30 seconds 10-15 minutes 1 ml every 30 seconds 15-30 minutes 2 ml every 5 minutes Plasmid About 14 protoplasts treated with DNA
The cells were collected by centrifugation at 7 × g for 10 minutes, resuspended in N6 medium containing 0.3 M mannitol (Rice Table III), and incubated at room temperature in the dark at 50 rpm in an orbital incubator. Each protoplast mixture sample was treated with a fungal elicitor prepared from the cell wall of Pyrichlaria oryzae . The preparation of fungal elicitors is described in Rice Table VI below. Table VI: Preparation of fungal elicitor Maintenance medium: Cornmeal agar (Difco ^TM 03
86-01-3) ； Plate Growth medium: Liquid culture Cornmeal culture solution: Cornmeal 5 in 800 ml of purified water
Mix 0 g; refrigerate the mixture overnight; then 60
Heat at 0 ° C. for 1 hour; Filter solution; Bring solution to 1 liter; Autoclave sterilize for 20 minutes. Autoclave all media and equipment twice (120 ° C / 20
Minutes). Allow plate / liquid medium to sit for at least 1 week and inspect for contamination. Retention of culture: Take 1 x 0.6 mm cork borer disc and place on cornmeal agar dish. A sample is taken from the edge of the mycelium that grew black. Inoculation into growth medium; cork borer disc 5 × 0.6 mm
Is taken from the edge of the growing mycelium / 50 ml medium (250 ml conical flask). Keep in the dark at 23 ° C. Mycelia are collected after 1 month. Preparation of crude elicitor from Pyricularia oryzae hyphae wall 1. The hyphae are homogenized in a Waring blender for 60 seconds (5 ml water per gram hyphal wet weight). Filter the homogenate through a coarse sintered glass filter *; retain the residue. (Caution, it is often necessary to wash the glass filter with concentrated nitric acid and then with purified water, because it can clog the eyes). 2. Repeat step 1 three more times *. 3. Once homogenized with chloroform: methanol (1: 1) mixture (1 g of mycelium per 5 ml of liquid); filter *. 4. Homogenize once with acetone (5 ml of acetone)
Per hypha 1 g); filtered. 5. Air dry at room temperature. This treatment leaves a hyphal wall fraction. 6. 5 g of the wall are suspended in 100 ml of water and sterilized in an autoclave at 121 ° C for 1/2 hour. 7. The autoclaved suspension is filtered through a coarse sintered glass filter. 8. Concentrate by freeze-drying (freeze-drying). 9. Resuspend in sterile water (about 10 ml). 10. Perform carbohydrate and protein assay. * This varies. Do the final acetone step until the filtrate is clear. Reference: Ayers et al., Plant Physiol. 1976, 57:76.
0. Dixon, Planta, 1981, 157: 272. Transformation of a fungal elicitor at a concentration of about 60 or 80 μg / ml 22
(80 μg) or 36 (60 μg) after the addition,
Incubation was carried out for another 20 hours. This 4
After 2 or 56 hours of incubation, control and treated samples were treated as described below.

1. Transient Expression Assays After incubation for 40 hours after transformation, rice PAL
The rice protoplasts transformed with the -GUS fusion construct were collected by centrifugation in microfuge for about 30 seconds. 50 μl of GUS extraction buffer (Je
fferson, 1987, Plant Mol. Biol. Rep. 5: 387:
-405). An additional 150 μl of GUS extraction buffer was added and the protoplasts were frozen in liquid nitrogen and then thawed at room temperature. Repeat this operation to mix the protoplast mixture in a microfuge.
Centrifuge for 0 minutes at 4 ° C. The GUS activity was determined by incubating the protoplast extract (100 μl) with 0.6 ml MUG buffer containing 20% methanol according to the procedure of Jefferson (1987, supra). Briefly, the GUS activity assay uses 4-from the fluorescent substrate 4-methylumbelliferyl glucuronide (MUG).
Fluorometric assay measuring the production of methylumbelliferyl. Protein concentration was determined using the reagents obtained from Bio-Rad according to the Bradford protein assay. 2. Results Protoplasts were rice PAL-GUS fusion constructs rPAL2.2 and rPAL4. Each containing a translational fusion between the rice PAL promoter and the GUS gene.
3 were transformed separately. Extracts of these protoplasts were assayed for GUS activity. The results of these assays are shown in Table VII of rice. Rice Table VII: Rice PAL-GUS fusion construct rPAL2.
Rice protoplasts transformed with 2 GUS activity GUS activity% increase by elicitor (pmol / min / mg) ^a Experiment 1: no DNA control 26-rPAL2.2 786-rPAL2.2 + elicitor 862 9.8% rPAL4.386 -RPAL4.3 + elicitor 70-Experiment 2: rPAL2.2 1,542-rPAL2.2 + elicitor 1,707 10.7% Experiment 3: rPAL2.2 + 2,464-rPAL2.2 + elicitor 2,683 8.8% rPAL2 .2 + elicitor 2,655 7.7% a. These values are the average of the data obtained at different time points in the enzyme assay. a. Background GUS expression Higher levels of uninduced GUS activity than in untransformed uninduced control protoplasts were measured in protoplasts transformed with plasmid DNA but not elicitor. High levels of GUS are due to some level of constitutive and / or inducible (induction other than elicitor induced) GUS expression in experimentally transformed protoplasts and successful transfer of the construct into said protoplasts. Suggests that you have done it. The difference in background GUS levels of protoplasts transformed with rPAL2.2 and rPAL4.3 was
As a result of the difference in transformation efficiency, the difference between the rice PAL promoter and the GUS gene in the construct, or the difference in the responsiveness of the two most likely rice PAL promoters to other possible inducers. Can also be b. Elicitor-Induced GUS Expression The elicitor-treated protoplasts transformed with rPAL2.2 averaged 9.8% higher G than the activity of untreated protoplasts transformed with the same construct.
It produced US activity. In the elicitor-treated protoplasts transformed by this mechanism compared to untreated protoplasts transformed by rPAL4.3, GU
There was no significant increase in S activity. These data are
It suggests that the λrPAL-3 promoter is inducible by the fungal elicitor evaluated herein, while the λrPAL-4 promoter is not.

Example IX: Rice Generation of transgenic rice plants A. Isolation of Protoplasts, Transformation, and Regeneration of Transgenic Rice Plants Rice seeds ( Oryza sativa ) are available from the US Department of Agriculture National Small Grains Collection, US
DA). Callus cultures were prepared according to Kyozuka et al. (Molecular Gene End Genetics (Mol. Gen.
Genet.), 1987, 206: 408-413). Induced callus was maintained and grown on two different media: MS2 media (Kyozuka et al., 1987) and N6 media. The formulations for these media are shown above. Rice suspension culture was prepared by using two suspension media N6
Derived from rice callus in medium and R2 medium. MS2
Rice callus grown in medium was induced in R2 medium to produce N6
Rice callus grown in medium was induced in N6 medium. This R2 medium is as follows: Rice Table VIII: Rice suspension R2 medium Constituent amount KNO ₃ 404 4.00 mg / l NaH ₂ PO ₄ .H ₂ O 275.98 mg / l (NH ₄ ) ₂ SO ₄ 330 .25 mg / l MgSO ₄ .7H ₂ O 246.48 mg / l CaCl ₂ .2H ₂ O 147.02 mg / l MnSO ₄ .7H ₂ O 1.54 mg / l ZnSO ₄ .7H ₂ O 2.20 mg / l H ₃ BO ₃ 2.86 mg / l CuSO ₄ .5H ₂ O 0.196 mg / l Na ₂ MoO ₄ .2H ₂ O 0.11 mg / l FeSO ₄ .7H ₂ O 10.24 mg / l Na ₂ -EDTA 7.25 mg / l thiamine · HCl 0.1 mg / l pyridoxine · HCl 0.5 mg / l nicotinic acid 0.5 mg / l glycine 2.0 mg / l 2,4-dichlorophenoxyacetic acid 2.0 mg / l myo-inositol 100.0 mg / l Sugar 30.0 g / l Casein hydrolyzate 3.0 g / l pH 5. 8 The induction culture was cultivated in the dark in a swirling culture machine at a speed of about 50 rpm.
Incubated at 7 ° C. Rice suspension cultures were subcultured weekly. At each subculture, 2.5 g fresh weight cells were transferred to 25 ml fresh suspension medium in a 250 ml flask. A two-fold increase in proliferation was observed over a 7 day growth period. 13 g of the fresh cell weight of suspension on the 5th day (2nd month of induction) induced with R2 medium was added to 100 ml of a protoplast washing solution (pH 6.0) containing 2% cellulase RS and 0.2% pectolyase Y-23 at room temperature. Speed about 50
Incubation was carried out for 3 hours and 45 minutes in the dark in a rotary incubator at rpm. The enzyme protoplast mixture is passed through a 300 mesh tissue sieve to remove debris, then about 10
Centrifuge at 0 × g for 10 minutes at room temperature. The pelleted protoplasts were washed twice by resuspending them in about 35 ml of the protoplast washing solution and centrifuging at about 100 × g for 10 minutes. Protoplasts are Oryza sa
Purified as described above for tiva IR-54 (see rice Example VIII.A). The Percoll protoplast gradient was centrifuged at about 200 xg for 15 minutes at room temperature. Protoplasts were taken from the top of 25% Percoll solution and transferred to 30 ml of protoplast wash solution. Resuspend in 30 ml of protoplast wash solution,
After each resuspension at approximately 100 × g for 10 minutes, centrifugation and washing were performed twice. Protoplasts were resuspended in protoplast wash solution at a density of approximately 3 × 10 ⁷ protoplasts per ml. Transformation with PEG was performed using Oryza sativa IR-5
4 was performed using the transformation method as described above. Protoplasts treated with plasmid DNA are
It was collected by centrifugation at about 100 xg for 15 minutes, resuspended in 1 ml of R2 protoplast medium, and containing 2.5% sea plaque agarose (equivalent to R2 medium except that 0.4 M sucrose was included) R2 Mix with an equal volume of protoplast medium,
Transferred to a 35 mm x 15 mm sterile petri dish. Protoplast-containing solidified agarose was cut into 4 blocks, and induced by N6 medium, and passed through a 30-mesh tissue sieve. Transferred to a sterile petri dish. Cultures were incubated in the orbital incubator at a speed of approximately 50 rpm in the dark at room temperature. After 10 days, the rice protoplast-containing agarose block was transferred to a new plate and washed with the R2 medium to completely remove Sasanishiki suspended cells. Add agarose block to 20 ml of R2
It was cultured in a 100 mm x 150 mm petri dish containing protoplast medium. The main culture was incubated at room temperature in the dark on a swirl culture machine at a speed of about 40 rpm. After 4 days, the protoplast-derived colony-containing agarose block was treated with 50
Transferred to R2 protoplast medium containing μg / ml hygromycin B. The main culture was incubated at room temperature in the dark in a swirl culture machine at a speed of about 30 rpm. After 11 days, protoplast-induced rice callus was visible. The rice callus-containing agarose block was treated with 50 μg / ml hygromycin B-containing N6 soft agarose medium (N6 basal medium, 2
mg / 1 2,4-dichlorophenoxyacetic acid, 6% sucrose and 0.25% sigma type I agarose (Sigma Type
I Agarose), pH 5.7. 4,000 main culture
Incubated at 27 ° C for 16 hours under lux light. After 10 days, the rice callus on the agarose block had a diameter of about 1 mm and had 50 μm as N6 soft agarose medium.
Transferred to N6 medium containing g / ml hygromycin B, but this medium contained 0.5% Sigma Type I agarose. After 2 weeks, when the rice callus became about 2-3 mm in diameter, they were transferred to regeneration medium containing 50 μg / ml hygromycin B. This regeneration medium is N6 inorganic salt, Murashige and Skoo
g vitamins, 6% sucrose, 1% sigma type I agarose and 2 μg / ml kinetin. When sprouts are 3 cm or longer, they are rooted in hormone-free medium containing 50 μg / ml hygromycin B containing 1% sigma type I agarose. When regenerated, this transgenic rice plant was used, for example, in a pesticide screen to identify inducers of these promoters.

Rice Example X Use of transgenic rice plants as a pesticide screen Transgenic rice was enhanced by germinating seeds in a suitable tissue medium containing a suitable concentration of hygromycin to produce a PAL-GUS fusion. Ensure that the construct is present in all tissues of this transgenic plant. Once this transgenic plant reaches the proper size, for example 3-6 inches in length,
They are transplanted to soil and kept under standard greenhouse conditions.
For use in pesticide screening assays, the size and / or size of plants that can be maintained under greenhouse conditions and that are resistant to pathogens of interest, eg Pyricularia oryzae
Or grow to stages. The plants are treated by dipping in leaf spray or soil with a wide range of concentrations of unknown chemicals or chemicals. In parallel, additional plants were exposed to water as a negative control, or known elicitors such as probenazole (Iwata et al., Anals.
Of Fight Pathology Society, Japan (Ann. P
hytopath. Soc. Japan), 1980, 46: 297-30.
Expose to 6). Leaf tissue was removed after 6-24 hours of treatment, and Jefferson et al. (Plant Mol. Biol. Rep., 1987,
5: 387-405) was used to assay GUS activity. A positive result is indicated by a level of GUS activity that is significantly higher than the activity observed in the negative control. A positive result suggests and identifies a chemical capable of inducing the rice PAL promoter.

Example XI: Assay Use of 3SR for Identification of Inducible Promoters from Rice Genomic DNA A. 3SR 3SR reaction (self-retaining sequence replication) is a method for in vitro amplification of specific RNA sequences (Guatelli et al., 19
90, Proc. Natl. Acad. Sci. USA. 87: 1874-187
8). The synthesis of products, which are predominantly RNA, is provided by the action of AMV reverse transcriptase and T7 RNA polymerase in the presence of ribo and deoxyribonucleotides. The region to be amplified is specified by a pair of RNA primers, one of which has a T7 RNA binding site and is complementary (antisense) to the target mRNA. The second primer is an mRNA in a region about 200-300 bp away from the first primer.
Is the same as (sense). The 3SR technique is shown in FIG. According to the present technique, the target RNA sequence is converted by the reverse transcriptase into an RNA / DNA duplex in the region initially specified by the first primer. This double strand is the RNas present on the AMV reverse transcriptase.
Attacked by eH. RNase H destroys the RNA template but leaves the cDNA unchanged.
This cDNA is used as a template for the synthesis of the second cDNA strand using reverse transcriptase and a second primer. The resulting double-stranded DNA template acts as a substrate for T7 RNA polymerase, producing multiple copies of antisense RNA that hybridizes to primer 2 and double-stranded cDNA.
Initiate the second cycle of synthesis. This causes RNA transcription to occur again, and this antisense RNA allows recycling of the 3SR reaction until cycling is stopped due to some limitation in the reaction. This target RNA is amplified 10 ^{6 to} 10 ⁹ times. This example uses the 3SR technology to generate the bean genome DN.
3 illustrates the identification of A-derived inducible promoters. B. Tissue Culture Conditions Bean ( Phaseolus vulgaris Canadian Wonder) suspension culture (obtained from Dr. C. Lamb) modified Schenk and Hildeb
It was maintained and grown on rand medium (SH medium) (below, Assay Table IX). The main culture was subcultured weekly. At each subculture, 6 g fresh weight of bean suspension cells were transferred to 100 ml of fresh SH medium. The main culture was incubated at 27 ° C in the dark in a swirl culture machine at a speed of about 125 rpm.

C. Bean PAL-1 amplification PAL induction experiments were subcultured on bean cell suspension cultures 7
Went a day later. 2 g of the culture was treated under the following conditions for 3 hours: (1) According to rice table IX, Collectoric hum lindemuth
20 μg / ml of fungal elicitor isolated from cell wall of ianum race Alpha: (2) E. coli strain DH5α
(BRL, Betheda, MD); (3) Agrobacterium tume
faciens strain C58 (Dr. Maaten Chrispeels, UCSD,
La Jolla, CA); (4) Clavibacter michiganese pv M
ichiganese (American Type Culture Collection, Rock
ville, MD); (5) Xanthomonas Campestris pv malvac
earum (ATCC, Rockville, MD): (6) Pseudomona
s syringae pv tomato (ATCC, Rockville, MD);
(7) Pseudonomas syringae pv tabaci (ATCC, Rock
ville, MD): (8) Erwinia carotovora subsp. caro
tovora (ATCC, Rockville, MD); (9) SH medium (negative control, t = 3); and (10) untreated, immediately frozen (t = 0). All bacteria were added at a concentration of approximately 10 ⁷ -10 ⁸ cells / ml. Total nucleic acid was isolated from 0.2-0.3 g of each sample using the following protocol: (1) Frozen tissue was crushed into a fine powder in a mortar and pestle; (2) 450 μl of powdered tissue TN buffer (0.1M NaCl, 0.01
M Tris. PH 9.0, 1 mM EDTA) and 450μ
l phenol: chloroform (1: 1),
Vortex until thawed; (3) Slurry was centrifuged in microfuge for 10 minutes; (4) Aqueous layer removed and precipitated with EtOH according to standard protocol of Maniatis et al. (1982); (5) ) Nucleic acids were collected by centrifugation and the concentration was determined by spectrophotometry at 260 nm; (6) Final concentration was 0.6 μg / ml.
Was prepared. Total nucleic acid was subjected to 3SR amplification using primers derived from the bean PAL-1 gene sequence (Edwards
1985. Pro. Natl. Acad. Sci.). The sequences of the primers are shown as SEQ ID NOS: 10 and 12. (T7 RNA
The binding site for polymerase 1 in primer 1 is shown as SEQ ID NO: 11). A total nucleic acid concentration of about 0.6 μg was used for the 3SR reaction, the exact details of which are:
It is shown in Assay Table X. 3SR reaction products were analyzed using the dot blot procedure (Schleier and Schuell
, Keene, NH). 2 μl of reaction was added to DM5 (2.6 m
M Tris, pH 8.0, 0.26 mM EDTA, 10xS
SC, 7.4% formaldehyde) and heated to 55 ° C. for 20 minutes, then placed on ice and then placed on nitrocellulose in a dot blot analyzer according to the manufacturer's instructions. .. Nucleic acid is UV
Immobilized on nitrocellulose by cross-linking (Stratagene, La Jolla, CA). The blot was probed with an oligonucleotide derived from the bean PAL-1 gene (Edwards et al., 1985, et al.). This sequence was identical to the nRNA (sense) strand. The probe is shown as SEQ ID NO: 13. Maniatis the oligonucleotide
Et al., 1982, et al., End labeled with T4 kinase with γ ³² P-ATP (BRL, Betheda, MD).
Blot at 2 × 10 ⁶ cpm / ml for 1 hour, 5 × SSP
E, hybridized in 4xBP (2% BSA, 2% polyvinylpyrrolidone-40). The filters were washed 3 times in room temperature for 5 min each in 1 × SSPE, 1% SDS and 1 hour at 42 ° C. in 1 × SSPE, 1% SDS. PA more in the induced sample than in the uninduced sample
L hybridizable was detected. Therefore,
This data suggests that PAL-1 is a gene inducible by phytopathogenic bacteria. further,
It can be concluded that 3SR ^™ is a rapid and sensitive technique and can be used for the identification of inducers of plant defense genes.

Assay Table IX: Schenk and Hildebrandt SH Medium Composition Amount mg / ml KNO ₂ 2500 0.00 NaH ₂ PO ₄ .H ₂ O 300.00 MgSO ₄ .7H ₂ O 400.00 CaCl ₂ .2H ₂ O 225.00 Na ₂ -EDTA 20.00 FeSO ₄ / 7H ₂ O 13.90 MnSO ₄ / 7H ₂ O ₁ 2.50 H ₃ BO ₂ 5.00 ZnSO ₄ / 7H ₂ O 1.00 KI 1.00 CuSO ₄ / 5H ₂ O 0.20 Na ₂ MoO ₄ .2H ₂ O 0.10 Thiamine.HCl 15.00 Pyridoxine.HCl 1.50 Nicotinic acid 15.0 2,4-Dichlorophenoxyacetic acid 0.440 P-chlorophenoxyacetic acid 2.100 Kinetin 0.105 Sucrose 34,000.00 Myo-inositol 1,000.00 pH 5.8 Assay Table X: 3SR Protocol 5x Reaction Buffer 20.0 μl 200 mM Tris.HCl, pH 8. 150mM MgCl ₂ 100mM KCl 20mM spermidine 50mM dithiothreitol dNTP's (25mM) (Promega, Madison, WI) 4.0μl rNTP's (25mM) ( Aldrich (Aldrich) Milwaukee, WI) 20.0μl DMSO (Sigma, St Louis, MO) 1.0 μl sorbitol (66.7% w / v) (Sigma, St. Louis, MO) 22.5 μl primer 1 5.0 μl primer 2 5.0 μl water 8.5 μl target nucleic acid 5.0 μl 65 Heated sample for 1 minute at 37 ° C Transfer for 1 minute at 37 ° C Place on ice Add 1 µl of AMV reverse transcriptase (20u) (Life Sciences, St. Petersburg, FL) 1 µl of T7 RNA polymerase (50u) (Stratagene) , La Jolla, CA) Leave the reaction at 42 ° C for 1 hour to stop amplification by placing on ice Transcription of DNA sequence in plant host It can be controlled,
And responds directly or indirectly to inducible or exogenous elicitors and / or damage.

[0041]

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 245 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism: Solanum tuberosum strain name:. cv Desiree direct origin clone name: pPAL-1 sequence GATCCTCTTC AGAAACCAAA GCAAGCATCG TTATGCTCTC CGAACATCTC CACAATGGCT 60 TGGCCCTCAA ATTGAAGTCA TACGCGCAGC AACTAAGATG ATTGAGAGGG AGATTAACTC 120 AGTGAACGAC AATCCATTGA TCGATGTATC AAGAAACAAG GCCTTGCACG GTGGCAACTT 180 TCAAGGCACC CATATGGTGT GTCATGGATA ATACAGATTG GCCTGCATCA TAGGAATGAT 240 GTTTG 245 SEQ ID NO: 2 Sequence length: 271 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism: Solanum tuberosum Strain: cv. Desiree Direct origin Clone name: pPAL-2 sequence CTTGGGATTA ATCTCAGCCA GGAAAACAGC TGAGGCTGTT GATATCTTGA AGCTAATGTC 60 ATCAACCTAT CTCGTGGCGC TTTGCCAAGC TATAGACTT A CGGCATTTGG AGGAGAACTT 120 GAAGAGTGCT GTCAAGAACA CAGTTAGCCA AGTAGCTAAG AGAACTTTGA CAATGGGTGC 180 TAATGGGGAA CTTCATCCAG CAAGATTCTG TGAGAAGGAA TTGCTTCGAG TCGTGGATAG 240 GGAATACTTG STR: GLAAT ACT TG: C3 271 Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism name: Solanum tuberosum Strain name: cv. Desiree Direct origin Clone name: pPAL-3 Sequence GAATTCCTTG GGATTAATCT CAGCCAGGAA AACAGCCGAG GCTGTTGATA TCTTGAAGCT 60 AATGTCATCA ACCTAT GAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180 GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTCTGCGAA AAGGAATTGC TTCGAGTCGT 240 GGACAGGGAA TACTTGTTTG CCTATGCAGA TGACCCC 277 SEQ ID NO: 276 SEQ ID NO: 276 Sequence: SEQ ID NO: 276 Sequence: SEQ ID NO: 276 Genomic DNA Hypothetical: No Antisense Scan: No Origin: Organism Name: Solanum tuberosum strain name:. Cv Desiree direct origin clone name: pPAL-4 sequence GAATTCCTTG GGCTTAATCT CAGCCAGGAA AACAGCTGAG GCTGTTGATA TCTTGAAGCT 60 AATGTCATCA ACCTATCTCG TGGCGCTTTG CCAAGCTATA GACTTACGGC ATTTGGAGGA 120 GAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180 GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTTTGCGAA AAGGAATTGC TTCGAGTCGT 240 GGACAGGGAA TACTTGTTTG CCTATGCAGA TGACCC 276 SEQ ID NO: 5 Sequence length: 300 Sequence type: Nucleic acid Strand number: Single-stranded topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin : organism: Solanum tuberosum strain name:. cv Desiree direct origin clone name: pPAL-6a sequence AAGCTTGGAC TATGGTTTCA AGGGAGCTGA AATCGCGATG GCTTCTTACT GCTCGGAACT 60 TCAATTCTTG GCAAATCCAG TGACCAACCA TGTTCAGAGT GCCGAGCAAC ACAACCAAGA 120 TGTGAACTCC TTAGGCTTAA TCTCAGCAAG GAAAACAGCT GAGGCTGTCG ACATCTTAAA 180 GCTAATGTCA TCAACCTATC TCGTGGCACT TTGCCAAGCT ATAGACTTGA GGCATTTG GA 240 GGAGAACTTG AAGAGTGTTG TCAAGAACAC AGTTAGCCAA GTAGCTAAGA GACTTTGACA 300 SEQ ID NO: 6 Sequence length: 260 Sequence type: Nucleic acid Strand number: Single stranded Topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin : organism: Solanum tuberosum strain name:. cv Desiree direct origin clone name: pPAL-6b sequence TGGTGAACTT CATCCAGCAA GATTCTGCGA GAAGGAATTG CTTCGAGTCG TAGACAGGGA 60 ATACTTGTTT ACCTATGCTG ATGACCCCTG CAGCTCCACC TATCCTTTGA TGCAGAAGCT 120 GAGACAGGTC CTTGTTGATC ATGCAATGAA GAATGGTGAA AGTGAGAAGA ATATCAACAG 180 CTCAATCTTC CAAAAGATTG GAGCTTTCGA GGACGAATTA AATGCTGTGT TGCCTAAAGA 240 AGTTGAGAGT GCAAGAGCTC 260 SEQ ID NO: 7 Sequence length: 278 Sequence type: Nucleic acid Strand number: Single-stranded Topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism name: Solanum tuberosum Strain name: cv. Desiree Direct origin Clone name: pPAL-8 Sequence GAATTCCTTG GGATTAATCT CAGCCAGGAA AA CAGCCGAG GCTGTCGATA TCTTGAAGCT 60 AATGTCATCA ACCTATCTCG TGGCGCTTTG CCAAGCTATA GACTTGAGGC ATTTGGAGGA 120 AAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180 GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTCTGCGAA AAGGAATTGC TTCGAGTCGT 240 GGACAGGGAA TACTTGTTTG CCTATGCAGA TGATCCCC 278 SEQ ID NO: 8 SEQ Length: type 2338 sequence: the number of nucleic acid strands: Single Strand Topology: Linear Sequence Type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism: Oryza sativa Strain: cv. Desiree Direct origin Clone: rPAL-2 Sequence CTCTCCTTGT CCCGGCCAAG TCGGCCGAGC TCGAATTCCA ATGATAGTTA TGTTTGATTT 60 TTACAATCCC AATGACAATA AAGAGTATGC GGCGGACGAG TCGTAGAGGA GTATAGTGGC 120 AGTGTTTGAC GGGTTTTCTA AAAATTATAA AAAACATGAA ACCCAACGAG ACAATAAACT 180 CTAAAAACTA TAAGGTTCAA ATTTTAAAGG TTCGGGCTTC TAAAAAATAA AAAAATAAAC 240 CCTAATGATA ATCATATTTG ATTTTTAAAA TCTCAATGAC AATAAAGAAG GGCGGCAGCG 300 GGCGGGCCGT AGAGGAGTAC AGTGGCAAAG CAACTGACGG TTTGGCAGGA CTTCTAGAAA 360 GTAA AAAATG AACCCGAATG ATAATTATGT TCGATTTTTA AAATCCCAAT GATAATAAAT 420 AGAAAAGACA ATTGACGGGG CATAGAGGAG TATAATGACA GCGTTTGACG GGACTTATAG 480 AAATTATAAA AACGAAACCC AACGAGACAC TAAACTCTAA AAACTATAAG ATCCTATTTT 540 TAAAGGTTTC AAGGAGAATG AATAGAAATA GTGGTAGATT GAGCAAGCAA ATAAAAAATG 600 ATATGAGAAA AGTAAGACGT AGCAGCTGGT GTGACTTTAA AAACCATATA ATTAGAAATA 660 TGGAGATGAT AAGGTTTGGT CTTTCAAAGT CTTAAGACAA CGAAATAGCT ATTTAATAAA 720 TTTTAAGCAA AATCATACTT AAAAAATATA TAATTTTGTT TGTGTACTAG CCGCGCAGTT 780 GCGCGGGCCA CCAGCTAGTT GAGAGTATAA TTAACTTTTT TTCTTTAAAA TATACACAAT 840 AAACTATATT TTTTAAAAGA TTTTCTGTCG CACAGACATT ATACTAGTTC TGAGAAAAAA 900 CTGTCTATAT TTTCTCAGTC AAGTCAGGTG TGTATTGCGC AGAACGAAAG CTCGGAGAGA 960 ATACTAGCAC TTGGTATGAA CCATTAGGAC TTGCTAAAGA CAGATGAAAG GGTTATGCCA 1020 ACACCAGTTT TTGTCGGGGG ACTGGTGACC GCGCAATTGG ATTGGTACCT CTCTTGTGCC 1080 GTAGTTTCCC CCCTCATGCA CCCCCAAAAC CCCAGAAAAA TTTTGTTGTT TGTACACAGA 1140 CCGCTTGACC ATCAGCCCAT CACCTACGTG CGGAGAACCA ATGACCTATG GAATATGTAA 1200 CTAGAACACA AAACCTAAA C AAACGTGTTG CACGAAGTAG AAAGCGATAG AGAAAGATAA 1260 GCCGGGGGAC CAAGGAAATG ATATCTGGAT ATGAGTTCAC AGCCCTTCCA GATAAACGGA 1320 CGCCCGGACG AAACGAAAGA CGACGAGTCG AGGACCGTCA GCAGCCGCAG AAACGGCGAG 1380 AGACGCCCCC AAGCCAAACG TGGCTTCGTG GCGTGACGAC GCGAGGTGTT TCACGCCCCG 1440 TATCCCCCCG CGCCGCGCTG CCGCGTGCAA CTCTCTCTCT CTTCCCCCGC ATGCACTCCC 1500 GCCACTGCCC GCCGCCCGCA TCGCTCCGCT CCCCCGAGCC CAACCGCCAC AGGGCACGCC 1560 ACGACCACCA CGAAACCTCT ACGTAGCCAC ACGCCCACCC GGCCCGTAGT TGCGGTCCCA 1620 AACTCGTCGC GCCGGCACAC CAATCCCGTG GTCAACCCAA CCGGCCACAC CGAACCCACA 1680 CTCCCCACTC CCACCCATCC TGCGCCTCCT ATTTAAACTC CCCACAACTC CCTCCATTCC 1740 CCTCCAAGAG CAAAGCCACT GCAGCTTCCA TATCCCCGGC TCTTCCGCAC ACACAACTCC 1800 TCCACCTCCA TCGGGAGCAA ACCGCTCGAG CAACCACCAC TCGTTACAGC TAGACATCGA 1860 TCTCCCCTCT CGTTCGCCGT TCCGATGGAG TGCGAGAACG GGCACGTCGC CCCCGCCGCC 1920 AACGGCAGCA GCCTGTGCGT GGCTAAGCCG CGTGCCGACC CGCTCAACTG GGGGAAGGCG 1980 GCGGAGGAGC TGTCCGGGAG CCATCTGGAC GCGGTGAAGC GCATGGTGGA GGAGTACCGC 2040 AGGCCCGTGG TGACGATCGA GGGC GCCAGC CTGACCATCG CGCAGGTCGC GGCGGTGGCC 2100 TCCGCCGGCG CCGCCAGGGT GGAGCTCGAC GAGTCCGCCC GCGGCCGCGT CAAGGCCAGC 2160 AGCGACTGGG TCATGAACAG CATGATGAAC GGCACCCACA GCTACGGCGT CACCACCGGC 2220 TTCGGCGCCA CCTCCCACCG GAGGACCAAG GAGGGCGGCG CGCTCCAGCG AGAGCTTATC 2280 CGGTAAGAAG CCGCAAGAGT TTGCTGTTCG TCTGGTGAGA GCTTGTGTGG ATCAGAGG 2338 SEQ ID NO: 9 SEQ Length: 2001 sequence type: nucleic acid strands Number: Single-stranded Topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin: Organism: Oryza sativa Direct origin Clone name: rPAL-4 Sequence TGTCCGTCGA CCCAGATCTG GGTCGACCTG CAGGTCAACG GATCTTCTAC TGGCGTTCCA 60 CCTGCAAGTAAG AGCTAGCATA CATCCACCAC GTGAGCCGCC 120 GGGGCAGTGG GATGCCGACG TCGCGCACAA CAACATCAAG AGCACGAACA TACTGCTCGC 180 AAAGCCGGCA AGGCATGGCT GCGCGGGGGGCGCGGCGCGGGGCGCGTCGCGTCCCTACGG 240GCCGCCGCGA GCTCGGCGTCGCGTCGCACGCGGCGCGCGCGCCGCGGCGCGCGCGCCGCGGCGC CAAGCGAG 360 CTTCCCAACG CGACGCCGCC GTGCCCACCA GCTGCGGCCA CGGTTGCGTC ATCCCCCTTG 420 GCGAAACCAG CGGGCACAGC AACAGCAGCG GGGAAAAGAT GAGCCGGGCA GCTGTGGTAG 480 CAATTGTGGC CGGGGATTTC GCCGGTACAG GTTGAGGCGA TACAGAAGAA GAGAGGAGAG 540 AGAAGGAAGA AGATGGAAAA TGAAGAAGAT GATGGAAAAC GTGATGGTAG TGGTATGATC 600 GTAGTTTTGT AAAATTTCGA TGGCACGACT ACGAATAGAT AAATTTAATT ATAATGGTAT 660 TTTTCTGAAT AGACAAATTT ACAATGGCAT GGACCAATTA ACCCTACCTC TTTCCCATGT 720 GGAGAGTATG CAAGCATGCA ACAACTAGAA AAGATACTCA TGATAGTTAA ACTCCAAATA 780 GTTTTTTCTT GCAAATTACA TATCCGATCA ACAATCCGAT TATACCATTG TATTCGTTGT 840 AATTAAATCT TATAACAAGA TCTTACAAAG ATTATATTTT GATAAAAAAA ATTATATATG 900 TTGTAATTAA TTATATATGT AAGTTACTTT ATCGTATATA TAAATTACTT CTAGATTTAA 960 TTAAATTATA TTTTGGACAT TTGAAAATTT TATTTTATTG GTTAGTTCTA TAATGTGTTC 1020 ATTAAATTTA ATTTCTAGAC AGTCTTATGT TTTTATCCCC ATAATAGATT TGTTTATAAT 1080 GTCGTGACAA GTTACTTTAT TTTTATGTTA CTTCTATACT TATATGCAAA TTACATTTAG 1140 GCTTTATTGA ATTTACTTTT ATATGTCTAA GAAGTAATTT AATGAAATCT AAAAATAATT 1200 CAG ATATATT ATAAAAGTAA TTTGTATTTT TATAAAAATT ATAGCTATAA ATATTCAATT 1260 GTTACGAACA ATAGTGTTAT CGGATCGTAA ATTGGATGAG TAGTTTAATA GAAATTTTTA 1320 TTTGAATCAT AGGTGGGAGG GATATATTTT TCTACTTGCT TTATGGCCTA GTAGTATCGA 1380 GATAAACATT AAGGCTGTGT TTAGTTCACA CCAAAATTGG AAGTTTGGTT GAAATTGGAA 1440 CGATGTGACG GAAAAGTTAG AAGTTTGTGT GTGTAGGAAA GGTTTTGATG TGATGGAAAA 1500 GTTAGGAAGT TTGAAGAATT ATTTTGTAAC TAAACACGGC GTAAGAGGTC TCTTTGATTT 1560 AGATTTTGCA TAAAACAAGG GACGGTCCCG CTCTTGCTAC TTATTTAAGC ACCCCCCTCA 1620 GTAGTCCTGA CTCCAACAAG CTCCACCGCA AAGATCCTCT GTTAGCTGGA CGACCTGTGG 1680 ACTGCGGTAC GTGGCGCTGC GAGCAATGGA GTGTGAGACC GGTCTGGTCG ACCGTCCCCT 1740 CAACGGCGAC CCCTTGTACT GGGGCAAGGC GGCGGAGGGT CTTGCGGGGA GCCACCTCGA 1800 CGAGGTGAAG AGGATGGTGG TGGAGTACCG CGCGCCCGCT GGTGAAGATC GACGGCGCCA 1860 TGCTCAGCGT CGCCAAGGTG GCAGCCGTCG CTGGCGAGGC CGCCCGGGTG CAGGTGGTGC 1920 TGGACGAATC CGCACGACCC CGCCTGGAGG CTAGTCGCGA GTGGGTCTTC GACAGCACCA 1980 TGAACGGCAC CGACACGTAC A 2001 SEQ ID NO: 10 sequence Length: 54 array type : Number of nucleic acid strands: single-stranded topology: linear sequence type: cDNA hypothetical: No antisense: Yes origin: organism name: Phaseolus vulgaris sequence AATTTAATAC GACTCACTAT AGAAACAATA GGAAGCCATG GCAATTTCAG CTCC 54 SEQ ID NO: 11 sequence length: 25 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Genomic DNA Hypothetical: No Antisense: No Origin: Synthetic sequence AATTTAATAC GACTCACTAT AGAAA 25 SEQ ID NO: 12 Sequence length: 29 Sequence Type: nucleic acid number of strands: single-stranded topology: linear sequence type: cDNA hypothetical: No antisense: No origin: synthetic organism name: Phaseolus vulgaris sequence GAGAGAGATT AACTCCGTGA ATGACAACC 29 SEQ ID NO: 13 sequence length: 24 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: cDNA Hypothetical: No Antisense: No Origin Biological name: Phaseolus vulgaris Sequence TCTACAACAA CGGTCTGCCT TCAA 24

[0042]

[Brief description of drawings]

FIG. 1 is a schematic diagram of the S. Tuberosum (tube
rosum) cv. 1 shows the restricted restriction map of 6 potato PAL gene clones isolated from Desiree genomic library.

[Explanation of symbols]

Clone 1 has λpPAL-1, clone 3 has λpPAL
-3, clone 4 has λpPAL-4, clone 6 has λp
PAL-6, clone 7 is λpPAL-7, clone 8
Represents λpPAL-8. B is BamHI, E is Eco
RI, H is Hind III, K is KpnI, S is SstI
Represents. The parentheses around the symbol indicate that there is a restriction enzyme cleavage site but its exact location is unknown.
The square frame is subcoulombized and S. Tuberosum (tube
rosum) cv. The region of each clone containing sequences that hybridize to Desiree cDNA clones pPAL-3 and pPAL-21 is shown. The orientation of the putative PAL gene is shown in a box.

FIG. 2 is a schematic diagram of S. tubersum cv. 1 shows a restriction map of 7 potato PAL gene clones isolated from a Desiree genomic library.

[Explanation of symbols]

Clone 1 has λpPAL-1 and clone 2 has λpPAL
-2, clone 3 has λpPAL-3, clone 4 has λp
PAL-4, clone 6 is λpPAL-6, clone 7
Represents λpPAL-7 and clone 8 represents λpPAL-8. B is BamHI, BglII is BglII, E is Eco
RI, H is Hind III, P is PatI, S is SstI
Represents. The parentheses around the symbol indicate that there is a restriction enzyme cleavage site, but its exact location is unknown.
The orientation of the PAL gene is shown below each map.

FIG. 3 is, rice Oryza sativa (Oryza sati
va) Three rice PALs isolated from a genomic library
The restriction enzyme maps of clones are shown.

[Explanation of symbols]

Clone 2 has λrPAL-2 and clone 4 has λrPAL
-4, clone 10 represents λrPAL-10. Boxes indicate the regions subcloned into the pUC vector. The arrow on the map indicates the direction of the PAL gene within this genomic clone. E is Ec
oRI, H represents Hind III and S represents SstI.

FIG. 4 is a schematic diagram showing a self-retaining sequence replication system.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Fen-Fuen, Lin USA 92056, California, USA 92056, Oushanside Avenida Segovia 1841 (72) Inventor Tei Eric Mirkov, California 92128, San Diego, Creswick Kout 12202 (72) Inventor Jana Gaybin Coryle United States California 92120, San Diego, Danbury Way 6232 (72) Inventor Poula Kay Shawneck United States California 92014, Del Mar, Apto Ipotohuino Drive 13760

Claims (33)

[Claims] 1. A promoter of a plant gene encoding a potato or rice phenylalanine ammonia lyase (PAL). 2. λpPAL-1, λpPAL-2, λp
PAL-3, λpPAL-4, λpPAL-6, λpP
AL-7, λpPAL-8, λrPAL-2, λrPA
The PAL promoter of claim 1 derived from L-4 or λrPAL-10. 3. The transcription of a DNA sequence can be controlled in plant protoplasts, plant cells, plant callus, plant tissues, seedlings, immature plants, mature plants or seeds,
And the promoter of claim 1 or 2, which is inducible or responds directly or indirectly to an exogenous elicitor or damage. 4. The promoter according to any one of claims 1 to 3, which is directly or indirectly bound to at least one DNA sequence encoding a protein that gives rise to a trait. 5. Herbicides, fungi, viruses, bacteria, insects,
A DNA encoding a protein, an enzyme or a production of a reporter compound which causes resistance or resistance to nematodes or arachnids, secondary metabolite production, male or female infertility, and the like. 3. The promoter according to any one of 3 above. 6. Chloramphenicol acetyl transferase (CAT), neomycin phosphotransferase (NPT), novaline synthase (NO)
S), octopine synthase (OCS), β-1,3-
Glucuronidase (GUS), acetohydroxy acid synthase (AHAS), β-galactosidase (βGA
L), or D encoding luciferase (LUX)
The promoter according to any one of claims 1 to 3, which is bound to NA. 7. Glutathione reduced type, homoglutathione reduced type, glycan elicitors, hexa (β-D-glucopyranosyl) -D-glycitols, lipid elicitors, arachidonic acid, eicosapentaenoic acid, glycoprotein elicitors, fungi Induced by elicitors, mercury chloride, viruses, fungi, bacteria and insects,
Alternatively, the promoter according to any one of claims 1 to 3, which directly or indirectly reacts with them. 8. A transgenic plant protoplast, a plant callus, a plant tissue, a developing plantlet, an immature plant, a mature plant and a seed, which contains the promoter according to any one of claims 1 to 7. 9. The transgenic plant protoplast, plant callus, plant tissue, developmental plantlet, immature plant, mature plant and seed according to claim 8, wherein the plant is Solanum tuberosum . 10. The plant is Oryza sativa or Oryza in
The transgenic plant protoplast, plant callus, plant tissue, developmental plantlet , immature plant, mature plant and seed of claim 8, which is dica . 11. λpPAL-1, λpPAL-2, λ
pPAL-3, λpPAL-4, λpPAL-6 / 7,
A chimeric DNA sequence consisting of a PAL promoter derived from λpPAL-8, λrPAL-2, λrPAL-4 or λrPAL-10 and a part of the PAL gene coding region. 12. The chimeric DNA sequence according to claim 11, which is operably linked to at least one other peptide structural gene. 13. A structural gene having chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS),
The chimeric DNA according to claim 12, which encodes a reporter protein of β-1,3-glucuronidase (GUS), acetohydroxy acid synthase (AHAS), β-galactosidase (βGAL), and luciferase (LUX). .. 14. A plant cell, plant tissue, plant or seed transformed with the chimeric DNA according to any one of claims 11 to 13. 15. The plant cell, plant tissue, plant or seed according to claim 14, wherein the promoter is the potato PAL promoter and the plant is Solanum . 16. The plant cell, plant tissue, plant or seed according to claim 15, wherein the plant is Solanum tuberosum . 17. The plant cell, plant tissue, plant or seed according to claim 14, wherein the promoter is a rice PAL promoter and the plant is Oryza . 18. The plant is Oryza sativa or Oryza indi.
The plant cell, plant tissue, plant or seed according to claim 17, which is ca. 19. A probe consisting of at least 10 nucleotides, which consists of the DNAs of SEQ ID NOs: 1 to 9 or fragments thereof, or mRNA corresponding thereto. 20. DNAs complementary to the DNAs shown in SEQ ID NOs: 1 to 9 or fragments thereof, or mRNAs corresponding thereto
An antisense probe consisting of at least 10 nucleotides. 21. A method for inducing transcription of a chimeric DNA in transgenic potato or rice, which comprises directly or indirectly inducing a host containing the chimeric DNA comprising at least one PAL promoter derived from potato or rice and a structural gene. Chimera D in a potato or rice host characterized in that it is contacted with an exogenous elicitor
A method for inducing transcription of NA. 22. A method for inducing transcription of a chimeric DNA contained in a transgenic plant, wherein the chimeric DNA is operably linked to at least one PAL promoter derived from potato or rice to at least one structural gene. Wherein the promoter is inducible by the elicitor or reacts directly or indirectly to the elicitor, and wherein the transgenic plant is contacted with the elicitor directly or indirectly to the exogenous elicitor. And a method for inducing transcription of a chimeric DNA. 23. A method of cloning an elicitor-inducible plant gene promoter, comprising: a) contacting a plant with elicitor; b) isolating RNA from the plant; c) preparing a cDNA library from the RNA; d) searching this library with a probe consisting of a nucleotide sequence from the transcriptional coding or non-coding region of the gene of interest; e) the probe or cD hybridized with it.
NA is used to search a plant genomic library, f) identify segments of genomic DNA, and g) antisense RNA from genomic DNA segments.
Generating and labeling transcripts h) hybridizing to mRNA from elicitor-treated and non-elicitor-treated plants using the labeled antisense RNA transcript as a probe, i) subjecting the hybridized mixture to RNase j ) Identifies antisense RNA transcripts protected from degradation by elicitor-treated RNA but not RNA-degraded from non-elicitor-treated plants, and k) protected only by RNA from elicitor-treated plants. A method for cloning an elicitor-inducible plant promoter, which comprises isolating the promoter from a genomic clone producing an antisense RNA transcript. 24. A method for cloning elicitor-inducible promoters, comprising the steps of: a) searching a plant genomic library with a probe consisting of a nucleotide sequence from a transcriptional coding or non-coding region of a gene of interest; Identify the hybridized genomic DNA segment, and c) antisense R from this genomic DNA segment.
Generating and labeling an NA transcript, d) hybridizing to mRNA from elicitor-treated and non-elicitor-treated plants using the labeled antisense RNA transcript as a probe, and e) RNaseing the hybridized mixture. And f) identify antisense RNA transcripts that are protected from degradation by elicitor-treated RNA, but are not protected from degradation by RNA from non-elicitor-treated plants, and g) protected only by RNA from elicitor-treated plants. A method for cloning elicitor-inducible promoters, which comprises isolating the promoter from a genomic clone producing an antisense RNA transcript. 25. The DNA according to SEQ ID NOS: 1 to 9. 26. A method according to claim 25, wherein the sequences are operably linked to at least one structural gene and at least one terminator sequence.
Item DNA. 27. A protein, enzyme or reporter in which the structural gene causes resistance or resistance to a herbicide, fungus, virus, bacterium, insect, nematode or arachnid, secondary metabolite production, male or female infertility. 27. The DNA according to claim 26, which is a gene encoding a DNA encoding the production of a compound. 28. The structural gene comprises chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OC).
S), β-1,3-gel clonidase (GUS), acetohydroxy acid synthase (AHAS), β-galactosidase (βGAL), and luciferase (LU).
DNA according to claim 27, characterized in that it encodes X). 29. A method for selecting an elicitor which induces a PAL promoter, which comprises transforming a host with a chimeric DNA sequence consisting of a PAL promoter and a reporter gene or a vector containing the chimeric DNA sequence to transform the elicitor Contacting the transformed host,
A method for selecting an elicitor which induces a PAL promoter, which comprises selecting one that causes expression of the reporter gene. 30. A method for selecting an organism that induces transcription of a plant defense gene, comprising a reporter structural gene and a potato or rice P operably linked thereto.
Inducing transcription of a plant defense gene, which comprises producing a transgenic plant containing a chimeric DNA sequence consisting of an AL promoter, contacting the organism with the transgenic plant, and selecting one that causes expression of a reporter structural gene How to select the organism to do. 31. A method for selecting an organism capable of inducing transcription of a defense gene, which comprises exposing a transgenic plant containing a plant defense gene promoter operably linked to a reporter gene to the organism of interest. Let
It is possible to induce transcription of a protection gene, which is characterized by monitoring the expression of the reporter gene and selecting an organism that induces the transcription of the reporter gene as an inducible chimeric DNA containing a plant defense gene or its promoters. How to select organisms. 32. The method according to claim 31, wherein the organism is a virus, bacterium, fungus or insect. 33. A method for selecting a pesticide capable of inducing plant gene expression, comprising: (a) isolating RNA from plant material not exposed to a test pesticide; and (b) encoding the plant gene from this RNA. Identified RNA, (c)
Amplifying the identified RNA using 3SR technology,
(D) exposing the same plant material as the plant material from which the RNA was isolated to a test pesticide; (e) isolating the RNA of the exposed plant material; (f) extracting from this RNA the above-mentioned (b) Identify RNA encoded by plant genes,
(G) Amplifying the RNA of (e) above using 3SR technologies, (h) comparing the amplified products from (c) and (g) above, (i) from (c) above (g) above A method for selecting a pesticide capable of inducing plant gene expression, wherein a pesticide that strongly induces amplification is selected as a pesticide capable of inducing plant gene expression.