CN1247569A - Transgenic plants with altered sterol biosynthesis pathway - Google Patents

Abstract

The composition of phytosterols in transgenic plants is adjusted to render the plants resistant to insects, nematodes, fungi and/or other harsh environmental factors and/or to enhance the nutritional value of the plants. The recombinant DNA molecules of the present invention encode RNA or protein sequences that affect the expression or activity of sterol biosynthetic enzymes, thereby altering the sterol profile of plants. Transforming the DNA molecule into plant cell, and regenerating transgenic plant with changed sterol component based on the transformed plant cell.

Description

Transgenic plants with altered sterol biosynthesis pathway

The present invention relates broadly to plant genetic engineering. More specifically, it relates to manipulating the level and/or activity of endogenous phytosterol components as a strategy to minimize crop damage from plant insects or other pests and/or to enhance plant nutrition value.

Background of the Invention

Harsh environmental factors such as drought, severe cold, weeds and crop-eating organisms can cause harm to agricultural production. Traditional methods of killing weeds and parasites rely almost entirely on chemical herbicides, pesticides and fungicides. However, the widespread use of these agricultural chemicals has led to the development of resistance. In fact, insects have been reported to be resistant to most major classes of insecticides, including organophosphates, chlorinated hydrocarbons, and carbamates.

Sterols comprise a class of essentially natural compounds that are required to some extent by all eukaryotic organisms. As shown in the figure below, they have an identical tetracyclosteroid core and a side chain. Some sterols play structural roles in cell membranes, while others are essential during development.

Plants are capable of producing more than 250 different phytosterols (Akisha et al., 1992). As many as 60 sterols have been identified in a single species, Zeamays (maize), (Guo et al., 1995). However, insects, fungi and nematodes, as well as many other non-sterol-producing parasites, do not desynthesize all the sterols they need. Instead, they meet their nutritional needs for sterols by eating plants. This fact has been exploited to develop commercial agrochemicals such as triazoles, pyrimidines and diazosterols, which work by interfering with sterol production in parasitic organisms.

Recently, advances in molecular biology have enabled people to introduce beneficial traits into plants through genetic engineering. Several forms of insect resistance traits have been introduced into plants through genetic engineering methods. For example, a transgenic plant expressing a foreign gene encoding a Bacillus thuringiensis (Bt) endotoxin acquires the ability to kill pests that feed on it. Unfortunately, such methods are only effective against specific endotoxin-sensitive insects. There is a continuing need in the agricultural industry for alternative pest control strategies that are broadly effective against a broad range of pests/pathogens.

Summary of Invention

The present invention broadly relates to methods of altering the composition, levels and/or metabolism of sterols by genetically engineering plants. The method can enhance the plant's natural insect resistance, drought resistance and cold resistance, and/or can improve the nutritional/health value of the plant.

According to one aspect of the present invention, the recombinant DNA molecules provided include:

A promoter, which causes the production of RNA sequences in plants, the promoter is operably linked to

A DNA coding sequence that encodes an enzyme capable of binding primary sterols and producing secondary sterols, operably linked to

A 3' untranslated region that causes polyadenylation of the 3' end of an RNA sequence; wherein said promoter is heterologous to said DNA sequence.

DNA coding sequences encoding enzymes that bind primary sterols and generate secondary sterols can be in sense or antisense orientation. Thus, a DNA molecule of the invention can encode a non-translated RNA molecule (eg, antisense or cosuppression) or a protein molecule. The RNA or protein so produced selectively targets the expression and/or activity of sterol biosynthetic enzymes to affect the profile of phytosterol components in the plant to produce desired changes.

Therefore, according to another aspect of the present invention, there is provided a method of altering the composition of phytosterols, thereby enhancing the resistance of plants to insects, nematodes and Pythium fungi. This aspect of the invention improves insect and disease resistance in plants by altering the composition and/or distribution profile of certain phytosterols. This method of transgenic plants overcomes many of the drawbacks inherent in the use of agrochemicals, as foreign substances introduced into plants can ultimately select for new insect resistance mechanisms. The invention not only retains the advantages in the use of agricultural chemicals, but also overcomes many disadvantages of agricultural chemicals. With respect to the existing basic pathways of pests and pathogens, the present invention reduces the possibility of evolution of mechanisms that block the pathways.

In this regard, the sterol composition of plants was altered by increasing the content of the following unavailable sterols: 4-methylsterol, 9β,19-cyclopropylsterol, ^Δ7 -sterol, ^Δ8 -sterol, 14α-methanol Alkysterols, Δ ²³⁽²⁴⁾ -24-alkyl sterols, Δ ²⁴⁽²⁵⁾ , 24-alkyl sterols or Δ ²⁵⁽²⁷⁾ , 24-alkyl sterols. An alternative approach is to modify the sterol composition to have lower levels of sterols containing ^Δ5 groups.

Another aspect of the invention relates to the production in plants of sterols that impart drought and cold resistance to the plants.

Another aspect of the invention relates to altering the sterol profile in plants to increase the levels of cholesterylsterol therein.

Typically, the above aspects of the present invention are achieved by changing the expression and/or activity of sterol-related enzymes, preferably S-adenosyl-L-methionine-Δ ²⁴ -sterol methyltransferase (SMT _I and SMT _II ), C-4 demethylase, cycloeucalenolto obtusifoliol isomerase, 14α-methyl demethylase, ^Δ8 to ^Δ7 -isomerase , Δ ⁷ -sterol-C-5-desaturase or 24,25-reductase.

Another aspect of the invention relates to transgenic plants having altered levels of selected sterols, the plants being produced by introducing a recombinant DNA molecule of the invention into the genome of a plant cell and selecting cells expressing the molecule. Transgenic plants are regenerated from the transformed plant cells, and plants containing the recombinant DNA develop to maturity. Plants expressing the recombinant DNA are then identified, and plants whose sterol profile meets the requirements of the present invention are selected and propagated.

Brief description of attached drawings

The following drawings constitute a part of this specification and are written to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of the following drawings in combination with the detailed description of specific embodiments presented herein.

What Fig. 1 shows is to use the cofactor of isotope-labeled substrate, carry out the result that SMT enzyme carries out HPLC radiation counting B group and mass spectrometry A group to determine the result obtained;

What Fig. 2 provided is to be used for measuring six kinds of inhibitors of SMT enzyme;

Figure 3 shows the activity of SMT during seedling development;

Figure 4 shows the pathway of sterol end products during seedling development;

Figure 5 shows the yeast SMT gene sequence (group B; SEQ ID NO: 1) and the amino acid sequence (group A; SEQ ID NO: 2) deduced according to the predicted conserved region highlighted;

Figure 6 shows the Arabidopsis SMT gene (group B; SEQ ID NO: 3) and the deduced amino acid sequence (group A; SEQ ID NO: 4);

Figure 7 shows the ERG6 construct prepared with the pUC18cpexp expression cassette;

What Fig. 8 provided is the sequence (SEQ ID NO:5) of yeast SMT gene. The underlined sequences were used as primers for screening transgenic tomato genomic DNA; and

Figure 9 presents the resulting structures assayed for phytosterols in Spodoptera zea, which were found to be available or unavailable.

What Fig. 10 (SEQ ID NO: 6) provided is the nucleotide sequence and the aminoacid sequence of corn SMT gene.

Description of Illustrative Embodiments Phytosterols

The phytosterol metabolic pathway consists of enzymes acting on the cyclic sterol core and side chains of the tetracyclic structure. In higher vascular plants, the main pathway starts with cycloartenol (I):

Ending with Δ ⁵ -24-alkylsterols, of which sitosterol (II), stigmasterol (III) and campesterol (IV) predominate:

The number of alternative pathways is large enough that as many as 60 or more different sterols can be produced in a single plant. These alternative pathways differ by tissue-specific and developmentally-specific genetic programs.

Inhibitors of sterol biosynthesis have been used to study sterol metabolism. These inhibitors include several commercially available fungicides that block the sterol metabolic pathway in phytopathogenic fungi, thereby inhibiting the growth of the fungi. The steps of the major metabolic pathways described below were identified using metabolic inhibitors. The main pathway consists of 12 chemical transformations as described below.

In reaction 1, S-adenosyl-L-methionine-sterol-C-24 methyltransferase (SMT _I ) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine Conversion to the C-24 center of the sterol side chain. The cyclized sterols are characterized by functional groups that undergo transformation.

This is the first of two methyl conversion reactions and is the specific and rate-limiting step in the sterologenic pathway in plants. A different SMT enzyme, SMT _II , catalyzes the conversion of cycloartenol to Δ ²³⁽²⁴⁾ -24-alkylsterol, cyclosadol (Guo et al., 1996).

Reaction 2 involves demethylation at C-4. This is the first of several demethylation reactions on the steroid nucleus.

Reaction 3 involves opening the cyclopropyl ring at C-9(10) with cycloeucalyptol-obtusylol isomerase (COI) with simultaneous formation of a double bond at C-8.

Reaction 4 involves demethylation at C-14, demethylation at C-14 and formation of a double bond at C-14.

Reaction 5 is catalyzed by ^Δ14 reductase.

Reaction 6 includes Δ ⁸ -to Δ ⁷ -isomerase catalyzed reaction to generate Δ ⁷ group

Reaction 7 is the second methylation at the C-24 position of the sterol side chain. The reaction is catalyzed by SMT _I , the same enzyme that starts the main pathway.

Reaction 8 involves a C-4 demethylase that generates 4,4-norsterol.

Reaction 9 involves a ^Δ5 desaturase that generates a double bond at the C-5 position of the ring of the tetracyclic structure.

The product of reaction 9 is then catalyzed in reaction 10 by Δ ⁷ -reductase to remove the double bond at C-7.

Reaction 11 involves Δ ^{24(28 )} - to Δ ^{24(25 )} -isomerases that alter the side chain structure. (It is believed that this reaction would start directly from the product of reaction 5 if the kinetics were more favorable.)

Reaction 12: After stereoselective reduction of the Δ ²⁴⁽²⁵⁾ double bond at the C-24 position, sitosterol (II) is produced. In addition to the main pathway for sterol biosynthesis, a developmental program has been identified that regulates the expression of SMT enzymes. Enzyme studies have shown that there are two different SMT enzymes (SMT _I and SMT _II ) in maize, and their expression depends on the tissue and stage of differentiation. The leaf blade mainly contains 24-ethyl sterol (produced by the activity of SMT _I ), while the leaf sheath mainly contains 24-methyl sterol (produced by the activity of SMT _II ). These sterols are the products of the reaction of these two different SMT enzymes with the same starting material, cycloartenol.

The first enzyme, SMT _I, produces Δ ²⁴⁽²⁸⁾ -methylene sterol and the second enzyme produces Δ ²³⁽²⁴⁾ -methyl sterol (V). The first isomer produces an available sterol (ie insects, Pythium fungi and nematodes are able to use them to complete their own life cycle). The second isomer produces a non-available sterol (ie insects, Pythium fungi and nematodes cannot use them to complete their own life cycle). Therefore, it is able to inhibit the expression of the first isoform so as to cause the accumulation of unavailable Δ ²³⁽²⁴⁾ -methylsterol.

重组DNA分子：Therefore, sterols accumulated in tissues contain a double bond at C-23 (VI) and a methyl group at C-24.

Recombinant DNA molecules:

In order to obtain desired changes in sterol composition, the present invention provides a recombinant DNA molecule that can be used to produce transgenic plants. The recombinant DNA molecule of the present invention generally includes a promoter region capable of causing RNA sequence production in plants, a structural DNA sequence and a 3' untranslated region.

A region of a gene called a "promoter" regulates the transcription of DNA into mRNA. The promoter region contains a base sequence, which guides RNA polymerase to bind to the DNA sense strand or antisense strand, and uses the DNA sense strand as a template to generate a corresponding mRNA strand complementary to the sense strand. The process of producing mRNA from DNA as a template is often referred to as "expression" or "transcription" of a gene.

In the recombinant DNA molecules of the present invention, heterologous recombination between the promoter and the DNA coding sequence is generally preferred. With respect to a promoter, the term "heterologous" means that the DNA coding sequence in the recombinant DNA molecule of the invention is not derived from the same gene to which the promoter was originally linked.

Promoters come from a wide variety of sources, such as plants and plant viruses. The particular promoter selected for use in the embodiments of the present invention should preferably be such that it induces expression sufficient to allow for the desired change in the sterol profile in the plant.

A number of promoters active in plants have been described in the literature and can be used in the DNA molecules of the present invention. These promoters include, for example, the cauliflower mosaic virus (CaMV) 35S promoter (Odell et al., 1985), the Scrophulariaceae mosaic virus (FMV) 35S (Sanger et al. 1990), the sugarcane baculovirus promoter (Bouhida et al., 1993), the commelina yellow mottle virus promoter (Medberry and Olsewski 1993), the light-inducible promoter derived from the small subunit of ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984 ), rice cytoplasmic triose phosphate isomerase (TPI) promoter (Xu et al. 1994), Arabidopsis adenine phosphoribosyltransferase (APRT) promoter (Moffatt et al., 1994), rice actin 1 gene promoter (Zhong et al. 1996), mannopine synthase and octopine synthase promoters (Ni et al. 1995). Various types of DNA constructs created using all these promoters have been expressed in plants.

Typically, recombinant DNA molecules also contain a 5' untranslated leader sequence. This sequence can be derived from the promoter chosen to express the gene and, if desired, the leader sequence can be modified to increase translation of the mRNA. The 5' untranslated region may also be derived from viral RNAs, suitable eukaryotic genes or synthetic gene sequences.

As discussed further below, the structural DNA sequence of the recombinant DNA molecules of the invention will result in desirable changes in the sterol profile of the plant.

The 3' untranslated region of the recombinant DNA molecule of the present invention can be obtained from various genes expressible by plant cells. For example, the 3' UTR of the frequently used nopaline synthase (Fraley et al. 1983), the 3' UTR of ssRUBISCO from pea (Coruzzi et al. 1994) and the 7S seed storage protein gene from soybean 3' untranslated region (Schuler et al. 1982). The 3' untranslated region of the recombinant DNA molecule contains a polyadenylation signal sequence, which functions in plants to add adenylate nucleotides to the 3' end of the RNA.

Recombinant DNA molecules of the present invention may include other regulatory sequences or combinations thereof known to those skilled in the art as desired. For example, intronic sequences in recombinant DNA molecules are often used to produce transgenic plants for increased expression levels. Examples of plant introns suitable for plant expression include: maize hsp70 intron, rice actin 1 intron, maize ADH 1 intron, Arabidopsis SSU intron, petunia EPSPS Introns and other introns known to those skilled in the art. Transformation and regeneration of plants

The double-stranded DNA molecule of the invention can be inserted into the genome of a plant using any suitable method. A number of plant transformation methods have been reported, including Agrobacterium-mediated transformation, transformation using liposomes, electroporation, chemical transformation that increases the uptake of cell-free DNA, release of cell-free DNA by particle bombardment, transformation using viruses or pollen, etc. wait.

After the cells (protoplasts) are transformed, the selection of the method for the regeneration step is not strict. (cabbage, radish, rapeseed, etc.), Cucurbitaceae (melons and cucumbers), Gramineae (wheat, rice, corn, etc.) plan. The method of transformation and regeneration of dicotyledonous plants mainly obtains transgenic plants by using Agrobacterium tumefaciens. US Patent 5,569,834; US Patent 5,416,011; Christou et al (1988)), Brassica (US Patent 5,463,174), peanut (Cheng et al (1996)); Papaya (Yang et al (1996)) and pea (Schroeder et al (1993) ); De Kathen and Jacobsen et al. (1990)) and other species of plants.

In addition, the transformation of monocots using electroporation, particle bombardment, and Agrobacterium has now been reported.

Transformation and plant regeneration have been successful in asparagus (Bytebier et al. (1987)), barley (Wan and Lemaux (1994)), maize (Rhodes et al. (1988); Gordon-Kamm et al. (1990); Fromm et al (1990); Koziel et al (1993); Armstrong et al (1995)), oats (Somers et al (1992);), orchard grasses (Horn et al (1988)), rice (Toriyama et al (1988); Battraw and Hall (1990); Christou et al (1991); ), rye (Bryant et al (1987)), sugarcane (Bower and Brich (1992)), tall fescue (Wang et al (1992)) and wheat (Vasil (1992); Weeks et al (1993)).

For reviews of methods for plant transformation and/or regeneration see Richie and Hodges (1993) or Hinchee et al. (1994). Enhancing plant insect resistance by altering phytosterol components

In insects, a range of phytosterols were tested and found that many of them did not support insect growth, ie they were not available. These unavailable sterols include 9,19-cyclopropyl sterol. It was further confirmed that the new Δ ²³⁽²⁴⁾ - and Δ ²⁴⁽²⁵⁾ -alkenes and Δ ²⁵⁽²⁷⁾ -alkyl sterols also failed to support growth and maturation of insects. The experiments were carried out in cotton bollworms (H. zea) grown on synthetic media that did not contain any sterols other than those used in the experiments. It has now been found that if the ratio between available sterols and unavailable sterols is 1:9 or less, insects cannot develop normally. In fact, even at a ratio of 1:1, there are deleterious effects on insect development.

The metabolism of insects, nematodes and Pythium fungi is limited by the availability of major phytosterols. These insects cannot utilize sterols with the following groups: C-4 methyl, 9β,19-cyclopropyl or ^Δ8 groups. Furthermore, nematodes and insects cannot utilize 14-α-methyl-sterols, and some insects, including Lepidoptera, Diptera, and Coleoptera, cannot utilize Δ24-containing sterols due to in vivo mechanisms ⁽²⁵⁾ , Δ ²³⁽²⁴⁾ or Δ ²⁵⁽²⁷⁾ and other groups of C-24 alkyl sterols. Some insects cannot utilize sterols missing the ^Δ5 group. As a result, the increase of these sterols in plants provides harmful sterols to pests from feeding channels.

When the DNA molecule of the present invention is expressed in a transgenic plant, it will result in a change in the composition/distribution of the sterols contained in the plant. In a preferred embodiment, the DNA molecule results in the accumulation of sterols that are not available to insects and other pests, thereby enhancing the plant's ability to resist these organisms. This can be achieved by a number of methods, eg, overexpression, antisense, co-suppression, etc. Typically, the genes of interest for the DNA molecules of the invention are those endogenous genes encoding enzymes selected from the kinetically favored sterol biosynthetic pathways.

Preferably in this embodiment, targeted inhibition of sterol biosynthetic enzyme gene expression and/or translation is performed. For example, the inhibitory effect can be achieved through the following ideas: antisense molecules, ribozymes or co-suppressive RNA molecules complementary to endogenous target genes are prepared by constructing the DNA molecules of the present invention. The manipulation of such targeted inhibition of gene expression is well known to those skilled in the art (for a comprehensive review, see Bird et al., 1991; Schuch, 1991; Gibson et al., 1997).

A preferred target for inhibition is S-adenosyl-L-methionine- ^Δ24(25) -sterol methyltransferase (SMT). Directed binding of an antisense or co-suppression construct to the gene effectively inhibits the expression of SMT, resulting in the accumulation of unavailable sterols.

Other genes in the phytosterol conversion pathway may also be targeted in this or other embodiments of the invention in order to alter the compositional profile of sterols in plants. While the preferred gene to target depends on the application, the approach is the same: express an RNA or protein molecule that alters the sterol composition in a desired manner.

Therefore, in addition to SMT, other preferred intracellular targets that can lead to sterol alterations include:

(i) C-4 demethylase: This enzyme is involved in the removal of the two methyl groups at the C-4 position and appears in Reaction 2 and Reaction 8 in the description section. A single protein is responsible for both reactions. Blocking this enzyme will result in 4,4-dimethylsterols such as cycloartenol, 24(28)-methylenecycloartenol, or neosterols such as 24-dihydrolanosterol (structure 18 in Figure 9) accumulation. This can be achieved by suppressing this gene in the plant.

(ii) Cyclocineol-obtusylol isomerase (COI) and ^Δ8 to ^Δ7 -isomerase: These two enzymes occur in reactions 3 and 6 of the synthetic pathway. Certain fungicides are known to block these two enzymes, resulting in the accumulation of 9β,19-cyclopropylsterol. Locusts that feed on these plants are known to be abnormally developed and depleted of cholesterol and ecdysteroids. This suggests that if either of these two enzymes is disturbed or inhibited, the phytosterols are altered such that they do not support insect development (Coste et al., 1987).

(iii) C-14 demethylase: This is reaction 4 in the synthetic pathway. There are several fungicides and plant growth regulators that block this reaction step in fungi and plants. In plants, this blockade leads to depletion of normal Δ ⁵ -sterols and accumulation of 9β,19-cyclopropyl sterol, 14α-methyl sterol and Δ ⁸ -sterol. These accumulated sterols are intermediates in the main sterol pathway. They are also unavailable sterols. Studies with chemical inhibitors have shown that plants that accumulate these intermediates are tolerant to water and cold. Therefore, inhibiting the activity of this enzyme through genetic manipulation is also a useful strategy.

(iv) Δ ⁷ -sterol-C-5-desaturase: This is reaction 9 in the synthetic pathway. Inhibition of this enzyme results in depletion of Δ ⁵ -sterols and increase in Δ ⁷ -sterols. Certain insects are known to be unable to metabolize Δ ⁷ -sterols to ecdysteroids. Therefore, the accumulation of Δ ⁷ -sterols in plants also becomes a pathway to produce unavailable sterols. Moreover, the substitution of Δ ⁷ -sterols for Δ ⁵ -sterols in plant membranes did not cause any morphological changes in plant development.

(v) C-24 reductase: This is the last step in the conversion of phytosterols (reaction 12) during the formation of sitosterol, the major Δ ⁵ -sterol in plants. Interfering with or inhibiting the gene encoding this enzyme will result in

Accumulation of Δ24 ⁽²⁵⁾ -24-alkyl sterols, which are also not available.

A number of genes encoding these preferred phytosterol biosynthetic enzymes have now been isolated from yeast and are those targeted by the present invention (see Lees et al., 1997 for a full review). Some such genes have also been isolated from plants. For example, SMT genes isolated from soybean (Shi et al., 1996), Arabidopsis (Husselstein et al., 1996; Bouvier-Nave et al., 1997), tobacco and castor plant (Bouvier-Nave et al., 1997) and maize (Grabenok et al., 1997). Other phytosterol biosynthesis genes that have been isolated include ^Δ7 -sterol-C-5-desaturase from Arabidopsis (Gachote et al., 1996) and cycloartenol synthase from Arabidopsis (Corey et al., 1993).

In addition to these existing genes, genes encoding sterol biosynthetic enzymes can be easily obtained from desired sources by methods known to those skilled in the art. For example, using a gene or cDNA isolated from one source as a hybridization probe to isolate homologous sequences from other sources. It should be noted, however, that regardless of the source of the sterol biosynthesis genes in the targeting construct, the DNA molecules of the present invention should be active in many species of plants. A method using yeast ERG6 Successful demonstration of antisense constructs altering the sterol profile in tomato.

Preferably, the following enzymes in sterol metabolism are targeted: S-adenosyl-L-methionine- ^Δ24 -sterol methyltransferase, C-4 demethylase, cycloeucalyptol-oblateol -isomerase, 14α-methyl demethylase, Δ ⁸ -to Δ ⁷ -isomerase, Δ ⁷ -sterol-C-5-desaturase or 24,25-reductase.

Preferably, in plants produced according to this embodiment, the content of certain unavailable sterols is increased, in particular, 4-methylsterol, 9β,19-cyclopropylsterol, ^Δ7 -sterol, ^Δ8 -sterol, 14α-methylsterol, Δ23 ⁽²⁴⁾ -24-alkylsterol, Δ24 ⁽²⁵⁾ ,24-alkylsterol or Δ25 ⁽²⁷⁾ ,24-alkylsterol. An alternative approach is to alter the sterol composition to reduce the level of sterols containing the ^Δ5 group.

According to this embodiment of the present invention, the crops for which insect resistance is preferably provided include: corn (corn borer, earworm, armyworm), rice, sorghum, forest trees, potatoes, tomato (Mangnatha) and cantaloupe. Moss vegetables.

Crops for which nematode resistance is preferably provided according to this embodiment of the invention include: soybean (H. sojae), tomato (Metroderma nematode), sugar beet and squash.

Crops for which antifungal properties are preferably provided according to this embodiment of the invention include: maize, rice, wheat, sorghum, soybean (Phytophthora root), sunflower, forest trees, fruits and berries, potato (late blight), tomato (late blight), sugar beet, squash and brassica vegetables. Phytosterols as Cholesterol Lowering Agents

Studies in animals and humans have shown that phytosterols can lower serum and/or plasma total cholesterol and low-density lipoprotein (LDL) cholesterol (Ling and Jones, 1995). In this regard, transgenic plants with altered sterol profile are beneficial for establishing a dietary regimen to control cholesterol and prevent cardiovascular disease.

Studies on the structure-specific effects of single phytosterols have shown that saturated phytosterols such as sitostanol are more effective than unsaturated compounds such as sitosterol in lowering cholesterol. Another structural feature that appears to be at play is the esterification of phytosterols. Some studies have shown that ferrulate esters of sitosterol, sitostanol or cycloartenol are more effective than the corresponding free sterols in lowering serum cholesterol (Meittinen and Vanhanen, 1994).

In the diet, some natural sources of phytosterols are rice bran oil, corn fiber oil, and soybean oil. Rice bran and corn fiber are by far the richest sources of phytosterols. Soy phytosterols are by-products of the oil refining process. Making these and other plants more nutritious phytosterols could help develop new foods that improve human health and nutrition.

Accordingly, in another embodiment, the present invention relates to increasing cholesterol-lowering sterols in transgenic plants. For example, the conversion of cycloartenol in developing seeds is inhibited by antisense, co-suppression or ribozyme-mediated inhibition of SMT expression using the recombinant DNA molecules of the invention, resulting in the accumulation of this sterol in the seed oil . Conversely, the SMT gene is overexpressed in order to increase the content of sitosterol.

Preferred crops for use in this embodiment of the invention include: sunflower, corn, soybean, Brassica oilseed and cotton. Enhancing plant tolerance to harsh environments by altering phytosterols

Another embodiment of the present invention derives from the fact that certain sterols are associated with reduced water permeability of cell membranes. Therefore, manipulation of sterols should provide a proven method of preventing, or at least minimizing, the damage caused by drought.

Several studies of chemical inhibitors of sterol biosynthesis have shown that treated plants exhibit secondary physiological responses, including tolerance to harsh environments such as drought and frost (Fletcher, 1988). The occurrence of these physiological reactions is mainly due to the increased levels of hormones such as abscisic acid. However, changes in membrane fluidity are also thought to be responsible for tolerance to harsh environments (Steponkus, 1984).

Membrane fluidity is controlled by several factors, such as the types of sterols and fatty acids and their ratios in the membrane. As far as is known, the type of sterol is the most important of these factors. The main function of sterols is to buffer mutations in membrane fluidity. They can more specifically affect the activity of membrane-bound enzymes. The biosynthesis of sterols can be disrupted by the use of inhibitors, leading to the depletion of terminal sterols and the accumulation of intermediates, thereby altering membrane function.

There is evidence that inhibition of sterol biosynthesis in plants leads to increased abscisic acid levels and stomatal closure (Hareuser, C. et al. 1990 J. Plant Physiol. 137:201-207). It is unclear how this pathway is mediated. However, it has been well documented that altering phytosterols can lead to plant tolerance to several harsh environments, most likely mediated by elevated levels of abscisic acid. Furthermore, in all of these studies using chemical inhibitors of sterol biosynthesis, the sterols that accumulated were precisely those sterols identified as unavailable by the present invention, namely, 9β,19-cyclopropylsterol, 14α-methylsterol and Δ ⁸ -sterols. Therefore, the present invention generates unavailable sterols through various genetic manipulation strategies, which can not only protect plants from pests and pathogens, but also enable plants to resist harsh environments such as drought and severe cold. In this regard, preferred increased sterols include ^Δ5-24 alkyl sterols such as 24-methylcholesta-5,23-dienol and cycloartenol.

Preferred crops for use in this embodiment of the invention include: corn, wheat, rice, sorghum, soybeans, Brassica oilseeds (rapeseed, canola), sunflowers, palms, peanuts, cotton, forest trees, fruits, berries , nuts, potatoes, tomatoes, sugar beets, sugar cane, gourds (pumpkins, melons, cucumbers, watermelons, zucchini), Brassica vegetables, alfalfa, ornamental crops, turfgrasses, peanuts, tea and coffee.

The following examples illustrate preferred embodiments of the invention. Those skilled in the art should recognize that the techniques disclosed in the following examples may represent the techniques disclosed by the inventors, in order to better implement the present invention, and thus may be considered as the preferred mode of implementing the present invention. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a similar or equivalent result without departing from the spirit and scope of the invention. Unless otherwise specified, all the techniques discussed in the above part of the description and used in the following examples can be operated according to the general methods in standard molecular biology and biochemistry well known to those skilled in the art (specific content See, for example, Sambrook et al., 1989).

Examples Example 1. Phytosterols

Sterol isomers were extracted from cotton and separated into homogeneous forms by chromatography. Identification of novel phytosterols with side chains not available to insects.

Sterols were found to be structurally specific by mass spectrometry and ¹ H and ¹³ C nuclear magnetic resonance (NMR) experiments (Table 1) (Guo et al., 1995).

Initial studies showed that 4-day-old maize produced mono- or di-alkylated sterols at C-24. Maize can produce those sterols because the isolated 24(28)-methylene and 24(28)-ethylene sterols were obtained from maize seedling tissue and their structures were determined by mass and proton NMR Resonance spectroscopy was identified.

Table 1

Sterol components of corn

MS ^a TLC ^a Sterols ^bd Vegetable source ^c

(M ⁺ ) (Rf) cycloartenol 426 0.29 st, c, g, r, sh, b,

P24 (28) -Aniah-ring wood 440 0.29 ST, C, G, R, SH, B, pineapple alkyl Pcyclosadol 440 0.29 ST, G, SH, SH Ringyl 440 0.29 ST cyclosol*428 0.29 SH24 -Methylcycloartanol 442 0.29 g24(28)-methylene 440 0.29 shparkeol*α-amyrin (triterpene) 426 0.29 st, c, g, r, sh, bβ-amyrin (triterpene) Terpene) 424 0.29 st, c, g, r, sh, b4 α,14 α-Dimethylergot 424 0.25 st, c, g, r, sh, b ster-7,24(28)-dienol 4 -Methyl-7-ene cholestanol 400 0.25 g, sh24-methylene 4-methyl-7-ene 412 0.25 c, g, r, sh, b, p, cholestanol I 2 - 4 4-methyl-7-enechol 414 0.25 g, sh stanol 24-ethyl 4-methyl-7-ene 428 0.25 g cholestanol cycloeucalyptol 426 0.25 sh c, g, r, 426 0.25 c, g, r, sh, b, p dihydroobtol 428 0.25 sh31-norlanosterol

412 0.25 sh (norlanosterol)*4α-methyl wheat horns 甾-

412 0.25 b8,24(28)-Dienol*4α-methylergoster-412 0.25 c,g,sh7(E)-23-Dienol 4α-methylergoster-7(Z)-412 0.25 sh23-dienol* α ₁ -sitosterol 426 0.25 c,g,r,sh,b α ₁ -isositosterol* 426 0.25 sh4α,14α-dimethylergot 426 0.25 c,sh sterol-8(E)- 23-Dienol 4α, 14α-Dimethyl-Mai 426 0.25 sh Angistan-8(Z)-23-Dienol* 4α, 14α-Dimethyl-24-442 0.25 Sh Ethyl-Cholesteryl- 8-enol*4α,14α-dimethyl-9,19-cycloergost-23-ene 426 0.25 c,sh alcohol 4α-methyl-cholesta-8(9),14(15),24( 28)- 410 0.25 sh Trienol* Cholesta-5,22-dienol* 384 0.18 sh Cholester-7-enol* 386 0.16 b Cholesta-8(9)-enol* 386 0.18 b Cholesterol Sterol 386 0.18 st, c, g, sh, b, p Cholesterol 388 0.16 st Brassicasterol 398 0.18 st, sh

st, c, g, sh, b, r, 24-methylene-cholesterol 398 0.18 t, p-ergosta-5(E)-23-diene 398 0.18 st, c, g, sh, b, r Alcohol Codisterol 398 0.18 st, sh ergosta-7(E)-23-di-ene 398 0.16 st, c, sh alcohol 24-methylene-cholest-7-ene 398 0.16 st, c, sh, p-alcohol 24-methylene-zymosterol 398 0.18 p campesterol 400 0.18 st, c, g, sh, b, r,

t, p24-epicampesterol 400 0.18 st, c, g, sh, b, r, (epicampesterol) p-ergosta-(E)-23-enol** 400 0.16 ster-cholesteryl-sh14α-methyl ene 400 0.16 sh alcohol*ergosta-7-enol 400 0.16 st, cergosta-8(9)-enol* 400 0.18 sh ergostatanol , β-cholesteryl 402 sh-2 4c 0.16 410 0.18 sh5,22,25-trienol 14α-methylergosterol- 412 0.18 sh8,25-dienol*14α-methylergosterol- 412 0.18 sh8,24(28)-dienol*stigmasterol -7,25-dienol 412 0.16 sh stigmaster-8,25-dienol* 412 0.18 sh24β-ethyl-cholesteryl-5,25- 412 0.18 st,sh stigmaster-5,23 -dienol 412 0.18 sh fucosterol 412 0.18 st, g, sh isofucosterol 412 0.18 st, c, g, sh, b, r,

t,p24-Ethylcholesterol- 412 0.18 st,sh5,24(25)-dienol avenatrol 412 0.16 st,c,sh25-methyl-24-methylene-cholesterol 412 0.18 sterol sh sterol *beans 412 0.18 st, c, g, sh, b, r,

T, P, Pedin-7-hydol 412 0.16 C Bean Radium-22-Antol 414 0.16 ST, SH14α-methyl-based wheat horn-8 414 0.18 SH (9) -Diotanol glycol 414 0.18 ST, C. g, sh, b, r,

t, p

Stigmasterol 416 0.16 st, sh

^a MS, mass spectrometry; TLC, thin layer chromatography

^b* new zeosterol; **new natural sterol

^c St, sprout; c, cloeoptile; g, germ oil (germ oil);

sh, sheath; b, leaf r, root; t, ear; p, pollen

^d gives the customary or system name

The biosynthetic process of sterols was analyzed to determine the relationship between the precursors and products of sterols. Detecting developmental regulation of sterol metabolism by comparing different maize tissues. The results showed that the sterols in leaves mainly included 24-ethyl sterols, eg, sitosterol, while the leaf sheaths mainly contained 24-methyl sterols, eg, 24-methyl-cholesta-5,23-dienol.

Four [3- ³ H]24-methylsterol isomers cultured from 8-day-old blanched leaf sheath tissue were used for uptake-capture experiments, and the results showed that: Δ ²⁴⁽²⁸⁾ -methylene sterol and Δ ²⁴⁽²⁵⁾ -24-methylsterol is the precursor of 24α-methylsterol and 24β-methylsterol, while ^Δ23(24) -24-methylsterol and Δ25 ⁽²⁷⁾ -24-methylsterol Is the end product of the sterol pathway.

The results show that a single SMT _I enzyme is responsible for catalyzing the two-step methylation reaction, a critical link between cycloartenol (the starting material of the pathway) and ^Δ5-24 -alkylphytosterol (the final product of the pathway). The slow reaction step is the methylation reaction step, which is regulated by the feedback of 24-ethyl sterol. During plant growth and maturation, the SMT _I enzyme regulates the type and amount of phytosterols produced from cycloartenol. This finding contradicts the generally accepted view regarding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). This enzymatic step occurs early in the isoprenoid pathway to sterols and is considered the rate-limiting reaction in phytosterol biosynthesis. The present findings suggest that the role of HMGR is simply to control the flow of carbon into the sterol pathway.

During seedling development after seed imbibition, studies on the expression of microsomal HMGR activity and microsomal SMT enzyme activity (Fig. 3C and 3D) showed that: (1) SMT activity was related to sterol synthesis and plant growth; When being 100mM, sitosterol and 24 (28)-methylene-cycloartanol all do not affect the activity of HMGR, and the activity of this explanation HMGR has nothing to do with the generation of plant growth or sterol; And (3) the effect of phytosterol transformation The rate is related to the activity of SMT _I enzyme catalyzing the first methylation and second methylation reactions, but not to the activity of HMGR.

These results suggest that sterol biosynthesis is negatively regulated during early shoot development following seed imbibition. The sterols accumulated in the 3-day-old sprouts were transported from the sterols originating in the seeds. Subsequent seedling development in maize results in a positive regulation of phytosterol synthesis. Carbon flow directed into the phytosterol pathway: The rate of Δ ⁵ -24-alkyl sterol synthesis should meet the increasing demand of membrane synthesis. Cycloartenol and related C-4 methylated sterols are converted to Δ ⁵ -end products. The key slow reaction step is the methylation of cycloartenol, which is the first conversion step in phytosterol synthesis.

Figure 4 summarizes the pathways leading to the kinetically favorable end products ^Δ5-24 -alkylsterols during seedling development into leaves and sheaths under dark-grown conditions. Expression of SMT enzyme activity and data on sterol specificity early in the process of leaf and sheath formation suggest that maize synthesizes at least two distinct SMT enzymes: SMT _I catalyzes the subsequent methyl conversion to ^Δ24(28) -methylene base sterol and Δ ²⁴⁽²⁸⁾ -ethylene sterol, while SMT _II catalyzes methyl conversion to Δ ²³⁽²⁴⁾ -24-methyl sterol. Identification of sterols required for embodiment 2 plant growth

The phytosterols identified in Example 1 were individually tested for their ability to support growth. Due to the lack of phytosterol mutants available for this study, the yeast sterol auxotroph GL-7 was grown in the presence of the sterols identified in Example 1, see above (Li, 1996). This yeast mutant was used as a model system because of its ability to take up sterols from the culture medium, incorporate the tested sterols into the lipid bilayer of the cell membrane, and proliferate. Cell proliferation was measured in the presence and absence of hormone levels of ergosterol, the major zymosterol.

Sterols can be classified according to their effects on growth processes. Growth-activating sterols include ergosterol. Sterols including cholesterol and sitosterol that are migrated to membrane and cellular structural components without affecting cell growth rate (Nes et al., 1993). The enzymatic research of embodiment 3 sterol converting enzyme

To illustrate the enzymatic basis for the sterols identified in Example 1, the sterol specificity of microsome-bound soluble SMTase from 4-day-old maize seedlings was determined. When using a microsome-bound enzyme system we found that cycloartenol was the preferred sterol acceptor and that 24(28)-methylene-4-methyl-7-encholestanol was methylated to generate 24 (28)-Ethylidene-4-methyl-7-encholestanol. Table 2 summarizes the specificity of soluble SMTases from maize seedlings for various sterol substrates.

Table 2

Sterol Specificity of (S)-Adenosyl-L-Methionine: D ²⁴ -Sterol Methyltransferase Substrate Enzyme Activity Relative to Cycloartenol Methyl

(DPM/Min) The % vitality ring of Antonol 37,515 100 (C1) Wool Teltol 24,384 65 (C1) Parkeol 6,002 16 (C1) 31-reduced wool alcohol 18,757 50 (C1) 24-dehydrolytic alkyl alcohol 8,253 2222 (C1) yeast alcohol 5,252 14 (C1) 4α-methyl yeast alcohol 10,504 28 (C1) 14α-methyl yeast alcohol 3,376 9 (C1) 3-deoxymal alcohol BG 0 (C1) biler-8-hydol BG 0(C1)24(28)methylene-4-methyl-7- 3,800 10(C2)enecholestanol 4α-methylergosta- 1,500 4(C2)8,24(28)-diene Alcohol phyllol BG 0(C2)cycloeucalyptol BG 0(C2)ergoster-8,24(28)-dienol BG 0(C2)ergoster-7,24(28)-dienol BG 0(C2)ergoster-7,24(28)-dienol 0(C2)ergosta-5,24(28)-dienol BG 0(C2)24(28)-methylene-cycloginol BG 0(C2)rodanol

There was little difference in relative binding efficiencies ( _Km ) for sterols in the microsome-bound soluble enzyme systems studied. There was a difference in the apparent V _max for each substrate, but this was expected since the levels of protein and total endogenous sterols varied during enzymatic solubilization. The properties of the soluble SMTase from 4-day-old maize were similar to those of the microsomal-bound SMTase from sunflower.

Cycloartenol and [Methyl- ^3H ]-AdoMet were incubated with soluble enzymes from 4-day-old shoots to illustrate the first methyl conversion reaction. In a study of the operation of the methylation mechanism in maize, [27 ¹³ C]-lanosterol was used to determine the methylation mechanism for the production of 24(28)-methylene sterol in 4-day-old shoots (Guo et al., 1996 ).

In experiments incubating cycloartenol or lanosterol, neither sterol receptor molecule was methylated to a second methyl product (Nes et al., 1991; Venkatramesh et al., 1996). If SMT is a single type of protein, there may be two binding sites on this enzyme.

The maize SMT protein is a tetramer composed of 4 subunits with a size of 39 kDa. A bifunctional sterol methylation (SMT) enzyme was partially purified from 4-day-old blanched maize sprouts using the following procedure:

(i) non-ionic detergents dissolve microsome-bound SMT enzymes;

(ii) gel filtration fractionation of the dissolved protein to generate an active fraction with an apparent natural molecular weight of about 156 kd; and

(iii) Hydroxyapatite chromatography was performed on the active fraction.

Both methylation activities were co-purified approximately 200-fold.

Figure 1 shows the results obtained by HPLC radiometric counting (Figure 1B) and mass spectrometry (Figure 1A) for the determination of reaction products in 50 collected samples from 24(28)-methylene- 4-Methyl-7-encholestanol assay for soluble SMTase (4-day-old seedlings). During this incubation, the conversion of 24(28)-methylene-4-methyl-7-ene cholestanol to 24(28)-ethylidene-4-methyl-7-ene cholestanol was demonstrated. Second methyl conversion of alkanols. Thus, SMTases from 4-day-old maize shoots catalyze successive first and second methylation reactions of the appropriate sterol receptor molecules.

Table 3 shows the effect of a series of substrate and transition state analogues on the first and second methyl conversion reactions.

table 3

Substrate and transition state analogue inhibitor pairs

(S)-Adenosyl-L-methionine: effect of Δ ²⁴ -sterol methyltransferase activity.

Entry site relative to the first methyl conversion K _i of the methyl conversion relative to the second inhibitor (entry)

no.* K _i campesterol 1 NA NA 24(28)-methylene-cyclo 2 20 μM NA artenol 26,27-cyclopropylidene ring 3 25 μM NA sterol 24-(R,S)- 25-cycloimine-lanosterol 4 55nM 55μM Z-24(28)-Ethylidene-4-methyl-7-encholestanol 5 NA 75μM Sitosterol 6 NA 100μM

*Numbers represent structures in Figure 2

Various inhibitors were tested with soluble SMTase from 4-day-old maize seedlings (Figure 2). The fact that some inhibitors were unable to affect the methylation activity of the two sterol substrates suggests that the SMT enzyme has two binding sites.

SMT catalyzes two consecutive transmethylation reactions from coenzyme (S)-adenosyl-L-methionine to different substrates: generation of cycloartenol (Δ ²⁴ -4,4-dimethylsterol) Km is 20mM and Vmax is 4pmol/min/mg protein, and generates 24(28)-methylene-4-methyl-7-ene cholestanol (Δ ⁷ , 24(28)-4-monomethyl Km and Vmax were 11 μM and 1 pmol/min/mg protein, respectively. Thus, cycloartenol is the preferred substrate for the first methylation reaction, while 24(28)-methylene-4-methyl-7-encholestanol is the preferred substrate for the second methylation reaction. Substrate is preferred. Zymosterol (Δ ^8,24 -4-desmethylsterol) is the preferred sterol substrate of yeast SMT enzyme, and it is also a rare sterol substrate for the first methylation reaction.

Substrate specificity and inhibition studies have shown that of the two binding sites on the SMT enzyme, site I catalyzes the conversion of the first methyl group to 24(28)-methylene sterol; while site II catalyzes the second Methyl conversion yields 24(28)-ethylene sterol.

For example, sitosterol (24α-ethylcholesterol), the major end-product of zeosterol production in maize leaf tissue, inhibits the second methylation transition (100 μM K _i ) without affecting the first; rapeseed oil The sterol (24α-methylcholesterol) does not inhibit both the first and second methylation transformations; 24(28)-methylenecycloartanol, a cycloartanol transmethylation The product was not methylated; 24(28)-methylene-cycloartanol inhibited the first methyl conversion (20 μM K _i ), but not the second. 26,27-Cyclopropylidene cycloartenol does not bind to yeast SMTase, it is a potent competitive inhibitor of the first methyl conversion reaction, while it does not affect the second methyl conversion.

The second alkylation reaction was inhibited by product inhibition from 24(28)-ethylidene-4-methyl-7-encholestanol, but the first methylation conversion was not affected. The transition state analog 24-(R,S)-25-cycloimine-lanosterol inhibits the first and second methylation reactions with a similar _K value of 55 nM, showing a non-competitive dynamic mode. In the initial enzyme-substrate interaction, the sterol character of the substrate appears to be typical of other plant SMT enzymes, ie a nucleophilic group is required at the C-3 and C-24 positions. The value of coenzyme K _m equal to 5 μM is the same for the first and second methylation reactions. Embodiment 4 is from the SMT gene of yeast

The yeast SMT gene ERG6 was derived from the yeast ERG6 genomic fragment pRG 458/erg6 (FIG. 5B; SEQ ID NO: 1).

The cloned ERG6 gene was expressed in Escherichia coli. Enzymatic studies showed that the recombinant protein was a sterol biomethylase, which also demonstrated that the recombinant protein was similar in kinetic properties to the naturally occurring enzyme in yeast. Unlike plant SMT which prefers cycloartenol, yeast SMT prefers zymosterol as a substrate, which is a ^Δ24-4 -desmethylsterol.

After successfully overexpressing this active protein in Escherichia coli with PET23a(+) vector with T7 promoter, the molecular weight of yeast SMT monomer was determined to be 43KD. Overexpressed proteins were visualized on SDS-PAGE gels by Coomassie blue staining and Western blotting using a yeast SMT polyclonal antibody. This recombinant protein has now been purified using the system described above.

From the deduced yeast SMT amino acid sequence (Fig. 5A; SEQ ID NO: 2), the potential AdoMet binding motif is presumed to be the first conserved region (YEYGWGS) identified in Fig. 5A, according to Example 3. Mechanistic analysis of the described biomethylation, tryptophan (W) was identified as the binding site. By site-directed mutagenesis of the ERG6 gene, alanine was used instead of tryptophan. The mutated DNA was also cloned into PET23a(+) for overexpression in E. coli. The mutant protein is inactive under conditions where the wild-type protein is active.

The above strategy provides a way to introduce inactivated SMT proteins into plants to alter phytosterols. The introduction of non-functional SMT monomers can inhibit the activity of SMT, for example, affect the formation of functional SMT enzyme complexes in cells, resulting in the formation of unavailable sterols. For example, inhibition of the first SMT _I reactivity results in SMT _II activity catalyzing the production of the product ^Δ23(24) -24-alkylsterol. Conversely, inhibition of the second SMTI reaction will lead to the formation of Δ24 ⁽²⁵⁾ -24-alkylsterols. Example 5 SMT gene from Arabidopsis

The SMT gene of Arabidopsis was cloned and sequenced (FIG. 6; SEQ ID NO: 3). The gene was overexpressed in E. coli, Arabidopsis SMTs were partially purified and characterized by specific stereochemistry.

The Arabidopsis SMT gene was amplified by PCR from a cDNA library. The primers used were designed according to the full-length cDNA sequence (accession number X89867) extracted from the gene bank. The amplified product is the full-length Arabidopsis SMT gene, and then the gene product is subcloned into a T/A cloning vector and sequenced. From this sequence data an open reading frame (ORF) is determined. An Nde I site was created by PCR-mediated directed mutagenesis at the ATG start codon. According to the method for cloning the ERG6 gene in Example 4, the full-length ORF containing an Nde I site at the beginning and a BamHI site at the end was cloned into the PET23a(+) vector. The activity of the recombinant protein converts both cycloartenol and 24(28)-methylene-4-methyl-7-encholestanol, respectively, into their respective alkylated products (Tong et al., 1997). When the substrate is cycloartenol, only one product, 24(28)-methylene-cycloartanol, is generated, which is the reaction catalyzed by SMT _I in Figure 4. The enzymatic data in Example 3 were further confirmed because a single gene product was able to metabolize both sterol substrates. Furthermore, because the metabolism of cycloartenol catalyzed by the recombinant plant SMT produces only one product, which is the product of SMT _I , this shows that another product cyclosadol (structural formula 6 in Figure 4) is produced by another different gene The encoded isomer (SMT _II ) is catalyzed. Embodiment 6 is from the SMT gene of corn

The maize sterol methyltransferase (SMT) gene was isolated from a commercial maize cDNA library (Stratagene, La Jolla, CA). The SMT gene was amplified by polymerase chain reaction (PCR) using 5 ml of maize cDNA (equivalent to 5×10 ⁷ pfu) as a template. Since the cDNA library was constructed in the vector Uni-ZapXR (Stratagene), the T7 sequence in the vector was used as one of a pair of primers (3' end primer) in the PCR amplification reaction. The 5' end primer (2650-1) was designed according to the 2-20 nucleotide sequence of a deduced SMT fragment published in GenBank (T23297). According to the requirements of the instructions, 5 units of Taq polymerase produced by Promega were added to a total volume of 100 ml, and the reaction was carried out for 30 cycles. Take 1 ml of the PCR product of the above reaction as a template for the second round of PCR, wherein the primers used are T7 primers and primers designed according to the 250-268 nucleotide sequence in T23297. When the final reaction product was analyzed on 1% agarose gel, a band with a size of 1.3 kb was seen. This PCR band was then subcloned into plasmid pGEM-T (Promega) and sequenced.

In order to obtain the SMT gene at the 5' end, a pair of primers designed according to the 2-20 and 366-349 nucleotide sequences in T23297 were used for PCR amplification. A 366bp nucleotide fragment was obtained and sequenced. The sequence of this 366 bp fragment overlaps with the above 1.3 kb clone by 116 nucleotides. The two fragments were spliced together by PCR using primers 2650-1 and 3082-2. The latter primer was designed based on 20 nucleotides of the 1.3 kb fragment preceding the polyA sequence. These two PCR fragments of 366bp and 1.3kb were used as DNA templates. The reconstructed SMT gene was ligated into the PCR cloning vector pGEM-T and bidirectionally sequenced with ABI Prism DNA automatic sequencer (Model 310).

The cloned SMT cDNA is 1497 nucleotides, which has a coding region of 1032 nucleotides, which can encode 344 amino acids (Figure 10; SEQ ID No: 6). The initiation codon ATG is located at positions 66-68 of the nucleotide sequence. Before the start codon (ATG), there was a stop codon at positions 42-44, which indicated that the reconstructed SMT gene contained a complete 5' end. There is a polyA tail of 28 nucleotides at nucleotide 371 downstream of the stop codon, which indicates that the 3' end of the cDNA fragment is complete. Therefore, this cDNA clone is a clone of the full-length cDNA.

The amino acid sequence deduced from the cDNA clone contains 344 amino acids, encoding a polypeptide with a size of 38.8KD. The deduced amino acid sequence contains all three putatively conserved regions of methyltransferases (Kagan and Clarke, 1994. Arch. Biochem. biophys. 310:417-427): p. 104 - LDVGCGIGGP at position 114 (amino acid sequence), TLLDAVYA at position 167-174 and VLKPGQ at position 194-199. In addition, another conserved region of sterol methyltransferase identified by Nes (SFYEYGWGESFHFA, Guo et al., 1997. "Anti-fungal sterol biosynthesis inhibitors". "Subcellular Biochemistry" Volume 28: Cholesterol: "It is in Function and Metabolism in Biology and Medicine," edited by Robert Bittman. Plenum Press, New York) at Nos. 60-73.

The deduced maize SMT amino acid sequence was compared with other known SMT genes using GCG system (Gap and Bestfit). The deduced SMT amino acid sequence has 93.6% similarity to the independently isolated maize SMT sequence (GenBank U79669), 88.1% homology and 78.8% identity to soybean SMT (GenBank U43683), and part wheat SMT sequence (gene bank U60754) has 93.9% homology and 88.3% identity, has 58.8% homology and 39% identity with Arabidopsis (gene bank X89867), and yeast SMT (gene bank X89867) X74249) have 66.5% homology and 50.4% identity. The high similarity between the cDNA clone and SMT genes of other plant species confirmed that the cDNA clone was the full-length cDNA clone of maize SMT. Furthermore, since Grabenok et al. have functionally expressed the maize SMT gene in a yeast expression system and found that only ergosterol is a 24-alkyl sterol, this shows that the maize SMT gene isolated in my laboratory catalyzes the same This stereoselective C-methylation generates Δ24 ⁽²⁸⁾ , supporting the idea that maize synthesizes several distinct SMTases.

A similar strategy can be used to isolate cDNAs encoding SMT _II isoforms. In fact, the cDNA fragment isolated by the above method should be representative of both SMT _I and SMT _II , because the conserved region between them can be used to design primers. Example 7 SMT gene from Prototheca wickerhamii

Another example of a preferred SMT gene is the SMT gene of Prototheca wickerhamii. This yeast-like algae produces Δ25 ⁽²⁷⁾ -24-methylsterol as the major product of the transmethylation reaction. A preferred substrate is cycloartenol.

P. Wickerhamii microsome preparation studies have shown that the preferred substrate for SMT is cycloartenol. However, the preferred product is not 24(28)-methylene-cycloartenol but rather cycloartenol (VII), which is a ^Δ25(27) -24-alkylsterol and is an unavailable sterol .

In order to transform the phytosterol cycloartenol into the product cyclosaudonol, cloning the SMT gene will promote the transformation of the gene into plants, and the generation of cycloartenol will lead to the accumulation of unavailable sterols, namely Δ ²⁵⁽²⁷⁾ -24 - Accumulation of alkyl sterols. Prototheca SMT clone

Protothecawickerhamii cells were grown to mid-logarithmic growth in YPD-enriched medium (yeast extract-peptone-glucose). The pelleted cells were disrupted with 0.5 mm glass beads and a mini-Beadbeater (both Biospec, Bartlesville, OK) with the addition of Tri reagent. Isolate high-quality whole-cell RNA according to the instructions.

3'RACE (random amplification of cDNA ends) and 5'RACE were performed on whole-cell RNA using the reagents and methods provided in the kit produced by GibcoBRL. For 3'RACE, total cDNA is synthesized by reverse transcriptase after annealing of olig(dT)-containing primers to poly(A)-tailed RNAs in unfractionated total RNA. The above RNA template is degraded, and the synthesized cDNA is used as a template for polymerase chain reaction (PCR) amplification. The user-supplied primer "YEYGWG" (see primer design theory below) anneals to the cDNA and extends toward the 3' end of the gene under the guidance of Taq polymerase. The primer provided in the kit extending from the 3' end to the "YEYGWG" defined end anneals to a sequence containing 3 restriction enzyme recognition sites, and this sequence containing restriction enzyme recognition sites is the initial primer containing olig(dT) a part of. The second round of PCR amplification was to enrich cDNAs corresponding to the half-length sequence at the 3' end of SMT, and the primer pair used was the second "nested" primer (GCGVGG) and the 3' end primer provided by the kit. Another nested primer ("ATCHAP") has also been used in a similar fashion.

5' RACE was performed on whole-cell RNA. cDNA was synthesized by reverse transcriptase using the antisense primer "EWVMTDas". The cDNA is altered by adding a polydeoxycytidine "tail" at the 3' end with terminal deoxynucleotidyl transferase (TdT). Using this poly-C-tailed cDNA as a template, use the primer "EWVMTDas" and the poly-G-containing primer provided in the kit to carry out the initial PCR reaction. The second round of PCR reaction was carried out on the basis of the initial PCR reaction product. The primers used this time were the nested primer "ATCHAPas" and the primer provided by the kit that can anneal to part of the poly-G sequence. The G sequence contains restriction enzyme recognition sites. The purpose of this second round of PCR reaction is to enrich the SMT 5' end cDNA sequence.

The above 3'RACE and 5'RACE PCR amplification products were isolated from the gel and ligated into plasmid pPCRII (Invitrogen). Clones obtained after transformation into E. coli were identified by sequencing. An Apa I restriction site is contained in all plant DNA and yeast DNA that has been sequenced in the GCGVGG motif, present in both 3' and 5' cDNA clones. In this way, the 3' end half-length sequence and the 5' end half-length sequence of the SMT gene can be spliced together to form a complete full-length coding region. primer design theory

The first step in designing user-supplied primers was to examine several extremely highly conserved peptide motifs in sequenced plant and yeast SMTs. Among them were found shorter stretches of amino acid sequences that could be encoded by the smallest number of DNA sequences, these codons usually changing only the third (degenerate) base. According to the codon usage table found by Wada et al. (Nucleic Acids Res., Vol. 19, p1981, 1991), codons preferred by three different yeasts are present in the degenerate codons for each amino acid In the mix of , this is also expected. Thus, each primer designed by the user is a mixture of deoxynucleotides that define the internal termini of the PCR product. An additional requirement is that 4 or 5 of the 6 3'-terminal deoxynucleotides of each primer preferably match all species in which the G and/or C content is greater than 50%.

The first 3 of the primers given below are sense orientation and can anneal to antisense DNA (and original cDNA). The 4th and 5th primers are antisense primers that anneal to the sense strand of DNA of the SMT gene.

YE[Y/F/W]GWG (amino acids 81-86 in the yeast sequence; the amino acids in brackets are variable residues) is a part of the larger conserved region of SMT, and the primer "YEYGWG" is designed based on this sequence :

5’-TA[T/C]GA[A/G]T[A/G/T][T/G]GG[T/A/C]TGGGG-3’

(positions of degenerate nucleotides in parentheses)

The primer "GCGVGG" was designed by the DNA sequence encoding a part of the second conserved region (GCG[V/I]GG) located at the 129th to 134th yeast amino acid residues. The sequence of the primer "GCGVGG" is:

5'-GGATG[T/C]GG[T/A][G/A]T[T/C]GG[G/C]GG-3'.

The primer "ATCHAP" was designed on the basis of the DNA sequence encoding the highly conserved third conserved region (196-203 amino acid residues in yeast). The primer sequences are:

5'-GCCAC[A/G/T]TG[T/C]CA[C/T]GC[T/G/A]CC-3'.

Primer "EWVMDas" is an antisense primer used to synthesize the first strand of cDNA in 5'RACE experiment. It is designed on the basis of a small conserved region located at the 225th to 231st amino acid residues in yeast. The sequence of the primer is:

5'-TC[A/C/G]GTC[G/A]T[T/A/G][C/A][C/A]CCA[C/T]TC-3'.

Primer "ATCHAPas" is a nested antisense primer for 5'RACE experiment, its sequence is:

5'-GG[T/C/A]GC[A/G]TG[G/A]CA[A/C/T]GTGGC-3'Example 8 SMT genes from other plants

Using the Arabidopsis cDNA or the SMT sequence of another plant as a probe, a cDNA library of any crop of interest is screened and corresponding clones of appropriate size are isolated and sequenced. Methods for construction and screening of cDNA libraries are well known to those skilled in the art. As described in Example 6, suitable primer combinations can be easily designed based on the conserved region information of the known sequences of various SMT genes. To determine the identity of the sequences cloned by this method, they can be compared to known plant SMTase sequences and/or subjected to in vitro translation and biochemical evaluation. Example 9 Transformation of plants with ERG6 DNA

To obtain transgenic plants with altered sterol profile, a DNA fragment containing the open reading frame of the yeast SMT ERG6 gene isolated from a genomic clone was identified (Example 4). The ERG6 DNA was modified by PCR to contain NcoI restriction sites in either end of the open reading frame. This PCR method provides a way of thinking for realizing the frameshift mutation of the gene. This mutation causes the ERG6 gene transformed into the plant to be untranslated, but it is able to suppress endogenous tomato SMT by antisense or co-suppression mechanisms, depending on the nature of the construct.

The modified ERG6 DNA fragment was cloned into the pUC18cpexp expression cassette vector. Clones carrying ERG6 DNA in sense and antisense strands relative to the 35S promoter were propagated (Figure 7).

Digestion of these clones with HindIII generated an ERG6 construct comprising the 35S promoter and termination sequences flanking the ERG6 open reading frame. The fragment obtained by HindIII digestion was cloned into the binary vector pJTS246, which contains T-DNA edge recognition sequence and NPTII gene with kanamycin resistance.

The binary vector cloned with the sense or antisense ERG6 construct was transformed into Agrobacterium tumefaciens co-cultured with the cotyledons of tomato (Solanum lycoperisum) to obtain transformed plant cells. Transgenic plants were generated from calli formed on selective medium containing kanamycin.

Transgenic analysis was performed on leaves of control plants (without insert) and transgenic plants (with insert). DNA was extracted from leaf samples of each transformant and an additional non-transformed tomato plant. Quantitative analysis of DNA extracts was performed using A260 absorbance.

Aliquots containing 200 ng of DNA from each sample were used as templates to PCR amplify ERG6 fragments with oligonucleotide primers corresponding to the ERG6 sequence (underlined in Figure 8). Control samples for PCR reactions included a sample without template DNA and a sample containing sense and antisense ERG6 from the binary plasmid. PCR reactions were performed for 20 cycles under non-stringent conditions (annealing at 55[deg.] C. for 2 minutes per cycle) and each aliquot was electrophoresed on a 0.8% agarose gel.

Such primers were chosen to amplify a 1100 bp fragment in ERG6 DNA (Fig. 8). Like the control plasmid, all regenerated transgenic tomato plants (R ₀ ) carried the above fragment. There is also non-specific amplification in the above reactions because non-stringent conditions result in non-target DNA strands appearing in transformed plants and in non-transformed controls. However, the level of these amplifications was much lower than that of the target fragment. This further confirmed the presence of ERG6 DNA in the tomato genome.

Sterol analysis was performed on the unsaponifiable lipid fraction of leaf material from regenerated plants transformed with sense constructs and regenerated plants transformed with antisense constructs. The obtained results are listed in Table 4.

Table 4

Sterol components in tomato plants

   (% of total sterols)

Contain ERG6 Yes Contain ERG6 antisterol Control group

The insertion fragment of the righteousness segment of the plug-in fragment bile glycol 29 18 20 20 biliary 甾-7-hydol no 21 13 beanol 25 22 24 vallete alcohol 26 27 24 different rock alcohol (

As a result, it was confirmed that the ERG6 gene was incorporated into the transgenic plants, and the sterol composition of the transgenic plants was changed. Mass spectrometry was used to detect and characterize a novel sterol, cholest-7-enol, which was absent in leaves of control tomato plants.

The protocol for introducing new pathways in tomato plants by insertion of the yeast ERG6 gene is described below:

Since both the sense and antisense inserts of the ERG6 gene lead to the accumulation of cholest-7-enol (VIII), endogenous SMT activity may be inhibited in both cases. This would lead to a diversion of the carbon flow into an alternative minor pathway of phytosterol metabolism in which the first step in cycloartenol metabolism is the reduction of the double bond at C-24 by a reductase. The resulting sterol is cycloartanol (IV), which will then undergo the usual demethylation, isomerization, desaturation and reduction reactions to the cholest-7-enol form as in the main pathway . This is a Δ ⁷ -sterol and the double bond at C-5 is missing, which means that some insects will not be able to use this sterol to complete their life cycle.

The regenerated plants (R ₀ ) flower and bear fruit. The seeds were harvested and the next generation of plants (R ₁ ) were successfully cultured. The individual plants grown from the above seeds were analyzed by ELISA experiment of NPT2 protein to detect whether the selectable marker (NPT2) existed. Control analysis of sterol components was performed on 53 plants from 6 _R1 progeny and one non-transgenic plant. The sterol profile of these plants can be divided into 4 distinct groups or phenotypes:

The mean and standard deviation (Std) of sterols (percentage in total sterols) in the _R1 plants of the 4 types of progenies identified in Table 5. Phenotype 1 2 3 4

Average Average Average Average

Std Std Std Std Std

值              值             值            值甾醇胆甾醇         7.62    2.54    6.20   2.77    4.93   1.14   8.60   2.97菜油甾醇       4.17    3.15    16.60  11.24   4.50   1.95   6.60   4.83豆甾醇         13.14   3.13    12.80  5.26    8.86   1.41   22.60  1.14谷甾醇         11.48   2.86    11.60  2.19    9.57   1.87   16.60  3.91异岩藻甾醇     13.14 2.08 7.60 3.71 9.86 2.32 14.40 4.98B-Chanshin 12.52 3.90 9.75 5.91 10.36 3.95 8.80 1.79 Huan Antin Ally 31.76 5.67 49.36 4.91 28.8824 1.14 1.46.0 6.0 6.8 6.8 6.8 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0. 2.00 rotanol

In addition to the control non-transgenic plants, all _R1 plants that tested negative for the NPT2 marker (thus they were also non-transgenic segregants) also exhibited a normal phenotype (phenotype 1). _R1 plants that tested positive for the NPT2 marker (therefore they were segregants of the transgene) were assigned to all 4 classes. Statistical comparisons were performed for each sterol (inverse sine transformation with percent sterol levels; Student-Neuman-Keuls test, 5% significance level) and a qualitative summary of the results is given below:

Table 6 Comparison between Table 6 (Compared with Press Type 2, 3 and 4 Compared with normal phenotype 1) Holly Allymeter Pathomer 1 Panel Model Type 3 Pressing 4 Biltol Beanol Normal, Normal, Normal High Gwario Gyllatic Hylol Normal Low Normal β-Fragrant Main Normal Normal Normal Ring Antonol, Normal High, Normal 24 (28) -The nails of normally normal normal, normal, normal normal normal high

The distribution of plants across classes (i.e., non-transgenic control plants distributed only in normal classes, whereas transgenic plants were present in all 4 classes) was consistent with that expected for plants transformed with antisense or co-suppression constructs . Thus, different levels of repression between or within progeny result in different levels of expression of an altered sterol phenotype. Therefore, these results are consistent with the results of transformation of the repressed ERG6 gene. More specifically, phenotypes 2 and 3 accumulate intermediates, consistent with a partial inhibition of the activity of primary or secondary methylation of sterol methyltransferases in the biosynthetic pathway. Elevated levels of sitosterol and stigmasterol (normal end products) are contradictory to the inhibitory effect and require further study to explain.

Independent studies of a subpopulation of these progeny plants further supported the hypothesis that repression of the SMT gene could be observed in the transgenic lineage. Table 7 below presents the sterol composition of the non-transformed and non-transgenic segregants.

Table 7

Sterol components of control plants (non-transformed plants and non-transgenic segregants)

             G55(Non-transfer G62(Non-transfer

Average Standard Partial Phytosterol Non-transformed Gene Score Gene Score

Value Poor

ion) ion) sterol cholesterol 18 13 13 14.7 ^2.9Δ0 -cholesterol-tr. 1 1.014-α-methyl-Δ7- 5 5 5.0 0.0 cholesterol -Δ8- 3 1 1 1.7 1.2 cholesterol zymosterol 18 - - 18.0Δ ^{7, 24} - zymosterol 5 - - 5.024-methylene-cholesterol- 19 1 10.0 12.7 alcohol campesterol 2 8 3 4.3 3.2 chain sterol 2 - 2.0Δ ⁰ -camesterol - - 1 1.0 stigmasterol 18 20 25 21.0 3.6Δ ⁰ -stigmasterol - tr. 1 1.0 sitosterol 7 13 18 12.7 5.5Δ ⁰ -sitosterol- - tr. isofusterol 4 2 2 2.7 1.2 cycloartenol 7 19 29 18.3 11.02 4-methylene-cycloartenol 14-tr. Leaf alcohol 1 - tr. 1.0- means not detected; tr. means trace; ND means not sure. NSF is first chromatographed on a thin-layer chromatography plate, from which three bands corresponding to 4-demethyl, 4-monomethyl and 4,4-dimethyl sterol standard bands are eluted , and then further detected by 3% SE-30 column chromatography and GC-MS. The limit of detection was 0.1 mg sterols per leaf sample.

These sterols can be compared with the transgenic plants whose sterol composition is listed in Tables 8, 9 and 10.

Table 8

Sterol components phytosterols from transgenic plants of the G3 lineage G31 G32 G34 G35 G37 G38 G39 Sterol cholesterol 12 10 8 10 8 11 8Δ ⁰ -cholesterol 1 tr. 1 1 1 tr. 114-α-methyl-Δ7- Cholesterol 3 - - - - - 3Δ7-cholesterol- 8 6 13 11 1 -14-α-methyl-Δ8-cholesterol 1 2 2 - - - - zymosterol 10 5 12 - - - 8Δ ^{7, 24} - Zymosterol 2 - 1 - - - 124-methylene-cholesterol - - - - - - - campesterol 4 2 3 - - 1 1 strepsterol - - - - - - Δ ⁰ -camesterol - - - - - - -stigmasterol 16 14 12 20 16 16 6Δ ⁰ -stigmasterol 1 - - tr. - - -sitosterol 15 9 12 10 8 16 6Δ ⁰ -sitosterol 1 tr. - - - - -isofusterol 4 2 2 2 2 1 1 cycloartenol 26 41 36 40 44 41 41 24-methylene-cycloartenol 1 3 3 4 4 4 4 24-methylene-4-methyl-7-ene2 3 2 tr. 4 6 tr. cholestanol obtuse leaf alcohol 1 1 1 tr. 2 3 tr.- stands for not detected; tr. stands for trace amount; ND stands for indeterminate. NSF is first chromatographed on a thin-layer chromatography plate, from which three bands corresponding to 4-demethyl, 4-monomethyl and 4,4-dimethyl sterol standard bands are eluted , and then further detected by 3% SE-30 column chromatography and CC-MS. The limit of detection was 0.1 mg sterols per leaf sample.

Table 9

Sterol Component Phytosterol G51 G52 G53 G54 G56 G57 G58 G59 G510 Sterol Cholesterol 13 5 6 11 16 11 4 15 5Δ ⁰ -cholesterol 1 1 1 1 1 tr. tr. 1 114-α -methyl-Δ7- 1 3 1 2 6 5 2 4 1 cholesteryl Δ7-cholesterol- - - - - - - - -14-α-methyl-Δ8- - 1 tr. tr. 1 tr. tr. 1 1 cholesterol zymosterol- - - - - - - - - Δ ^{7, 24} - zymosterol - - - - - - - - -24-methylene-cholesterol - - 3 4 - 1 6 6 - campesterol 8 15 4 2 1 2 2 3 19sterol- - - - 2 - - - -Δ ⁰ -camesterol - 1 - - - - - - 1 stigmasterol 20 6 10 13 20 17 4 11 6Δ ⁰ -stigmasterol- - tr. tr. - tr. - - 1 sitosterol 21 11 7 9 9 8 3 11 1Δ ⁰ -sitosterol- tr. 1 tr. - tr. tr. 1 1 isofucosterol 1 1 1 1 1 8 1 2 1 cycloartenol 34 48 58 52 41 47 49 35 43 24-methylene-cycloartenol 1 4 6 5 1 1 28 10 14 alcohol 24-methylene-4-methyl 1 3 1 tr. - tr . - tr. 4-7-enecholestanol obtuse leaf alcohol tr. 1 1 tr. - tr. tr. tr. 1

- stands for not detected; tr. stands for trace; N.D. stands for uncertain. NSF is first chromatographed on a thin-layer chromatography plate, from which three bands corresponding to 4-demethyl, 4-monomethyl and 4,4-dimethyl sterol standard bands are eluted , and then further detected by 3% SE-30 column chromatography and GC-MS. The limit of detection was 0.1 mg sterols per leaf sample.

Table 10

Sterol components phytosterols from transgenic plants of the G3 lineage G63 G65 G66 G67 G68 G69 G610 Sterol cholesterol 7 7 9 8 5 6 7Δ ⁰ -cholesterol tr. 1 1 1 tr. tr. 114-α-methyl-Δ ⁷ - 2 2 5 1 1 3 1 cholesterol Δ ⁷ -cholesterol - - - - - - -14-α-methyl-Δ ⁸ - 1 1 1 1 tr. 1 1 cholesterol zymosterol - - - - - - ^-Δ7,24 -zymosterol- - - - - - -24-methylene-cholesterol 2 tr. tr. - - - - campesterol 18 3 1 3 20 1 3-sterol - - - - - -Δ ⁰ -camesterol tr. - tr. tr. 1 - -stigmasterol 10 7 11 8 5 6 7Δ ⁰ -stigmasterol tr. tr. 1 tr. tr. tr. tr. sitosterol 13 7 7 9 8 4 7Δ ⁰ -sitosterol tr. 1 tr. 1 tr. tr. tr. isofusterol 2 2 1 1 1 tr. 2 cycloartenol 30 61 61 61 39 72 70 6 20 7 1-Tenol 24-methylene-4-methanol 2 - - - - - -yl-7-encholestanol obtuse 1 - - - - - -

- stands for not detected; tr. stands for trace; N.D. stands for uncertain. NSF is first chromatographed on a thin-layer chromatography plate, from which three bands corresponding to 4-demethyl, 4-monomethyl and 4,4-dimethyl sterol standard bands are eluted , and then further detected by 3% SE-30 column chromatography and GC-MS. The limit of detection was 0.1 mg sterols per leaf sample.

These analyzes revealed that levels of cycloartenol were significantly elevated in many transgenic plants compared to controls. As described below in Example 10, levels of cycloartenol at or above levels of unavailable sterols required to produce deleterious effects on insects can be successfully achieved by this method. Furthermore, this result is consistent with the fact that the first methylation reaction catalyzed by SMT was successfully inhibited in vivo. Example 10 Utilization and Metabolism of Sterols by Spodoptera zea

Insects were fed several naturally isolated or synthetic sterols. One in vivo model used to study Spodoptera zea is to grow Spodoptera zea in a synthetic medium without sterols and to feed only experimental sterols. In this in vivo model, cycloartenol and several 24-methylsterol and 24-ethylsterol isomers were found to inhibit insect growth.

Two important zeosterols, 24-methylcholestan-5,23-dienol and 24-methylcholestan-5,25(27)-dienol, are non-available sterols. Like the Δ23 ⁽²⁴⁾ -24-alkenosterol and Δ25 ⁽²⁷⁾ -24-alkenosterol isomers, 9,19-cyclopropylsterol is also an unavailable sterol.

The relationship between the structure of sterols and their utilization in the insects was studied by feeding artificial diets with different sterol supplements to the armyworm. Establish a disease-free stocking colony with eggs of zea molasses.

The above-mentioned stocked insects were fed aseptically with a diet mainly composed of pinto beans. Moths were fed 10% sucrose. The culture temperature was maintained at 27±1°C, and other conditions included a photoperiod with a light-to-dark ratio of 14:10, a relative humidity of 40±10%, and artificial food supplemented with different sterols. The experimental food was sterol-free, except for agar that was contaminated with trace amounts of cholesterol.

Dissolve the sterol in acetone. An aliquot of the solution is then added to the sterol-free food in a mortar, mixing the contents of the food thoroughly and allowing the organic solvent to evaporate. Sterols were added to the medium at a concentration of 200 ppm (equivalent to 1 mg sterol per experimental vessel containing one insect).

By 20 days, the larvae of the armyworm had developed to the last stage of larval development (6th instar), after which the insects were ready to pupate. Single neonatal larvae were placed in experimental culture flasks and allowed to grow for 20 days. The fresh weight, length and molt stage of 20-day larvae were recorded.

In some experiments, larvae were allowed to grow for an additional 4 days to determine whether they would fully pupate and develop into moths.

When no sterols were added to the diet, the newborn larvae of the armyworm could not molt and develop into the second instar larvae. Some of these insects are able to tolerate for more than 15 days.

Separation of sterols, including long-chain fatty alcohols, from unsaponifiable lipid fractions extracted from larvae. In some forms of chromatography, these fatty alcohols can co-migrate with sterols and interfere with the quantification of sterols, especially cholesterol. Therefore, in order to determine the identity and content of cholesterol in insects, it is necessary to inject an aliquot of NSF into the column of HPLC and perform GC-MS determination on the fraction relative to cholesterol.

Larvae cannot grow and develop on sterol-free media. Δ ⁵ -sterol, cholesteryl substituted by hydrogen, methylene, E-ethylene or Z-ethylene, or a-ethyl or b-ethyl at the C-24 position of the side chain Sterols, 24(28)-methylenecholesterol, sitosterol, isofusterol, fucosterol, spongasterol and stigmasterol supported larval development to the late 6th instar. These sterols were defined as "available" sterols (Table 11 and Figure 9). At each incubation period, the major sterol recovered from larvae was cholesterol, suggesting that zeidae operates the typical 24-dealkylating sterol pathway of insects.

In contrast, derivatives of 3β-cholesterol with the following groups: 9β,19-cyclopropyl group, paired dimethyl groups at C-4 (for example, cycloartenol and lanosterol), Δ ⁸ -bond, or derivatives with side chain changes with the following groups: Δ ²³⁽²⁴⁾ -24-methyl or 24-ethyl, Δ ²⁴⁽²⁵⁾ -24-methyl or 24-ethyl or Δ ²⁵⁽²⁷⁾ -24β-Ethyl, none of them can satisfy the sterol requirement of zea exoderma. These sterols were defined as "unavailable" sterols (Table 11 and Figure 9).

The main sterol recovered from larvae developing on unavailable sterols was the experimental sterol added to the medium. Competition experiments with different ratios of cholesterol and 24,25-dihydrolanosterol (9/1 to 1/9 sterol mixture) showed that when the ratio between available sterols and unavailable sterols in the sterol mixture (Table 12) When the ratio is less than 1 to 1, the development of corn armyworm will be abnormal. Sterol absorption is related to the degree of sterol utilization and metabolism.

Table 11

Effects of sterols on the growth and metabolism of zea exigo moth

Sterol component ³ (total

                                           , 

% of sterols source of sterols

Stage ¹ to ² mg/insect

Comparison) Use of alcohol gallosterol 1 100 6 56 bile glycol 24- (28) Asian TS/biliary alcohol-bile glycol 2 100 6 59 (16/84)

ts/cholesterol fucosterol 4 100 6 71 ((10/90)

TS/chain alcohol/bile garden alcohol 3 100 6 52 sterol (8/14/78) Valley glycol 5 100 666 TS/biliary glycol

((20/80) Wear the sponge alcohol TS/biliary glycol (50/50) 6 100 6 43 ((14/84) (75/2

5)

TS/chain sterol/bile bean sterol 7 100 6 27 (15/1/84) Do not use sterol cholesterial-8-hydrolytic 13 5 3 ND 24-dehydrated pollen 14 5 3 0.6 TS/cholesterol alcohol ( 86/14) 24-methyl gallbladder-TS/bile glycol 5, 23-dilay alcohol 10 50 5 6 (80/20) 24-base biliary 甾-TS/bile glycol 5, 23-dilay glycol 12 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 3 3 (86/14) 24-methyl gallbladder-TS/biliary alcohol 5, 24-dilay alcohol 9 5 3 1 (65/35) 24-ethyl chillet-5, 23-2 11 10 3 ND ND enol ts/cholesterol clereosterol 8 20 3 3 3 (80/20)

                                                                                                                                                                           

(36/41/23) Ring Anton Alcohol 17 5 3 ND ND Wool Holly Alcohol 16 5 3 ND ND24-Dihydrous Wool 18 5 3 ND ND Telmac alcohol 1 Structure is open in Figure 9. 2 from 30mm in length 323mg of 323mg Cholesterol is produced by 20-day-old larvae to support growth.

Typically, 16 out of 20 larvae survive on cholesterol until they pupate and develop into

adult moth. The growth effect of the measured sterols is that when cholesterol supports growth

When the degree is specified as 100%, the degree to which the measured sterol supports growth and its relative value.

24-Methylcholesta-5,23-dienol supports pupation and adult moth formation. However, this

Some insects exhibit congenital deformities. Typically, insects grown in unavailable sterol

Weight less than 100mg, length 2-15mm, 6 out of 12 larvae survived until

Day 20. 3 After clearing the contents of the insect gut, use RP-HPLC and GC-MS to analyze the

sterols isolated from tissues. ND Not determined ts Total sterols

The highest efficiency of sterols absorbed and incorporated into tissues was 27-36 mg per insect, and the lowest efficiency was 0.6-6 mg per insect. These studies suggest that: (i) S. zea reduces the structural changes in the sterol core and side chains, (ii) the dealkylation pathway of phytosterols to cholesterol is highly position-selective and steric-selective, and ( iii) Maize produces several of the non-available sterols described herein.

Table 12

24-Dihydrolanosterol (not available) compared to Cholesterol (available)

Utilization of

                                                                                            , 

                                                                                                                   

Comparison) Use of alcohol bile glycol 1 100 6 56 biliary alcohol (100 %) (100 %) bile glycol/24, 25 biliary glycol // 24,25 hydrogen melterol 1/18 100 6 45 dihydrous alcohol alcohol alcohol alcohol (90:10) (93:7)cholesterol/24,25disterol/2 cholesterol/5

1/18 100 6 36 Dihydrous Hydrogenol Hydrogen Hydrolasm (88: 12) (70: 30) bile glycol

Biliary sterol/24,25/24,25 two 1/18 70 6 25 dihydrogen wool hydrogen alcohol (75: 25) (50: 50) biliary glycol biliary glycol // 24, 25/24, 25 two 1 1 /18 30 3 12 dihydrous alcohol hydrogen alcohol (50: 50) (30: 70) gallol/24, 25 two 1/18 10 3 ND ND hydrogen melterol (10: 90)

*Represents the structure in Figure 9.

The minimum dietary concentration of cholesterol required for larval growth and pupation was 0.01% of the experimental diet. This level of cholesterol does not support the rapid molting of the larvae as well as higher levels of cholesterol. However, when the proportion of cholesterol in the diet reached or exceeded 0.015%, the development speed of the larvae would be accelerated. Therefore, a slightly higher amount of dietary sterols (0.02%) ensured that a non-limiting amount of sterols (alone or in a mixture) could be obtained from the experimental diet, or the diet without any sterols was used as a control.

Trace amounts of cholesterol were present in all larvae treated with unavailable sterols, ranging from 80 to 350 ng per insect depending on the treatment. This cholesterol is likely derived from cholesterol carried by the eggs (we detected about 80 ng cholesterol per egg) and absorption of the trace levels of cholesterol originally present in the agar.

As the insects grow larger, the insects can accumulate the increasing cholesterol from the agar diet. Cholesterol obtained in this way can become a precursor of ecdysteroids. Two isomeric pairs, sitosterol/spongosterol and isofusterol/fucosterol, differ in their efficiencies in active metabolism supporting growth and generating cholesterol, suggesting stereoselective operation of the 24-demethylation pathway sex.

A comparative study of the developmental outcomes of moths produced by larvae of Spodoptera zea larvae. One larva was fed on available (cholesterol-treated) sterols, while the other larvae were fed on unavailable (24-methylcholesta-5,23-dienol-treated) sterols.

The majority of sterol-unavailable insects failed to develop past the 3rd instar (Table 11), suggesting that they were ineffective cholesterol substitutes and detrimental to growth and development. Some of the sterol-unfeedable insects pupated and developed into moths. However, the wings and legs of these moths are not fully developed.

Table 11 and Figure 9 show that the position of the sterol side chain and the double bond in the sterol core determines the developmental process controlled by the sterol. The inability of cholest-8-enol to support growth suggests that zeidae cannot convert 9β,19-cyclopropylsterol to ^Δ5 -sterol. Blocking this pathway results in the production of unavailable sterols. These results indicate for the first time that the substitution of several sterols synthesized in maize for cholesterol is inappropriate.

All of the components and methods disclosed and claimed herein can be made and performed without further experimentation in light of the present disclosure. The components and methods in the present invention have been described in preferred embodiments, obviously, those skilled in the art can make changes without departing from the spirit, spirit and scope of the present invention. More specifically, it will also be apparent that chemically and physiologically related reagents may be substituted for the reagents described herein to give the same or similar results. It is apparent to those skilled in the art that all such similar substitutions or modifications should fall within the spirit, spirit and scope of the invention as defined by the appended claims.

Bibliography Akisha et al., "Physiology and Biochemistry of Sterols" (GW Patterson and W. David Nes, Eds.) (1992) American Oil Chemists' Society Press, pp. 172-228. Armstrong et al., Field evaluation of corn borer control in 173 progeny of transgenic maize expressing an insecticidal protein from Bacillus thuringiensis, Crop Science (1995) vol. 35 pp. 550-557 Battraw and Hall, Transgenic Histochemical Analysis of CaMV 35S Promoter-β-Glucuronidase Gene Expression in Rice Plants, "Plant Molecular Biology" (Plant Mol.Biol.) (1990) Volume 15 pp.527-538Bird and Ray, used Antisense RNA manipulates gene expression in plants, "Biotechnology & Genetic Engineering Reviews" (Biotechnology & Genetic Engineering Reviews) 9: 207-27, 1991Bouhida et al., Complete sequence analysis of the genome of sugarcane baculovirus that can infect bananas and rice, " Journal General Virology (1993) Vol. 74 pp. 15-22 Bouvier-Nave et al. Eur. J. Biochem 246: 518-529 (1997) Bower and Brich, via Preparation of transgenic sugarcane by particle bombardment, "Plant J." (1992) Vol. 2 pp.409-416Bryant, transgenic rye-cereal genetic engineering successfully applied to cereal crops by injecting plasmid into flower tillers, "Bio "Technology Development Trends" (Trends Biotechnol.) Volume 15 pp.60-61 Bytebier et al., T-DNA organization in tumor cultures and transgenic plants of the monocotyledonous plant D. .Acad.Sci.) (1987) Volume 84 pp.5345-5349Christou et al., DNA-coated gold particles stably transform soybean callus, "Plant Physiol" (Plant Physiol) (1988) Volume 87 pp.671-674Christou et al. Production of transgenic rice (Oryza Staiva L.) plants from agriculturally important Indian and Japanese varieties by means of charged particles to facilitate entry of exogenous DNA into immature zygotic embryos, (1991) Vol. 9 pp. 957-962 Cheng et al., along Embryogenic tissue liquid phase trauma with emery to successfully transform papaya ringspot virus coat protein gene into papaya through Agrobacterium, "Plant Cell Reports" (Plant Cell Reports) (1996) Volume 16 pp.127-132Corey et al., "United States Proceedings of the National Academy of Sciences (Proc.Natl.Acad.Sci.) 90:11628-11632 (1993) Coruzzi et al. Expression of the pea L. nuclear gene encoding the ribulose-1,5-bisphosphate small subunit is tissue-specific Sex and light regulation, "European Molecular Biology Organization Journal" (EMBO J.) (1984) Volume 3 pp.1671-1679 Coste et al., "Proc. .643-647 (1987) De Kathen and Jacobsen, Transformation of pea by Agrobacterium tumefaciens with binary vectors and coinciding vectors, Plant Cell Reports (1990) vol. 9 pp.276- 279 Fletcher, RA et al., 1988, in: D. Berg et al., (Eds.) Inhibitors of Sterol Biosynthesis—Pharmacological and Agrochemical Aspects, Ellis Horwood, pp 321-331 Fraley et al., Expression of Bacterial Genes in Plant Cells, " Proceedings of the National Academy of Sciences of the United States of America (Proc.Natl.Acad.Sci.) (1983) Vol. 80 pp.4803-4807 Fromm et al., UCLA Monograph on Molecular Level Strategies for Crop Improvement, April 16-22. Keystone, CO. (1990 ) Gachotte et al., The Plant Journal 9:391-398 (1996) Gibson and Shillitone, Ribozymes, their function and strategies for use, Molecular Biotechnology 7(2):125 -37 (1997) Gordon-Kamm et al., Transformation of maize cells and regeneration of fertile transgenic plants, Plant cell, Vol. 2, pp603-618 (1990) Guo et al., Sterol synthesis in maize Developmental regulation of phytosterols, "Lipids" 30:3, p.203-219 (1995) Guo et al., Stereochemistry of hydrogen atom transfer from C-24 to C-25 during phytosterol biomethylation, " Journal of the American Chemical Association (J.Am.Chem.Soc.) 118, pp8507-8508 (1996) Guo et al., Biosynthesis of phytosterols: related to the biomethylation reaction to generate 24-alkenosterol isomers isotope effect, "Tetrahedron letters" (Tetrahedron letters) 37:38, pp.6823-6826 (1996) Grabenok et al., "Plant Mol.Biol.) 34: 891-896 (1997) Haeuser , C. et al., 1990 "Journal of Plant Physiology" (J.Plant Physiol.) 137:201-207Hinchee et al., Plant Cell and Tissue Culture (1994), pp.231-271, Vasil and Thorpe (eds.), Kluwer Academic PublishersHorn etc., from the transgenic plant of orchard grass duckweed generated from protoplasts, "Plant Cell Reports" (Plant Cell Reports) (1988) the seventh volume pp.469-472Husselstein etc., "European Biochemical Association Communications" ( FEBS Letters) 381:87-92 (1996) Koziel et al., Transgenic Maize for Control of Corn Borer, Abstract, ACS Annual Meeting, Chicago, II, August, 22-27, 1993. (1993) Li, S., S. Stereochemical studies of sterol metabolism by Cervesiae, MSThesis, TexasTech University (1996) pp.1-123 Medberry and Olszewski, Identification of cis-elements associated with Commelinas yellow mottle virus promoter activity, Plant Journal (1993) Vol. 3 pp.619-626 Moffatt et al., the gene encoding adenine phosphoribosyltransferase of Arabidopsis thaliana, "Gene" (Gene) (1994) Vol. 143 pp.211-216 Nes et al., (S)- Structural conditions required for substrate conversion of adenosyl-L-methionine: Δ ²⁴⁽²⁵⁾ -sterol methyltransferase, J.Bio.Chem. 266:23, pp.15202 -15212(1991) Nes et al., "Biochemistry and Biophysics Documents" Arch.Biochem Biophys.300, pp.724-733(1993) Nes et al., Utilization and Metabolism of Sterols by Spodoptera zea, "Lipids" ( Lipids) (1997) Vol. 32 No. 11 Ni et al., Length and tissue specificity of chimeric promoters from octopine and mannopine synthase genes, "Plant J." (1995) No. Vol. 7 pp.661-676 Odell et al. Identification of the DNA sequence determining the activity of the cauliflower mosaic virus 35S promoter, "Nature" (Nature) (1985) Vol. 313 pp.810-812 Ritchie and Hodges, in: Transgenic Plants (1993 ), Vol. 1, pp.147-178, Shain-Dow Kung (Ed.), Academic Press Inc. Rhodes et al., "Science" (Science) (1988) Vol. 240 p.204 Sanger et al., from "Scrophulariaceae Characterization of the strong promoter of Figwort mosaic virus: comparison with analogues of the cauliflower mosaic virus 35S promoter and the regulatable mannopine synthase promoter. "Plant Molecular Biology" (Plant Mol. Biol.) (1990) Vol. 14 pp.433-443 Sambrook, J., EFFritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Guide, Second Edition, (Cold Spring Laboratory) Cold Spring Harbor, New York Schroeder, etc., Transformation and regeneration of two cultivated varieties of pea, "Plant Physiol" (Plant Physiol) (1993) Vol. Function, "Symposia of the Society for Experimental Biology" (Symposia of the Society for Experimental Biology) 45: 117-27, 1991Schuler et al., "Nucleic Acids Res. (1982) Vol. 10 pp.8225-8244Shi et al. , "Journal of Biochemistry" (J.Bio.Chem.) (1996) 271: 9384-9389 Somers et al., "Bio/Technology" (Bio/Technology) (1992) Vol. 10 pp.1589-1594Steponkus, PL1984, "Plant Physiology Review Yearbook" (Annu.Rev.PlantPhysiol.) 35:543-584Tong et al., "tetrahedron letters" (tetrahedron letters) 38:35, pp.6115-6118 (1997) Toriyama et al., "Bio/Technology" (Bio /Technology) (1988) Vol. 6 p.10 Vasil et al., Bio/Technology (1992) Vol. 10 p.667-674 Venkatramesh et al., Biochimica Biophysica Acta 1299 (1996) 313-324 Wan and Lemaux, "Plant Physiol.) (1994) Vol. 104 pp.37-48Wang et al., "Bio/Technology" (1992) Vol. 10 p. 691Weeks et al., "Plant Physiol." (Plant Physiol.) (1993) Volume 102 pp.1077-1084Xu et al. The 5' sequence of rice triose phosphate isomerase gene controls the activity of β-glucuronidase in transgenic tobacco But it needs an intron when expressing in rice, "Plant Physiology" (Plant Physiology) (1994) Volume 106 pp.459-467Yang et al., "Plant Cell Reports" (Plant Cell Reports) 15: 459-464 ( 1996) Zhong et al., Functional activity analysis of a 1.4kb fragment of the 5' region of rice actin 1 gene in stable transgenic maize (Zea mays L.), Plant Science (1996) vol. 116 pp. 73-84

sequence listing

(1) General information:

(i) Applicant: NES, DAVID W.

(ii) Title of the invention: Transgenic plants with altered sterol components

(iii) Number of sequences: 6

(iv) Correspondence address:

(A) Recipient: ARNOLD, WHITE & DURKEE

(B) Street: P.O.BOX 4433

(C) City: HOUSTON

(D) State: TX

(E) Country: USA

(F) Zip code: 77210-4433

(v) in computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: Patent In Release#1.0, Version #1.30

(vi) The application materials:

(A) Application number: US 60/033,923

(B) Application date: 1996.12.26

(C) Classification:

(viii) Lawyer/Representative Information:

(A) Name: KAMMERER,

PATRICIA A.

(B) Registration number: 29,775

(C) Reference/registration number: MOBT148(ix) Electronic communication information:

(A) Tel: 713/787-1400

(B) Teletype: 713/787-1440 (2) SRQ ID NO: 1 Information (i) Serial characteristics

(A) Length: 1320 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D)拓扑结构：线型(xi)SRQ ID NO：1序列图：TTACTTTCGA TTTAAGTTTT ACATAATTTA AAAAAACAAG AATAAAATAA TAATATAGTA60GGCAGCATAA GATGAGTGAA ACAGAATTGA GAAAAAGACA GGCCCAATTC ACTAGGGAGT120TACATGGTGA TGATATTGGT AAAAAGACAG GTTTGAGTGC ATTGATGTCG AAGAACAACT180CTGCCCAAAA GGAAGCCGTT CAGAAGTACT TGAGAAATTG GGATGGTAGA ACCGATAAAG240ATGCCGAAGA ACGTCGTCTT GAGGATTATA ATGAAGCCAC ACATTCCTAC TATAACGTCG300TTACAGATTT CTATGAATAT GGTTGGGGTT CCTCTTTCCA TTTCAGCAGA TTTTATAAAG360GTGAGAGTTT CGCTGCCTCG ATAGCAAGAC ATGAACATTA TTTAGCTTAC AAGGCTGGTA420TTCAAAGAGG CGATTTAGTT CTCGACGTTG GTTGTGGTGT TGGGGGCCCA GCAAGAGAGA480TTGCAAGATT TACCGGTTGT AACGTCATCG GTCTAAACAA TAACGATTAC CAAATTGCCA540AGGCAAAATA TTACGCTAAA AAATACAATT TGAGTGACCA AATGGACTTT GTAAAGGGTG600ATTTCATGAA AATGGATTTC GAAGAAAACA CTTTCGACAA AGTTTATGCA ATTGAGGCCA660CATGTCACGC TCCAAAATTA GAAGGTGTAT ACAGCGAAAT CTACAAGGTT TTGAAACCGG720GTGGTACCTT TGCTGTTTAC GAATGGGTAA TGACTGATAA ATATGACGAA AACAATCCTG780AACATAGAAA GATCGCTTAT GAAATTGAAC TAGGTGATGG TATCCCAAAG ATGTTCCATG840TCGACGTGGC TAGGAAAGCA TTGAAGAACT GTGGTTTCGA AGTCCTCGTT AGCGAAGACC900TGGCGGACAA TGATGATGAA ATCCCTTGGT ATTACCCATT AACTGGTGAG TGGAAGTACG960TTCAAAACTT AGCTAATTTG GCCACATTTT TCAGAACTTC TTACTTGGGT AGACAATTTA1020CTACAGCAAT GGTTACTGTA ATGGAAAAAT TAGGTCTAGC CCCAGAAGGT TCCAAGGAAG1080TTACTGCTGC TCTAGAAAAT GCTGCGGTTG GTTTAGTTGC CGGTGGTAAG TCCAAGTTAT1140TCACTCCAAT GATGCTTTTC GTCGCTAGGA AGCCAGAAAA CGCCGAAACC CCCTCCCAAA1200CTTCCCAAGA AGCAACTCAA TAAATTCACT AGATCAATAA GATTCAAATA AAGCGCACGA1260TATATACCTA TTTTCCTATA TATGCAGATA AAAAGATAGC ACGTTCATTG CTAGCAGGCC1320(2)SRQ ID NO：2的信息(i)序列特征

(A) Length: 383 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Line type (XI) SRQ ID NO: 2 serial chart: Met Ser Glu ThR Glu Leu ARG LYS ARG Gln Ala Gln PHR ARG GLU1 5 10 15LEU HIS GLE GLY LYS LYS LYS THR GLY Leu Ser Ala Leu Met

20 25 30Ser Lys Asn Asn Ser Ala Gln Lys Glu Ala Val Gln Lys Tyr Leu Arg

35 40 45Asn Trp Asp Gly Arg Thr Asp Lys Asp Ala Glu Glu Arg Arg Leu Glu

50 55 60ASP TYR Asn Glu Ala Thr His Ser Tyr Tyr ASP PHE65 70 75 80thR GLU GLY TRP GLY SE PHE HIS PHE PHE TYR LYSS

85 90 95Gly Glu Ser Phe Ala Ala Ser Ile Ala Arg His Glu His Tyr Leu Ala

100 105 110Tyr Lys Ala Gly Ile Gln Arg Gly Asp Leu Val Leu Asp Val Gly Cys

115 120 125Gly Val Gly Gly Pro Ala Arg Glu Ile Ala Arg Phe Thr Gly Cys Asn

130 135 140VAL Ile Gly Leu asn Asn Asn Asp Tyr Gln Ile Ala Lys Ala Lys Ala Lys Tyr145 150 155 160ty Ala Lys Lysn Leu Ser as Met ASP PHE VAL LYS GLY

165 170 175Asp Phe Met Lys Met Asp Phe Glu Glu Asn Thr Phe Asp Lys Val Tyr

180 185 190Ala Ile Glu Ala Thr Cys His Ala Pro Lys Leu Glu Gly Val Tyr Ser

195 200 205Glu Ile Tyr Lys Val Leu Lys Pro Gly Gly Thr Phe Ala Val Tyr Glu

210 215 220TRP Val Met THR ASP LYS TYR ASP GLU ASN Pro Glu His ARG LYS225 230 235 240ile Ala Tyr Glu Leu Leu Gly Ile Pro Lys Met Phes Met Phes Met Phes Met PHE PHE HIS

245 250 255Val Asp Val Ala Arg Lys Ala Leu Lys Asn Cys Gly Phe Glu Val Leu

260 265 270Val Ser Glu Asp Leu Ala Asp Asn Asp Asp Glu Ile Pro Trp Tyr Tyr

275 280 285Pro Leu Thr Gly Glu Trp Lys Tyr Val Gln Asn Leu Ala Asn Leu Ala

290 295 300thr Phe Phe ARG THR Leu Gly ARG Gln PHR THR THR Ala Met305 315 320val THR Val Met Glu Gly Leu GLU GLU GLY Ser Lys Glu

325 330 335Val Thr Ala Ala Leu Glu Asn Ala Ala Val Gly Leu Val Ala Gly Gly

340 345 350Lys Ser Lys Leu Phe Thr Pro Met Met Leu Phe Val Ala Arg Lys Pro

355 360 365Glu Asn Ala Glu Thr Pro Ser Gln Thr Ser Gln Glu Ala Thr Gln

370 375 380(2) Information of SRQ ID NO: 3 (i) sequence characteristics

(A) Length: 1420 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D)拓扑结构：线型(xi)SRQ ID NO：3序列图：CTCTCTCTCT CTCTCTCTTG GTCTTCCTCA CTCTTAACGA AAATGGACTC TTTAACACTC60TTCTTCACCG GTGCACTCGT CGCCGTCGGT ATCTACTGGT TCCTCTGCGT TCTCGGTCCA120GCAGAGCGTA AAGGCAAACG AGCCGTAGAT CTCTCTGGTG GCTCAATCTC CGCCGAGAAA180GTCCAAGACA ACTACAAACA GTACTGGTCT TTCTTCCGCC GTCCAAAAGA AATCGAAACC240GCCGAGAAAG TTCCAGACTT CGTCGACACA TTCTACAATC TCGTCACCGA CATATACGAG300TGGGGATGGG GACAATCCTT CCACTTCTCA CCATCAATCC CCGGAAAATC TCACAAAGAC360GCCACGCGCC TCCACGAAGA GATGGCGGTA GATCTGATCC AAGTCAAACC TGGTCAAAAG420ATCCTAGACG TCGGATGCGG TGTCGGCGGT CCGATGCGAG CGATTGCATC TCACTCGCGA480GCAACGTAGT CGGGATTACA ATAAACGAGT ATCAGGTGAA CAGAGCTCGT CTCCACAATA540AGAAAGCTGG TCTCGACGCG CTTTGCGAGG TCGTGTGTGG TAACTTCCTC CAGATGCCGT600TCGATGACAA CAGTTTCGAC GGAGCTTATT CCATCGAAGC CACGTGTCAC GCGCCGAAGC660TGGAAGAAGT GTACGCAGAG ATCTACAGGG TGTTGAAACC CGGATCTATG TATGTGTCGT720ACGAGTGGGT TACGACGGAG AAATTTAAGG CGGAGGATGA CGAACACGTG GAGGTAATCC780AAGGGATTGA GAGAGGCGAT GCGTTACCAG GGCTTAGGGC TTACGTGGAT ATAGCTGAGA840CGGCTAAAAA GGTTGGGTTT GAGATAGTGA AGGAGAAGGA TCTGGCGAGT CCACCGGCTG900AGCCGTGGTG GACTAGGCTT AAGATGGGTA GGCTTGCTTA TTGGAGGAAT CACATTGTGG960TTCAGATTTT GTCAGCGGTT GGAGTTGCTC CTAAAGGAAC TGTTGATGTT CATGAGATGT1020TGTTTAAGAC TGCTGATTGT TTGACCAGAG GAGGTGAAAC CGGAATATTC TCTCCGATGC1080ATATGATTCT CTGCAGAAAA CCGGAGTCAC CGGAGGAGAG TTCTTGAGAA AGGTAGAAAG1140GAAACATCAC CGGAAAAAGT ATGGAGAATT TTCTCAATTT GTTTTTATTT TTAAGTTAAA1200TCAACTTGGT TATTGTACTA TTTTTGTGTT TTAATTTGGT TTGTGTTTCA AGAATTATTA1260GTTTTTTTTT GTTTTGTTGC ATATGAGAAT CTTACTCTTG ATTTCTCCGC CGTAGAGCCG1320GCGAGACATA GGGGATTATT AGTATTTTTA AGTGTGTTTA AGATTGATTA ACAAGTTAGT1380AAAATAAAAT GTACTTAGGT GTCGAAAAAA AAAGGAATTC1420(2)SRQ ID NO：4的信息(i ) sequence feature

(A) Length: 361 amino acids

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Line type (XI) SRQ ID NO: 4 Sequences: MET Asp Sero Thr Leu PHE PHE Thr Leu Val Ala Val Gly1 5 10 15ile Trp Phe Leu Gly Pro Ala Glu ARG Lys Gly Lys

20 25 30Arg Ala Val Asp Leu Ser Gly Gly Ser Ile Ser Ala Glu Lys Val Gln

35 40 45Asp Asn Tyr Lys Gln Tyr Trp Ser Phe Phe Arg Arg Pro Lys Glu Ile

50 55 60GLU Thr Ala Glu Lys Val Val Pro ASP PHE VAL ASP ThR PHE TYR Asn Leu65 70 75 80VAL THR GLU TRP GLY Gln Ser Phe His Phe Ser

85 90 95Pro Ser Ile Pro Gly Lys Ser His Lys Asp Ala Thr Arg Leu His Glu

100 105 110 Glu Met Ala Val Asp Leu Ile Gln Val Lys Pro Gly Gln Lys Ile Leu

115 120 125Asp Val Gly Cys Gly Val Gly Gly Pro Met Arg Ala Ile Ala Ser His

130 135 140SER ARA Asn Val Val Gly Ile THR Ile Asn Gln Val ASN145 150 155RG Ala ARG Leu His Lys Lys Ala Gly Leu Ala Leu Cys Glu Glu Glu Glu

165 170 175Val Val Cys Gly Asn Phe Leu Gln Met Pro Phe Asp Asp Asn Ser Phe

180 185 190Asp Gly Ala Tyr Ser Ile Glu Ala Thr Cys His Ala Pro Lys Leu Glu

195 200 205Glu Val Tyr Ala Glu Ile Tyr Arg Val Leu Lys Pro Gly Ser Met Tyr

210 215 220val Ser Tyr Glu TRP Val THR THR GLU LYS PHE LYS Ala Glu ASP ASP 2225 235 240GLU HIS Val GLN GLN GLU ARG GLY Ala Leu Prou Pro

245 250 255Gly Leu Arg Ala Tyr Val Asp Ile Ala Glu Thr Ala Lys Lys Val Gly

260 265 270Phe Glu Ile Val Lys Glu Lys Asp Leu Ala Ser Pro Pro Ala Glu Pro

275 280 285Trp Trp Thr Arg Leu Lys Met Gly Arg Leu Ala Tyr Trp Arg Asn His

290 295 300ile Val Val Val Gln Ile Leu Sering Val Gly Val Ala Pro Lys Gly ThR30 315 320val ASP Val His Glu Met Leu PHE LYS Leu ThR ARGR ARGR ARGR ARGR ARG

325 330 335Gly Gly Glu Thr Gly Ile Phe Ser Pro Met His Met Ile Leu Cys Arg

340 345 350Lys Pro Glu Ser Pro Glu Glu Ser Ser

                                360 (2) SRQ ID NO: 5 information (i) sequence characteristics

(A) Length: 1320 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D)拓扑结构：线型(xi)SRQ ID NO：5序列图：TTACTTTCGA TTTAAGTTTT ACATAATTTA AAAAAACAAG AATAAAATAA TAATATAGTA60GGCAGCATAA GATGAGTGAA ACAGAATTGA GAAAAAGACA GGCCCAATTC ACTAGGGAGT120TACATGGTGA TGATATTGGT AAAAAGACAG GTTTGAGTGC ATTGATGTCG AAGAACAACT180CTGCCCAAAA GGAAGCCGTT CAGAAGTACT TGAGAAATTG GGATGGTAGA ACCGATAAAG240ATGCCGAAGA ACGTCGTCTT GAGGATTATA ATGAAGCCAC ACATTCCTAC TATAACGTCG300TTACAGATTT CTATGAATAT GGTTGGGGTT CCTCTTTCCA TTTCAGCAGA TTTTATAAAG360GTGAGAGTTT CGCTGCCTCG ATAGCAAGAC ATGAACATTA TTTAGCTTAC AAGGCTGGTA420TTCAAAGAGG CGATTTAGTT CTCGACGTTG GTTGTGGTGT TGGGGGCCCA GCAAGAGAGA480TTGCAAGATT TACCGGTTGT AACGTCATCG GTCTAAACAA TAACGATTAC CAAATTGCCA540AGGCAAAATA TTACGCTAAA AAATACAATT TGAGTGACCA AATGGACTTT GTAAAGGGTG600ATTTCATGAA AATGGATTTC GAAGAAAACA CTTTCGACAA AGTTTATGCA ATTGAGGCCA660CATGTCACGC TCCAAAATTA GAAGGTGTAT ACAGCGAAAT CTACAAGGTT TTGAAACCGG720GTGGTACCTT TGCTGTTTAC GAATGGGTAA TGACTGATAA ATATGACGAA AACAATCCTG780AACATAGAAA GATCGCTTAT GAAATTGAAC TAGGTGATGG TATCCCAAAG ATGTTCCATG840TCGACGTGGC TAGGAAAGCA TTGAAGAACT GTGGTTTCGA AGTCCTCGTT AGCGAAGACC900TGGCGGACAA TGATGATGAA ATCCCTTGGT ATTACCCATT AACTGGTGAG TGGAAGTACG960TTCAAAACTT AGCTAATTTG GCCACATTTT TCAGAACTTC TTACTTGGGT AGACAATTTA1020CTACAGCAAT GGTTACTGTA ATGGAAAAAT TAGGTCTAGC CCCAGAAGGT TCCAAGGAAG1080TTACTGCTGC TCTAGAAAAT GCTGCGGTTG GTTTAGTTGC CGGTGGTAAG TCCAAGTTAT1140TCACTCCAAT GATGCTTTTC GTCGCTAGGA AGCCAGAAAA CGCCGAAACC CCCTCCCAAA1200CTTCCCAAGA AGCAACTCAA TAAATTCACT AGATCAATAA GATTCAAATA AAGCGCACGA1260TATATACCTA TTTTCCTATA TATGCAGATA AAAAGATAGC ACGTTCATTG CTAGCAGGCC1320(2)SRQ ID NO：6的信息(i)序列特征

(A) Length: 1497 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: Line type (ix) Features:

(A) Name/key: modified_base

(B) Location: 1419

(C) Other information: /modified_base=other/comment="A or C

或G或T”(xi)SRQ ID NO：6序列图：AGACTCTGGT TCTGACATGC AGCAATTATT GCAGGTGCAT TTGATCCGTC CCGGCCGCCT60ACACGATGTC CAAGTCGGGA GCGCTGGATC TTGCTTCTGG CCTCGGAGGG AAGATCAACA120AGGTGGAAGT CAAGTCGGCC GTCGATGAGT ATGAGAAATA TCATGGATAC TATGGAGGGA180AGGAGGAAGC AAGGAAGTCC AACTATACTG ATATGGTTAA TAAATACTAT GATCTTGCCA240CTAGCTTCTA TGAGTATGGT TGGGGTGAAT CCTTCCACTT TGCTCACAGA TGGAATGGAG300AATCCTTACG TGAAAGCATC AAGCGACATG AGCATTTTCT TGCCCTGCAA CTTGGTTTGA360AACCAGGAAT GAAGGTTTTA GATGTGGGCT GTGGAATAGG TGGACCACTG AGAGAAATTG420CAAGATTTAG CTCAACTTCA GTTACCGGAT TGAATAACCA CGAATACCAG ATAACCAGGG480GAAAGGAGCT CAACCGTTTA GCAGGAATTA GTGGAACATG TGATTTTGTC AAGGCGGACT540TCATGAAGAT GCCGTTCGAT GACACACTTT TGGATGCTGT TTACGCCATT GAGGCAACAT600GTCATGCACC TGATCCAGTT GGTTGCTACA AGGAGATATA TCGTGTGTTG AAGCCTGGCC660AGTGCTTTGC CGTGTACGAG TGGTGCGTTA CGGATCACTA TGATCCTAAC AATGCAACCC720ACAAAAGGAT CAAGGATGAA ATTGAGCTTG GCAATGGCCT GCCAGATATC AGAAGCACTC780CGCAATGTCT CCGGGCTCTA AAAGACGCCG GGTTTGACGT TGTTTGGGAT AAGGATCTTG840CTGAAGATTC TCCCTTGCCT TGGTACTTGC CCTTGGACTC CAGCCGATGC TCACTGAGTA900GCTTCCGTCG ACCTCCTGTC GGGACGCATG ATACCCGCAC AATGGTCAAG GCCCTGGAGT960ACGTTGGTCT TGCTCCGCAG GGCAGTGAGA GGTCTCTAGT TTTCCTGGAG AAGGCTGCAG1020AAGGGCTGGT AGAGGGCGGA AAGAAGGAGA TCTTCACGCC AATGTACTTC TTTTTTGTTC1080GGAAGCCTCT TCTGGAATGA GCTCTTGGAT CACCTTTTCA GAGAGAGAAG GCAAGTGGTC1140ATTTCGAAGA AGCCGAGGAG AGGGAACCTG GAATCAAGAA AACCTTCAGC TCTCCTGTGT1200AGGAGGAAAG TTAACGAACA GTGTAGTAAC TGTTCAGCTC TGTGTTTATT CAGTTGTTTT1260GCTGCTTGAG GTTATTCGTT TCTAGGTGGG GGTTGGAATC CTTTTCGCCA TAAACCTCTC1320AGTGGCATAA ATAAGATGGT TTGCATAAGA GTACTTCATG GATACCGTAA GGGCTACTAC1380TGAAAGAGAA ATGTTTAAGC AGCATGGTAT GTGAGCAANT AGTGATAATT ATTCCATCCT1440TTTTTTTAAT ATAAAGCAGG AGTTTTGTCA AAAAAAAAAA AAAAAAAAAA AAAAAAA1497

Claims (40)

1. a double chain DNA molecule comprises:

A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to

The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to

One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic.

2. the dna molecular in the claim 1, wherein said dna encoding sequence is a sense orientation.

3. the dna molecular in the claim 1, wherein said dna encoding sequence is an antisense orientation.

4. the dna molecular in the claim 1, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ ⁸-sterol, 14 Alpha-Methyl sterols, Δ ²³, 24-alkyl sterol, Δ ²⁴, 24-alkyl sterol and Δ ^{25 (27)}, 24-alkyl sterol.

5. the dna molecular in the claim 1, wherein said elementary sterol or secondary sterol lack Δ ⁵Group.

6. the dna molecular in the claim 1, the enzyme of wherein said dna encoding sequence encoding is selected from S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ ⁸To Δ ⁷-isomerase, Δ ⁷-C-5-desaturase and 24, the 25-reductase enzyme.

7. the dna molecular in the claim 1, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase (SMT).

8. according to the DNA in the claim 7, wherein said SMT is from plant or yeast.

9. according to the DNA in the claim 7, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.

10. according to the DNA in the claim 7, wherein said SMT is yeast ERG6.

11. transgenic plant that contain a double chain DNA molecule comprise:

A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to

The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to

One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic.

12. the plant in the claim 11, wherein said dna encoding sequence is a sense orientation.

13. the plant in the claim 11, wherein said dna encoding sequence is an antisense orientation.

14. the plant in the claim 11, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ ⁸-sterol, 14 Alpha-Methyl sterols, Δ ²³, 24-alkyl sterol, Δ ²⁴, 24-alkyl sterol and Δ ^{25 (27)}, 24-alkyl sterol.

15. the plant in the claim 11, wherein said elementary sterol or secondary sterol lack Δ ⁵Group.

16. the plant in the claim 11, the enzyme of wherein said dna encoding sequence encoding are selected from S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ ⁸To Δ ⁷-isomerase, Δ ⁷-C-5-desaturase and 24, the 25-reductase enzyme.

17. the plant in the claim 11, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase (SMT).

18. the plant in the claim 17, wherein said SMT is from plant or yeast.

19. the plant in the claim 17, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.

20. the plant in the claim 17, wherein said SMT is yeast ERG6.

21. the plant in the claim 11, this plant can be resisted insect, nematode or rotten mould fungi.

22. the plant in the claim 11, this plant has been improved the level of the sterol of norcholesterol.

23. the plant in the claim 22, wherein said sterol are cycloartenol or Sitosterol.

24. according to the plant in the claim 11, this plant can be resisted arid, high salt or severe cold.

25. according to the plant in the claim 11, this plant is tomato, corn or soybean.

26. a method of producing transgenic plant comprises:

(a) with the recombinant DNA molecules transformed plant cells that comprises following composition:

A promotor, it causes the generation of RNA sequence in the plant materials, and is operably connected to

The section of DNA encoding sequence, its coded endonuclease capable is in conjunction with elementary sterol and produce secondary sterol, and this sequence is operably connected to

One 3 ' end non-translational region, it causes RNA sequence 3 ' terminal polyadenylation; Wherein said promotor for described dna sequence dna for allogenic;

(b) screening contain recombinant DNA molecules conversion vegetable cell; And

(c) become transgenic plant by the vegetable cell regeneration that has transformed.

27. the method in the claim 26, wherein said dna encoding sequence is a sense orientation.

28. the method in the claim 26, wherein said dna encoding sequence is an antisense orientation.

29. the method in the claim 26, wherein said elementary sterol is selected from 4-methylsterol, 9 β, 19-cyclopropyl sterol, Δ ⁸-sterol, 14 Alpha-Methyl sterols, Δ ²³, 24-alkyl sterol, Δ ²⁴, 24-alkyl sterol and Δ ^{25 (27)}, 24-alkyl sterol.

30. the method in the claim 26, wherein said elementary sterol or secondary sterol lack Δ ⁵Group.

31. the method in the claim 26, the enzyme of wherein said dna encoding sequence encoding are selected from S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14 Alpha-Methyl demethylases, Δ ⁸To Δ ⁷-isomerase, Δ ⁷-C-5-desaturase and 24, the 25-reductase enzyme.

32. the method in the claim 26, wherein said dna encoding sequence encoding S-adenosine-L-methionine(Met)-Δ ^{24 (25)}-sterol methyltransgerase (SMT).

33. the method in the claim 32, wherein said SMT is from plant or yeast.

34. the method in the claim 32, wherein said SMT is from corn, mouseearcress or Prototheca wickerhamii.

35. the method in the claim 32, wherein said SMT is yeast ERG6.

36. the method in the claim 26, wherein said plant can be resisted insect, nematode or rotten mould fungi.

37. the method in the claim 26, wherein said plant has been improved the level of the sterol of norcholesterol.

38. the method in the claim 37, wherein said sterol are cycloartenol or Sitosterol.

39. the method in the claim 26, wherein said plant can be resisted arid, high salt or severe cold.

40. the method in the claim 26, wherein said plant are tomato, corn or soybean.

Images