EP0958370A1 - Transgenic plants with modified sterol biosynthetic pathways - Google Patents

Transgenic plants with modified sterol biosynthetic pathways

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Publication number
EP0958370A1
EP0958370A1 EP97953327A EP97953327A EP0958370A1 EP 0958370 A1 EP0958370 A1 EP 0958370A1 EP 97953327 A EP97953327 A EP 97953327A EP 97953327 A EP97953327 A EP 97953327A EP 0958370 A1 EP0958370 A1 EP 0958370A1
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Prior art keywords
sterol
plant
smt
dna
methyl
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German (de)
English (en)
French (fr)
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W. David Nes
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Monsanto Technology LLC
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Monsanto Co
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention broadly relates to plant genetic engineering. More particularly, it concerns the manipulation of the levels and/or activities of endogenous plant phytosterol compositions as a strategy for minimizing crop damage due to plant insects and other pests, and/or for improving the nutritional value of plants.
  • Sterols comprise a class of essential natural compounds required to some extent by all eukaryotic organisms. They have a common tetracyclic steroid nucleus and a side chain, as shown in the diagram below. Some sterols serve a structural role in cell membranes, while others are required during development.
  • Plants produce more than 250 different phytosterols (Akisha et al., 1992). As many as 60 sterols have been identified in the single species, Zea mays (corn) (Guo et al., 1995). However, insects, fungi and nematodes, as well as many other sterol-less parasitic organisms, do not synthesize all of their necessary sterols de novo. Rather, they satisfy their nutritional requirements for sterols by feeding on plants. This fact has been utilized in the development of commercial agrochemicals such as triazoles, pyrimidines and azasterols, which act by interfering with production of sterols within parasitic organisms.
  • the present invention broadly relates to approaches for genetically engineering plants to have altered sterol compositions, levels and/or metabolism. Such approaches can increase the plants natural insect resistance, can increase the plants resistance to drought and cold, and/or can improve the nutritional/health value of the plants.
  • recombinant DNA molecules comprising: a promoter which functions in plants to cause the production of an RNA sequence, operably linked to a DNA coding sequence encoding an enzyme which binds a first sterol and produces a second sterol, operably linked to a 3' non-translated region which causes the polyadenylation of the 3' end of the RNA sequence; wherein the promoter is heterologous with respect to the DNA sequence.
  • the DNA coding sequence encoding an enzyme which binds a first sterol and produces a second sterol can be in the sense or antisense orientation.
  • the DNA molecule of the invention can encode a non-translatable RNA molecule (e.g., antisense or cosuppression) or a protein molecule.
  • the RNA or protein so produced selectively targets the expression and/or activity of a sterol biosynthetic enzyme to affect a desired change in the phytosterol profile of the plant.
  • an approach for modifying the sterol composition of plants to increase their resistance to insects, nematodes, and pythiaceous fungi enhances the plant's ability to resist pests and disease by modifying the composition and/or distribution profile of certain phytosterols.
  • Such an approach overcomes many of the limitations inherent in the use of agrochemicals, or with transgenic plants where the foreign product introduced into the plant has the potential to eventually select for new mechanisms of resistance by the pest.
  • the present invention retains the benefits obtained through the use of agrochemicals, but avoids many of their disadvantages. By targeting an existing essential pathway in pests and pathogens, this invention reduces the likelihood of the evolution of mechanisms which circumvent this pathway.
  • Plant sterol composition is modified in this aspect by increasing the amount of non- utilizable sterols such as 4-methyl sterol, 9 ⁇ ,19-cyclopropyl sterol, ⁇ 7 -sterol, ⁇ 8 -sterol, 14 ⁇ -methyl sterol, ⁇ 23(24) -24-alkyl sterol, ⁇ 24(25) ,24-alkyl sterol or ⁇ 25(27) ,24-alkyl sterol.
  • sterol compositions can be modified to contain lower levels of sterols having a ⁇ 5 group.
  • Another aspect of the present invention relates to producing sterols in plants that confer resistance to drought and cold in plants.
  • Another aspect of the invention relates to altering the sterol profile of plants such that levels of cholesterol-lowering sterols are increased.
  • sterolic enzymes preferably S-adenosyl-L-methionine- ⁇ 24 -sterol methyl transferases (SMT T and SMT ⁇ ), C-4 demethylase, cycloeucalenol to obtusifoliol-isomerase, 14 ⁇ -methyl demethylase, ⁇ 8 to ⁇ 7 -isomerase, ⁇ 7 -sterol-C-5- desaturase, or 24,25-reductase.
  • SMT T and SMT ⁇ S-adenosyl-L-methionine- ⁇ 24 -sterol methyl transferases
  • C-4 demethylase cycloeucalenol to obtusifoliol-isomerase
  • 14 ⁇ -methyl demethylase ⁇ 8 to ⁇ 7 -isomerase
  • ⁇ 7 -sterol-C-5- desaturase or 24,25-reductase.
  • Another aspect of the invention is directed to transgenic plants having altered levels of selected sterols, produced by introducing recombinant DNA molecules of the invention into the genome of plant cells and selecting for cells expressing said molecule.
  • Transgenic plants are regenerated from the transformed plant cells and plants containing the recombinant DNA are grown to maturity. Plants expressing the recombinant DNA are identified and those having a desired sterol profile in accordance with the present invention are selected and propagated.
  • Fig. 1 shows HPLC radiocount (panel B) and mass spectrum (panel A) results of testing SMT enzyme with radiolabeled substrate co-factor;
  • Fig. 2 shows six inhibitors used to test the SMT enzyme
  • Fig. 3 shows SMT activity during seedling development
  • Fig. 4 shows the pathway of sterol end-products during development of seedlings
  • Fig. 5 shows the yeast SMT gene sequence (panel B; SEQ ID NO:l) and the deduced amino acid sequence (panel A; SEQ ID NO: 2) with the predicted conserved regions highlighted;
  • Fig. 6 shows the Arabidopsis SMT gene (panel B; SEQ ID NO: 3) and deduced amino acid (panel A; SEQ ID NO: 4) sequences;
  • Fig. 7 shows the ERG6 constructs prepared with pUC18cpexp expression cassette
  • Fig. 8 shows sequences of yeast SMT gene (SEQ ID NO: 5). Underlined sequences are those used as primers for screening genomic DNA from transgenic tomato plants; and
  • Fig. 9 shows structures of plant sterols tested on Heliothis zea and found to be utilizable or non-utilizable.
  • Figure 10 (SEQ ID NO: 6) shows the nucleotide and amino acid sequences of the corn SMT gene.
  • the phytosterol metabolic pathway consists of enzymes that act on the tetracyclic ring nucleus and the side chain.
  • the major pathway in advanced vascular plants starts from cycloartol (I):
  • the number of alternate pathways is sufficiently great to produce as many as 60 or more different sterols in a single plant. These alternate pathways vary according to tissue- and development-specific genetic programs.
  • the major pathway consists of the 12 chemical transformations as follows.
  • reaction 1 the enzyme S-adenosyl-L-methionine-sterol-C-24 methyl transferase -(SMTj) catalyzes the transfer of a methyl group from a cofactor, S-adenosyl-L- methionine, to the C-24 center of the sterol side chain.
  • SMTj the enzyme S-adenosyl-L-methionine-sterol-C-24 methyl transferase -(SMTj) catalyzes the transfer of a methyl group from a cofactor, S-adenosyl-L- methionine, to the C-24 center of the sterol side chain.
  • SMTj the enzyme catalyzes the transfer of a methyl group from a cofactor, S-adenosyl-L- methionine, to the C-24 center of the sterol side chain.
  • SMT ⁇ catalyzes the conversion of cycloartol to a ⁇ 23(24) 24-alkyl sterol, cyclosadol (Guo et al., 1996).
  • Reaction 2 involves a demethylation at C-4. This is the first of several demethylation reactions in the nucleus.
  • 24(28)-Methylenecycloartanol Reaction 3 involves opening the cyclopropyl ring at C-9(10) by the enzyme cycloeucalenol-obtusifoliol isomerase (COI), which also creates a double bond at C-8.
  • COI cycloeucalenol-obtusifoliol isomerase
  • Reaction 4 involves a demethylation at C-14 which removes the methyl group at C-14 and creates a double bond at C-14.
  • Reaction 5 is catalyzed by a ⁇ 14 reductase.
  • Reaction 7 is a second C- methylation of the sterol side chain.
  • the reaction is catalyzed by SMTj, the same enzyme that initiated the major pathway.
  • Reaction 8 involves a C-4 demethylase to generate a 4,4-desmethyl sterol.
  • -10- (citrastadienol) Reaction 9 involves a ⁇ desaturase, producing a double bond at C-5 in the tetracyclic ring.
  • reaction 9 The product of reaction 9 is then transformed in reaction 10 by a ⁇ 7 -reductase by removing the double bond at C-7.
  • Reaction 11 involves a ⁇ 24(28) - to ⁇ 24(25) -isomerase which modifies the side chain. (It is believed that this reaction would have proceeded from the product of reaction 5 if the kinetics were more favorable.)
  • Reaction 12 the ⁇ 24(25) double bond at C-24 is reduced stereoselectively to produce sitosterol (II).
  • SMT T and SMT ⁇ Two different SMT enzymes exist (SMT T and SMT ⁇ ) whose expression depends on the tissue and stage of differentiation. Blades mainly contain 24- ethyl sterols (resulting from the activity of SMT T ), whereas the sheaths contain mainly 24-methyl sterols (VI) (resulting from the activity of SMT ⁇ ). These sterols are the products of the two different SMT enzymes that react with the same starting material, cycloartenol.
  • the first enzyme, SMTi produces ⁇ 24(28) -methylene and the second enzyme produces ⁇ -methyl sterol (V).
  • the first isoform leads to a utilizable sterol (a sterol which can be utilized by insects, pythiaceous fungi, and nematodes to complete their life cycles).
  • the second isoform produces a non-utilizable sterol (a sterol which cannot be utilized by insects, pythiaceous fungi, and nematodes to complete their life cycles). Therefore, one could inhibit expression of the first isoform so as to cause accumulation of the non- utilizable ⁇ 23(24) -methyl sterols.
  • the sterols that accumulate in the tissue contain a double bond at C-23 (VI) and a methyl at C-24.
  • a recombinant DNA molecule of the invention generally comprises a promoter region capable of causing the production of an RNA sequence in plants, a structural DNA sequence, and a 3' non-translated region.
  • promoter region contains a sequence of bases that signals RNA polymerase to associate with the sense and antisense DNA strands and to use the sense strand as a template to make a corresponding strand of mRNA complimentary to the sense DNA strand.
  • This process of mRNA production using a DNA template is commonly referred to as gene "expression” or “transcription” .
  • the promoter is heterologous with respect to the DNA coding sequence.
  • heterologous with respect to a promoter means that the DNA coding sequence of a recombinant DNA molecule of the invention is not derived from the same gene to which the promoter is attached.
  • Promoters may be obtained from a variety of sources, such as plants and plant viruses.
  • the particular promoters selected for use in embodiments of the present invention should preferably be capable of causing the production of sufficient expression to affect the desired change in the sterol distribution profile of the plant.
  • CaMV cauliflower mosaic virus
  • FMV Figwort mosaic virus
  • the sugarcane baciUiform virus promoter (Bouhida et al., 1993), the commelina yellow mottle virus promoter (Medberry and Olsewski 1993), the light-inducible promoter from the small ⁇ subunit of the ribulose-l,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984), the rice cytosolic triosephosphate isomerase (TPI) promoter (Xu et al. 1994), the adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis (Moffatt et al. 1994), the rice actin 1 gene promoter (Zhong et al. 1996), the mannopine synthase and octopine synthase promoters (Ni et al. 1995). All of these promoters have been used to create various types of DNA constructs which have been expressed in plants.
  • Recombinant DNA molecules also typically contain a 5' non-translated leader sequence.
  • This sequence can be derived from the promoter selected to express the gene, and if desired, can be specifically modified so as to increase translation of the mRNA.
  • the 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from synthetic gene sequences.
  • the structural DNA sequence of the recombinant DNA molecule of the invention will cause the desired alteration in the sterol profile of the plant, as discussed further below.
  • the 3' non-translated region of a recombinant DNA molecule of the invention can be obtained from various genes which are expressed in plant cells. For example, the nopaline synthase 3' untranslated region (Fraley et al. 1983), the 3' untranslated region from pea ssRUBISCO (Coruzzi et al. 1994), and the 3' untranslated region from soybean 7S seed storage protein gene (Schuler et al. 1982) are frequently used.
  • the 3' non-translated region of a recombinant DNA molecules contains a polyadenylation signal which functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA.
  • intron sequences are frequently included in recombinant DNA molecules used for producing transgenic plants in order to enhance expression levels.
  • plant introns suitable for expression in plants can include maize hsp70 intron, rice actin 4 intron, maize ADH 1 intron, Arabidopsis SSU intron, Arabidopsis EPSPS intron, petunia EPSPS intron and others known to those skilled in the art.
  • a double stranded DNA molecule of the present invention can be inserted into the genome of a plant by any suitable method.
  • Numerous plant transformation methods have been described, including Agrobacterium-mediated transformation, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, transformation using viruses or pollen, etc.
  • Transformation of monocots using electroporation, particle bombardment, and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved, for example, in asparagus (Bytebier et. (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)), oat (Somers et al. (1992)), orchardgrass (Horn et al. (1988)), rice (Toriyama et al. (1988); Battraw and Hall (1990); Christou et al.
  • a series of phytosterols were tested in insects and many were found to be unable to support insect growth, i.e., were non-utilizable. These sterols included 9,19- cyclopropyl sterols. Furthermore, novel ⁇ (23(24) - and ⁇ 24(25) -alkene and ⁇ 25(27) -alkyl sterols were also determined to be unable to support insect growth and maturation. These were tested in vivo using Heliothis zea (a corn earworm), cultured on synthetic media that was sterol-free with the exception of added test sterols. It was found that if the ratio of utilizable to nonutilizable sterols was 1:9 or less, insects could not undergo normal develop. In fact, even at 1:1 ratios, insect development was adversely affected.
  • insects The metabolism of insects, nematodes and pythiaceous fungi is limited by the availability of major plant sterols. These pests cannot use a sterol with a C-4 methyl group; a 9 ⁇ , 19-cyclopropyl group, or a ⁇ group. Furthermore, nematodes and insects cannot utilize 14- ⁇ methyl-sterols, and some insects, including lepidoptera, diptera and coleoptera, cannot utilize C-24 alkyl sterols with ⁇ 24(25) , ⁇ 23(24) , or ⁇ 25(27) groups for mechanistic reasons. Some insects cannot utilize sterols lacking a ⁇ 5 group. Consequently, elevation of these sterols in plants would provide a detrimental dietary source of sterols for these pests.
  • the DNA molecule of the present invention when expressed in transgemc plants, will cause alterations in the composition/distribution of the sterols present in the plant.
  • the DNA molecule causes the accumulation of sterols that are non-utilizable by insects and other pests, so as to increase the plants resistance to the organisms. This can be accomplished, for example, by a number of approaches, including overexpression, antisense, cosuppression etc.
  • the DNA molecule of the invention will typically target an endogenous gene encoding an enzyme selected from the kinetically favored pathways of sterol biosynthesis.
  • gene expression and/or translation of a sterol biosynthetic enzyme is targeted for inhibition.
  • This inhibition can be achieved, for example, by engineering a DNA molecule of the invention to produce an antisense, ribozyme or cosuppression RNA molecule complementary to an endogenous gene being targeted.
  • Approaches for the targeted inhibition of gene expression are well known to the skilled individual (for reviews, see Bird et al., 1991; Schuch, 1991; Gibson et al., 1997)
  • a preferred target for inhibition is the S-adenosyl-L-methionine- ⁇ 24(25) -sterol methyl transferase (SMT) enzyme.
  • SMT S-adenosyl-L-methionine- ⁇ 24(25) -sterol methyl transferase
  • genes in the phytosterol transformation pathway can also be targeted in this and other embodiments of the invention in order to alter the profile of sterols in transgenic plants.
  • the preferred target will depend on the application, however the approach is the same, i.e., to express an RNA or protein molecule capable of modifying the sterol composition of the plant in a desirable manner.
  • C-4 demethylase This enzyme is involved in the removal of the two methyl groups at C-4 and represents reactions 2 and 8 in the description section. A single protein is responsible for both the reactions. Blocking this enzyme will lead to accumulation of 4,4-dimethyl sterols such as cycloartol, 24(28)-methylene eycloartenol or a novel sterol such as 24-dihydrolanosterol (structure 18 in Fig. 9). All these are nonutilizable sterols. This may be achieved through suppression of this gene in plants.
  • ⁇ 7 -sterol-C-5-desaturase This is reaction 9 in the pathway. Inhibition of this enzyme leads to a depletion of ⁇ 5 -sterols and an increase in ⁇ 7 -sterols. Certain insects are known to be unable to metabolize ⁇ 7 -sterols into ecdy steroids. Therefore, accumulation of ⁇ -sterols in plants can also provide a way to form non-utilizable sterols. Further, ⁇ 7 -sterols can replace ⁇ 5 -sterols in plant membranes without any morphological changes in plant development.
  • C-24 reductase This is the terminal step in phytosterol transformation (reaction -12) during the formation of sitosterol, the major ⁇ 5 -sterol in plants. Disruption or suppression of the gene encoding this enzyme would result in the accumulation of ⁇ 24(25) -24-alkyl sterols which are also non-utilizable.
  • genes encoding these preferred sterol biosynthetic enzymes to be targeted by the present invention have been isolated from yeast (for review, see Lees et al., 1997). Some have been isolated from plants. For example, SMT genes have been isolated from soybean (Shi et al., 1996), arabidopsis (Husselstein et al., 1996; Bouvier- Nave et al., 1997) tobacco and castor (Bouvier-Nave et al., 1997); and corn (Grabenok et al., 1997).
  • the gene encoding a sterol biosynthetic enzyme can be readily isolated from a desired source by approaches known to the skilled individual. For example, an isolated gene or cDNA from one source can be used as a hybridization probe for the isolation of homolgous sequences from other sources.
  • a DNA molecule of the invention should be active in numerous plant types, regardless of the source of the sterol biosynthetic gene used in the targeting construct, given the successful demonstration provided herein of using a yeast ERG6 antisense construct to alter the sterol profile in tomato.
  • the following sterolic metabolic enzymes are targeted for inhibition: S- adenosyl-L-methionine- ⁇ 24 -sterol methyl transferase, C-4 demethylase, cycloeucalenol to obtusifoliol-isomerase, 14 ⁇ -methyl demethylase, ⁇ 8 - to ⁇ 7 -isomerase, ⁇ 7 -sterol-C-5- desaturase, or a 24,25-reductase.
  • Plants produced according to this embodiment preferably have increased amounts of certain sterols that are non-utilizable, particularly 4-methyl sterol, 9 ⁇ ,19-cyclopropyl sterol, ⁇ 8 -sterol, ⁇ 7 -sterol, 14 ⁇ -methyl sterol, ⁇ 23(24) ,24-alkyl sterol, ⁇ 24(25) -24-alkyl •sterol or ⁇ 25(27) -24-alkyl sterol, or decreased levels of sterols having a ⁇ 5 group.
  • Preferred crops for use in providing insect resistance according to this embodiment of the invention include com (European com borer, com earworm, fall armyworm), rice, sorghum, forestry, potato, tomato (tomato hornworm), and vegetable brassicas.
  • Preferred crops for use in providing nematode resistance according to this embodiment of the invention include soybean (soybean cyst nematode), tomato (root knot nematode), sugarbeet and cucurbits.
  • Preferred crops for use in providing fungal resistance include corn, rice, wheat, surghum, soybean (Phytophthora root rot), sunflower, forestry, fruits and berries, potato (late blight), tomato (late blight), sugarbeet, cucurbits, and vegetable brassicas.
  • the present invention in another embodiment, relates to increasing cholesterol- lowering sterols in transgenic plants.
  • a recombinant DNA molecule of the invention the conversion of cycloartol in developing seeds can be inhibited, for example by antisense, cosuppression, or ribozyme-mediated inhibition of SMT expression, thereby leading to an accumulation of this sterol in seed oils.
  • the SMT gene can be overexpressed in order to increase the levels of sitosterol.
  • Preferred crops for use in accordance with this embodiment of the invention include sunflower, corn, soybean, oilseed brassicas and cotton.
  • Another embodiment of this invention derives from the fact that certain sterols are associated with reducing water permeability of membranes. For this reason, sterol manipulation should provide an effective means for preventing or at least minimizing drought induced damage.
  • nonutilizable sterols in plants through the various gene manipulation strategies described in this invention will not only protect the plants from pests and pathogens but also from environmental stresses such as drought and cold.
  • Preferred sterols to be elevated in this aspect include ⁇ 5 -24 alkyl sterols, such as 24-methyl cholesta-5,23-dienol, and cycloartenol.
  • Preferred crops for use in accordance with this embodiment of the invention include com, wheat, rice, sorghum, soybean, oilseed brassicas (rapeseed, canola), sunflower, palm, peanut, cotton, forestry, fruits, berries, nuts, potato, tomato, sugarbeet, sugarcane, cucurbits (squash, melons, cucumbers, watermelons, pumpkins), vegetable brassicas, alfalfa, ornamental crops, turfgrass, peanut, tea and coffee.
  • Sterol isomers were extracted from corn and were isolated to homogeneity using chromatographic methods. Novel phytosterols were identified with side chains that have been found to be non-utilizable in insects.
  • the sterols were structurally characterized by mass spectroscopy and 1H and 13 C nuclear magnetic resonance (NMR) (Table 1) (Guo et al, 1995).
  • Obtusifoliol 426 0.25 c, g, r, sh, b, p
  • Citrastadienol 426 0.25 c, g, r, sh, b
  • Campesterol 400 0.18 st, c, g, sh, b, r, t, p
  • Isofucosterol 412 0.18 st, c, g, sh, b, r, t, p
  • Stigmasterol 412 0.18 st, c, g, sh, b, r, t, p
  • Sitosterol 414 0.18 st, c, g, sh, b, r, t, p
  • sterols in blades contain mainly 24-ethyl sterols, e.g., sitosterol, while sheaths contained mainly 24-methyl sterols, e.g., 24-methyl-cholesta-5 ,23-dienol .
  • 3C and 3D show: (1) that SMT activity is correlated with sterol synthesis and plant growth; (2) neither sitosterol nor 24(28)-methylene cycloartanol at 100 mM affected HMGR activity, suggesting that HMGR activity does not correlate to growth or sterol production; and (3) the rate of phytosterol turnover correlates to the activities of the first and second methylation of SMT ! enzyme and not HMGR activity.
  • Fig. 4 summarizes the pathway to kinetically favored ⁇ 5 -24-alkyl sterol end products in com during development of the seedling into blades and sheaths under dark-grown conditions.
  • SMT enzyme activities during early blade and sheath formation, and sterol specificity data show that com synthesizes at least two different SMT enzymes: SMT ! catalyzes the successive methyl transfer to produce ⁇ 24(28) -methylene and ⁇ 24(28) -ethylidene sterols; and SMT ⁇ catalyzes the methyl transfer to ⁇ 23(24) -24-methyl sterols.
  • Example 1 The phytosterols identified in Example 1 were tested individually for their ability to support growth. In the absence of a plant sterol mutant for such studies the yeast sterol auxotroph, GL-7, was cultured in the presence of sterols identified according to Example 1, above (Li, 1996). This yeast mutant is used as a model system because it can take up sterols from the culture medium and incorporate the test sterol into the membrane lipid bilayer and proliferate. The amount of proliferation of the cells was measured in the presence and absence of hormonal levels of ergosterol, the major yeast sterol.
  • Sterols were classified according to their effect on growth. Those sterols sparking growth included ergosterol. Those sterols that migrated to membrane and cell structural components without affecting the rate of growth of the cells included cholesterol and sitosterol (Nes et al., 1993).
  • the sterol specificity of the microsome-bound and soluble SMT enzyme from 4-day com seedlings was determined in order to elucidate the enzymatic basis for the plant sterols identified in Example 1.
  • cycloartol is the preferred sterol acceptor and that 24(28)-methylene lophenol was methylated to produce 24(28)-ethylidenelophenol.
  • Table 2 summarizes the specificities to various sterol substrates using the soluble SMT enzyme from com seedlings.
  • the com SMT protein is a tetramer with 4 subunits of 39 kDa.
  • a bifunctional sterol- methylating (SMT) enzyme was partially purified from 4-day etiolated Zea mays (com) shoots by the following steps:
  • non-ionic detergent solubilization of the microsome-bound SMT enzyme (i) non-ionic detergent solubilization of the microsome-bound SMT enzyme; (ii) gel-filtration fractionation of the solubilized protein to produce active fractions with an apparent native molecular weight of circa 156 kd; and (iii) hydroxyapatite chromatography of active fractions.
  • Fig. 1 shows an HPLC-radiocount (Fig. IB) and mass spectrum (Fig. 1A) of the reaction product from 50 pooled assays from a soluble SMT enzyme (4-day seedlings) assayed with 24(28)-methylene lophenol. The second methyl transfer from 24(28)-methylene lophenol to 24(28)-ethylidene lophenol is demonstrated in this incubation.
  • SMT enzyme from 4-day corn shoots catalyzes the successive first and second methyl transfers of an appropriate sterol acceptor molecule.
  • Table 3 shows the effect of a series of substrate and transition state analogs on the first and second methyl transfer reactions.
  • cycloartol ( ⁇ 24 -4,4-dimethyl sterol) with 20 mM Km and 4 pmol/min mg protein Ymax; and 24(28)-methylene lophenol ( ⁇ 7 ,24(28)-4-monomethyl sterol) with 11 ⁇ M Km and 1 pmol/min/mg protein Vmax.
  • cycloartol was the preferred substrate for the first methylation reaction
  • 24(28)-methylene lophenol was the preferred sterol substrate for the second methylation reaction.
  • Zymosterol ( ⁇ 8 ' 4 -4-desmethyl sterol), a preferred sterol substrate of yeast SMT enzyme, was a poor sterol substrate of the first methylation reaction.
  • binding site I catalyzes a first methyl transfer to produce a 24(28)-methylene sterol
  • binding site II catalyzes the second methyl transfer to produce a 24(28)-ethylidene sterol.
  • sitosterol 24 ⁇ -ethyl cholesterol
  • campesterol 24 -methyl cholesterol
  • campesterol 24 -methyl cholesterol
  • 24(28)-methylenecycloartanol a product of cycloartanol transmethylation, was not methylated
  • 24(28)-methylenecycloartanol inhibited the first methyl transfer (20 ⁇ M K,) whereas it failed to inhibit the second methyl transfer.
  • 26,27-cyclopropylidene cycloartol which failed to bind to the yeast SMT enzyme, was a potent competitive inhibitor of the first methylation reaction (25 ⁇ M K,), while not affecting the second methyl transfer.
  • the second alkylation was inhibited by product inhibition from 24(28)-ethylidene lophenol (75 mM K,), while not affecting the first methyl transfer.
  • a transition state analog, 24-(R,S)-25-epiminolanosterol inhibited the first and second methylation reactions with a similar K, value of 55 nM and to exhibit a non-competitive type kinetic pattern.
  • the sterol features of the substrate in the initial enzyme-substrate interaction appears to be typical of other plant SMT enzymes, i.e., a requirement for nucleophilic groups at C-3 and C-24.
  • the 5 ⁇ M K m for the coenzyme was the same for the first and second methylation reactions.
  • the yeast SMT gene, ERG6 was derived from a yeast ERG6 genomic fragment, pRG458/erg6 (Fig. 5B; SEQ ID NO:l).
  • the cloned ERG6 gene was expressed in E. coli.
  • the recombinant protein was shown to be the sterol biomethylation enzyme by enzymatic study which proved that the kinetic properties were similar to that of the native enzyme in yeast.
  • zymosterol a ⁇ 24 -4-desmethyl sterol
  • the molecular weight of the yeast SMT monomer was confirmed to be 43 kD after successfully overexpressing the active protein in E. coli using a T7 promoter-based pET23a(+) vector.
  • the overexpressed protein was visualized on SDS-PAGE gel both by Coomassie blue staining and Westem blot using a yeast SMT polyclonal antibody.
  • the recombinant protein has also been purified from this system.
  • Fig. 5A From the deduced amino acid sequence of the yeast SMT (Fig. 5A; SEQ ID NO:2) the potential AdoMet binding motif was predicted as the first conserved region identified in Fig. 5A (YEYGWGS) and based on mechanistic analysis of biomethylation described in Example 3, the amino acid tryptophan (W) was determined to be the binding site. By -site-directed mutagenesis of the ERG6 gene this amino acid was replaced with alanine. The mutated DNA was also overexpressed in E. coli by cloning into pET23a( +). This protein was not active under conditions where the wild-type protein was active.
  • Such a strategy provides a means to alter phytosterols by introducing inactive SMT protein into plants.
  • the introduction of non-functional SMT monomers can result in the suppression of SMT activity, for example by affecting the ability of the cell to form a functional SMT enzyme complex, thereby leading to the formation of nonutilizable sterols.
  • suppressing the activity of the first SMT Z reaction will lead to formation of ⁇ 23(24) -24-alkyl sterols, products of SMT ⁇ activity.
  • suppressing the activity of the second SMT : reaction will lead to the formation of ⁇ 24 25) - 24-alkyl sterols.
  • the SMT gene from Arabidopsis was cloned and sequenced (Fig. 6; SEQ ID NO:3). This gene was overexpressed in E. coli. Arabidopsis SMT was partially purified and characterized in stereochemical detail.
  • the Arabidopsis SMT gene was amplified by PCR from a cDNA library.
  • the primers used were designed from the full-length cDNA sequence retrieved from the GeneBank (Accession number X89867).
  • the amplified product was the full-length Arabidopsis SMT gene which was sub-cloned into a T/A cloning vector and sequenced. From the sequence data the ORF was identified. A Nde I site was created at the ATG start codon through PCR mediated site-directed mutagenesis.
  • the full-length ORF containing a Nde 1 site at the start and a BamH I site at the stop was cloned into the pET23a(+) vector just as the ERG6 gene was in Example 4.
  • the recombinant protein was active in transforming both cycloartol and 24(28)-methylene lophenol to their respective alkyl products (Tong et al, 1997).
  • cycloartanol only one product was formed, which is 24(28)-methylene cycloartanol, i.e., SMTj in Fig. 4.
  • SMTj 24(28)-methylene cycloartanol
  • the com sterol methyl transferase (SMT) gene was isolated from a commercial com cDNA library (Stratagene, La Jolla, CA). Five microliters of com cDNA (equivalent to 5x10 pfu) were used as template in the amplification of the SMT gene by polymerase chain reaction (PCR). Because the cDNA library was constructed in the vector Uni-Zap
  • PCR amplification (3 'end primer).
  • the 5' end primer (2650-1) was designed from nucleotides 2-20 of a putative SMT fragment published in Gene Bank (T23297). Thirty cycles of PCR were conducted using five units of Taq polymerase from Promega in a total volume of 100 microliters, according to the manufacturer's instructions. One microliter of PCR product from this reaction was used as the template for a second round of PCR using the T7 primer and a primer designed from nucleotides 250-268 of T23297. When the resulting reaction products were analyzed on a 1 % agarose gel, a band of 1.3 kb was seen. This PCR band was subcloned into the plasmid pGEM-T
  • the cloned SMT cDNA was 1497 nucleotides, with a coding region of 1032 nucleotides, which encodes 344 amino acids (Figure 10; SEQ ID NO: 6).
  • the start codon, ATG was located at nucleotide 66-68.
  • a poly A tail of 28 nucleotides was located 371 nucleotides downstream of the stop codon, indicating the cDNA fragment was complete at 3 'end. Therefore, this cDNA clone is a full length cDNA clone.
  • the deduced amino acid sequence from this cDNA clone contains 344 amino acids, encoding a polypeptide of 38.8 kiloDaltons.
  • This deduced amino acid sequence contains all three of the proposed conservative regions for methyl transferase (Kagan and Clarke, 1994. Arch. Biochem. Biophys. 310: 417-427): LDVGCGIGGP at position 104-114 (amino acid sequence) and TLLDAVYA at position 167-174, and VLKPGQ at position 194-199.
  • Another conserved region for sterol methyl transferase proposed by Nes (SFYEYGWGESFHFA, Guo et al.,1997. Antifungal sterol biosynthesis inhibitors.
  • Cholesterol Its function and Metabolism in Biology and Medicine, edited by Robert Bittman. Plenum Press, New York
  • the deduced com SMT amino acids sequence was compared with amino acid sequences from other known SMT genes using GCG progams (Gap and Bestfit).
  • the deduced corn SMT amino acid sequence shared a 93.6% similarity with an independently isolated corn SMT sequence (Genbank U79669), 88.1 % homology, 78.8% identity with soybean SMT (Genbank U43683), and a 93.9% homology, 88.3% identity with partial wheat SMT sequence (Genbank U60754 ), 58.8% homology, 39% identity with Arabidopsis thaliana (Genbank X89867), and a 66.5% homology, 50.4% identity with -yeast SMT (Genbank X74249).
  • this cDNA clone is a full length SMT cDNA clone of Zea mays. Furthermore, since Grabenok et al. have functionally expressed their com SMT gene in a yeast expression system and found no 24-alkyl sterols other than ergosterol, this suggests that the com SMT gene isolated by my laboratory catalyzes the same stereoselective C-methylation to ⁇ 24(28) , thereby supporting the view that com synthesizes several different SMT enzymes.
  • cDNA fragments isolated by the described method should be representative of both SMTj and SMT ⁇ based on the conservation of the region from which the primers were derived.
  • SMT gene Another example of a preferred SMT gene is that from Prototheca wickerhamii. This yeast-like alga produces ⁇ 25 27) -24-methyl sterol as the main product of transmethylation.
  • the favored substrate is cycloartol.
  • the preferred substrate of the SMT is cycloartol.
  • the preferred product is not 24(28)- methylene cycloartol but cyclolaudenol (VII) which is a ⁇ 25(27) -24-alkyl sterol, a nonutilizable sterol.
  • Cloning the gene of this SMT will facilitate the introduction of this gene into plants in order to transform the plant sterol, cycloartol, into a product, cyclolaudenol, which will lead to the accumulation of nonutilizable sterols, viz., ⁇ 25(27) -24-alkyl sterols.
  • Prototheca wickerhamii cells are grown to mid log phase in YPD rich medium (yeast extract - peptone - dextrose). The pelleted cells are disrupted in the presence of Tri Reagent (MRC) using 0.5 mm glass beads and a mini-Beadbeater (both from Biospec Products, Bartlesville, OK). High quality total cellular RNA is isolated according to the manufacturer's instructions.
  • YPD rich medium yeast extract - peptone - dextrose
  • Total cellular RNA is subjected to 3' RACE (rapid amplification of cDNA ends) and 5' RACE using reagents and protocols found in kits obtained from GibcoBRL.
  • 3' RACE total cDNA is synthesized by the action of reverse transcriptase after annealing oiigo(dT)-containing primers to the poly(A)-tailed RNAs present in the unfractionated total RNA.
  • the RNA templates are degraded and the cDNA serves as template for polymerase chain reaction (PCR) amplification.
  • PCR polymerase chain reaction
  • the user-supplied primer "YEYGWG” (see Rationale for primer design below) anneals to the cDNA and is extended toward the 3' end of the gene under the direction of Taq polymerase.
  • the kit-supplied primer for extension from the 3' end to the terminus defined by the "YEYGWG” primer anneals to a sequence composed of three restriction endonuclease recognition sites that was part of the original oligo-dT containing primer.
  • Another nested primer (“ATCHAP”) has been similarly used.
  • RNA Total cellular RNA is also subjected to 5' RACE.
  • cDNA is synthesized by reverse transcriptase using the antisense primer "EWVMTDas".
  • cDNA is modified at the 3' end by the addition of a polydeoxycytidine "tail" using terminal deoxynucleotidyl transferase (TdT).
  • TdT terminal deoxynucleotidyl transferase
  • An initial PCR reaction is carried out using this C-tailed cDNA as template and the primers "EWVMTDas" and a kit-supplied poly-G containing primer.
  • a second PCR reaction is carried out on this PCR product using the nested primer "ATCHAPas” and a kit-supplied primer that anneals to a part of the poly-G primer that contains restriction enzyme recognition sites. This second PCR reaction enriches for 5' SMT cDNA sequences.
  • the 3' RACE and 5' RACE PCR products are isolated from gels and ligated into the plasmid pPCRII (Invitrogen). Clones obtained after transformation into E. coli are characterized by sequencing. An Apa I restriction site is present in the DNA of all plants and yeast that have been sequenced in the GCGVGG motif and is present in both the 3' and 5' cDNA clones. This allows splicing of the two 3' and 5' halves of the SMT gene together, completing the entire coding region.
  • the first step in designing the user-supplied primers was to examine the several very highly conserved peptide motifs in the SMTs of those plants and yeast that have been sequenced. Within these are found shorter stretches of amino acid sequences that can be encoded by a minimum number of DNA sequences, the codons of which usually only vary at the third (degenerate) base. It was also desirable that the codon preferred by 3 different yeast species according to codon usage tables found in Wada, et. al. (Nucleic Acids Res., vol 19, pi 981, 1991) be present in the mix of degenerate codons for each amino acid. Each user defined primer is thus a mixture of deoxynucleotides that defines an internal end of a PCR product. It was also reqiured that 4 or 5 of the 6 3' deoxynucleotides of each primer be perfectly matched in all species and had greater than
  • the first three primers described below are sense orientation primers that anneal to -antisense DNA (and the original cDNA).
  • the fourth and fifth primers are antisense primers that anneal to the sense DNA strand of the SMT gene.
  • YE[Y/F/W]GWG (amino acids 81-86 of the yeast sequence; nonidentical residues at a position are in brackets) was the part of a larger conserved region of SMT that was the basis for the "YEYGWG" primer:
  • GCGVGG The "GCGVGG” primer was suggested by the DNA sequence that encodes part of a second conserved domain (GCG[V/I]GG) at yeast amino acid residues 129-134.
  • the sequence of primer "GCGVGG” is:
  • Primer "ATCHAP” is based on the DNA sequence encoding a third highly conserved domain (yeast amino acids 196-203).
  • the primer sequence is:
  • EWVMTDas is an antisense primer for first strand cDNA synthesis in the 5' RACE experiment. It is based on the small conserved domain at yeast amino acid residues 225-231. The sequence is:
  • ATCHAPas is a nested antisense primer for the 5' RACE experiment with the sequence:
  • cDNA libraries from any crop of interest can be screened and corresponding clones of appropriate sizes can be isolated and sequenced.
  • cDNA library construction and screening methodologies are well known in the art.
  • appropriate primer combinations can be readily determined using information of the conserved regions of known sequences for various SMT genes. To confirm the identity of sequences cloned by this method, they can be compared with known plant SMT enzyme sequence and/or in vitro tranlsated and evaluated biochemically.
  • Example 4 To obtain transgenic plants with altered sterol profiles a DNA fragment containing the open reading frame of the SMT ERG6 gene of yeast isolated from a genomic clone was identified (Example 4).
  • the ERG6 DNA was modified by PCR to include restriction sites for Nco I on either end of the open reading frame. This PCR procedure gave ruse to a mutaion which introduced a frameshift in the gene. This mutation made the ERG6 gene introduced into the plant untranslatable, but capable of inhibiting the endogenous tomato SMT via antisense or co-suppression mechanisms, depending upon the nature of the construct.
  • the modified ERG6 DNA fragment was cloned into the pUCl ⁇ cpexp expression cassette vector. Clones with the ERG6 DNA in the sense as well as the antisense orientations to the 35S promoter were generated (Fig. 7).
  • Hind HI digestion of these clones gave rise to the ERG6 constructs that included the 35S promoter and termination sequences flanking the ERG6 open reading frame. These Hind III digested fragments were cloned to the binary vector pJTS246 that contains T-DNA border recognition sequences and the NPTII gene conferring kanamycin resistance.
  • the cloned binaries with either the sense or antisense ERG6 constructs were transformed into Agrobacterium tumefaciens which were cocultivated with cotyledons of tomato (Solanum lycoperiscum) to obtain transformed plant cells. From calli formed on selective medium containing kanamycin transgenic plants were produced.
  • the leaves from control (no inserts) and transgenic plants (with inserts) were analyzed for the transgene.
  • DNA was extracted from leaf samples of each of the transformants and an untransformed tomato plant. The DNA extracts were quantified by A260 absorbance.
  • ERG6 sequence (underlined in Fig. 8). Controls in the PCR included a sample with no template DNA and samples of the sense and antisense ERG6 containing binary plasmids.
  • PCR was performed under non-stringent conditions (55 °C annealing temperature for 2 min in each cycle) in 20 cycles and aliquots were electrophoresed on 0.8% agarose gels.
  • the primers were selected such that a 1100 bp fragment of the ERG6 DNA would be amplified (Fig. 8). All the regenerated transgemc tomato plants (Ro) carried this fragment as did the plasmid controls. There also is some non-specific amplification because of the non-stringent conditions leading to other bands appearing in the transformed plants and in the untransformed control. However, the level of these amplifications is significantly less than that of the target fragment. This confirms the presence of the ERG6 DNA in the tomato genome. Sterol analysis was performed on the nonsaponifiable lipid fraction of leaf material frqm -one regenerated plant transformed with the sense construct and one regenerated plant transformed with the antisense construct. The results are shown in Table 4.
  • a scheme for the new pathway introduced into the tomato plants due to the insertion of the yeast ERG6 gene is predicted to be as follows:
  • the regenerant (RQ) plants were allowed to flower and set fruit. Seeds were collected, and the following generation (R-T) was grown. Individual plants arising from seeds were assayed for the presence or absence of the selectable marker (NPT2) via ELISA assay for the NPT2 protein. Fifty-three plants from six Rj progeny and a nontransgenic plant were analyzed for sterol composition. The sterol profiles of these plants could be divided into four distinct groups, or phenotypes: Table 5
  • Plant Nontransformed G55 (nontransgenic G62 (nontransgenic Mean Std. segregant) segregant) Dev.
  • Obtusifoliol 1 - tr. 1.0 -dash lines means not detected; tr. is trace; N.D.-not determined.. NSF was chromatographed on TLC plates and bands matching 4-desmethyl-, 4-monomethyl and 4,4-dimethyl sterol standards were eluted from the plate and examined further by chromatography on 3% SE-30 columns and GC-MS. Limit of detection is 0.1 mg sterol per leaf sample. These controls can be compared with transgemc plants, the sterol composition of which are given in tables 8, 9, and 10. Table 8 Sterol composition of transgenic plants from line G3
  • NSF was chromatographed on TLC plates and bands matching 4-desmethyl-, 4- monomethyl and 4,4-dimethyl sterol standards were eluted from the plate and examined further by chromatography on 3% SE-30 columns and GC- MS. Limit of detection is 0.1 mg sterol per leaf sample.
  • Obtusifoliol tr. 1 1 tr. - tr. tr. tr. 1
  • NSF was chromatographed on TLC plates and bands matching 4-desmethyl-, 4-monomethyl and 4,4- dimethyl sterol standards were eluted from the plate and examined further by chromatography on 3% SE-30 columns and GC-MS. Limit of detection is 0.1 mg sterol per leaf sample.
  • NSF was chromatographed on TLC plates and bands matching 4-desmethyl-, 4- monomethyl and 4,4-dimethyl sterol standards were eluted from the plate and examined further by chromatography on 3% SE-30 columns and GC-MS. Limit of detection is 0.1 mg sterol per leaf sample.
  • H. zea (com earworm) was reared on an artificial diet treated with different sterol supplements to study the relation between sterol structure and utilization in insects. H. zea eggs were used to establish a disease-free stock colony.
  • the stock insects were reared using sterile procedures on a pinto bean-based diet. Moths were fed 10% sucrose. Cultures were maintained at 27 + l°C, at 40 ⁇ 10% relative humidity on a 14:10 light-dark photoperiod and an artificial diet was used to rear the insects on different sterol supplements.
  • the experimental diet contained agar, which is known to contain trace contamination of cholesterol, otherwise the experimental diet was sterol-free.
  • Sterols were solubilized in acetone. Aliquots of the solutions were added to the sterol- free diet in a mortar, the material mixed thoroughly with the diet, and the organic solvent allowed to evaporate. Sterols were supplied to the medium at 200 ppm (equivalent to 1 mg of sterol per experimental vessel containing one insect).
  • H. zea larva are in the final stage of larval development (sixth instar), after which the insects may pupate.
  • a single neonate larva was placed in an experimental culture vial and allowed to grow for 20 days. The fresh weight, length and instar stage of 20-day larva were recorded.
  • the larvae were allowed to grow for another 4 days to determine whether they could pupate properly and develop into moth forms. Neonate larvae of H. zea failed to molt to the second instar when sterol was absent from the diet. Some of these insects survived for more than 15 days.
  • Sterols isolated from the nonsaponifiable lipid fraction extracted from larvae contain long chain fatty alcohols. These fatty alcohols may comigrate with sterols during some forms of chromatography and interfere with sterol quantitation, particularly of cholesterol. Therefore, in order to confirm the identity and amount of cholesterol in the insect an aliquot of the NSF was injected into a HPLC column and the fraction corresponding to cholesterol was examined by GC-MS.
  • Larvae did not develop on a sterol-less medium.
  • These sterols are referred to as "utilizable" sterols (Table 11 and Fig. 9).
  • the major sterol recovered from the larvae was cholesterol, showing that H. zea operates a typical insect 24-dealkylation sterol pathway.
  • Fig. 9 Indicates the structures in Fig. 9.
  • the minimal dietary concentration of cholesterol necessary for larvae to grow and pupate is 0.01 % of the experimental diet. This level of cholesterol does not support a rapid rate of molting as did higher levels of cholesterol. However, diets of 0.015% cholesterol or more enhanced the rate of development of larvae. Therefore, a slightly higher amount of dietary sterol (0.02%) was used to insure that a non-limiting amount of sterol (alone or as a mixture) was available in the experimental diet, or no sterol was added to the diet to act as a control.
  • Table 11 and Fig. 9 show that the position of the double bond in the sterol side chain and nucleus is critical to sterol-controlled growth.
  • the inability of cholest-8-enol to support growth suggests that H. zea cannot transform 9 ⁇ ,19-cyclopropyl sterols to c o
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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  • Tyr Lys Ala Gly lie Gin Arg Gly Asp Leu Val Leu Asp Val Gly Cys 115 120 125
  • Ala lie Glu Ala Thr Cys His Ala Pro Lys Leu Glu Gly Val Tyr Ser 195 200 205
  • Glu lie Tyr Lys Val Leu Lys Pro Gly Gly Thr Phe Ala Val Tyr Glu 210 215 220 Trp Val Met Thr Asp Lys Tyr Asp Glu Asn Asn Pro Glu His Arg Lys 225 230 235 240 lie Ala Tyr Glu lie Glu Leu Gly Asp Gly lie Pro Lys Met Phe His 245 250 255
  • GAAACATCAC CGGAAAAAGT ATGGAGAATT TTCTCAATTT GTTTTTATTT TTAAGTTAAA 1200 TCAACTTGGT TATTGTACTA TTTTTGTGTT TTAATTTGGT TTGTGTTTCA AGAATTATTA 1260
  • CTAGCTTCTA TGAGTATGGT TGGGGTGAAT CCTTCCACTT TGCTCACAGA TGGAATGGAG 300 AATCCTTACG TGAAAGCATC AAGCGACATG AGCATTTTCT TGCCCTGCAA CTTGGTTTGA 360
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CA2276087A1 (en) 1998-10-15

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