OA17068A - Microsomal w6 oleate desaturases. - Google Patents

Abstract

The present invention relates to the field of plant molecular biology, more particularly Jatropha microsomal w6 oleate desaturases. The present invention also relates to Jatropha plants or plants of other oil crops having seeds with altered ratios of monosaturated and polyunsaturated fats. In particular, the present invention relates to Jatropha plants or plants of other oil crops where the plants exhibit elevated levels of oleic acid.

Description

Microsomal ω6 Oleate Desaturases

CROSS-REFERENCE TO RELATED APPLICATION

The présent application daims priority to International Patent Application No. PCT/SG2011/000197 filed on 27 May 2011. This application Is Incorporated herein by référencé.

SEQUENCE SUBMISSION

The présent application Is being filed along with a Sequence Listing In electronic format. The Sequence Listing Is entitled 2577208PCT2SequenceListing.txt, was created on 16 August 2011 and Is 127 kb In size. The Information In the electronic format of the Sequence Listing Is part of the présent application and Is Incorporated herein by référencé In Its entirety.

BACKGROUND OF THE INVENTION

The présent Invention relates to the field of plant molecular blology, more particulariy Jatropha microsomal ω6 oleate desaturases. The présent Invention also relates to Jatropha plants or plants of other oil crops having seeds with altered ratios of monosaturated and polyunsaturated fats. In particular, the présent Invention relates to Jatropha plants or plants of other oil crops where the plants exhibit elevated levels of oleic acid.

The publications and other materials used herein to lllumlnate the background of the Invention, and In particular, cases to provide additional details respecting the practice, are Incorporated by référencé, and for convenience are referenced In the following text by author and date and are listed alphabetically by author In the appended bibliography.

Plant oiis hâve many kinds of diverse applications. Novel vegetable oil compositions and Improved approaches to obtain oil compositions, from biosynthetic or naturel plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired. Plants, especlaliy spedes which syntheslze large amounts of oils In seeds, are an Important source of oils both for edible and Industrial uses (Lu et al.' Durrett et al., 2008).

One major usage for plant oil Is for food. Plant oils are mostly composed of five common fatty adds, namely palmltic acid (16:0), stearic acid (18:0), oleic add (18:1), linolelc add (18:2) and linoienic add (18:3) (Durrett et al., 2008). Oleic add Is a monounsaturated omega-9 and 18 carbon fatty acid found In various vegetable oils. tt Is consldered one of the healthier sources of oil and fat In food resources for human and animal. Diets In which oil consumption are high In oleic acid hâve been proven to downreguiate overall levels of chronlc human diseases such as cholestérol, arterlosclerosis and cardlovascular disease. Spedfically, oleic acid has been shown to ralse levels of high*denslty llpoprotelns (HDLs) known as “good cholestérol, while lowering low-density llpoprotelns (LDLs) also known as the bad cholestérol. Thus, the development of new and inexpensive sources of foods comprising healthler forms of fatty acid is désirable.

One emerging purpose for oll Is to serve as feedstock of renewable bioenergy in the form of biodiesel. The demand for use blodlesel, mainly cornes from vegetable oll, has soared along with govemment subsldies and mandates for the alternative fuel. Because there are various fatty acid composition of each types, the fuel propertles of biodiesel derived from a mixture of fatty acids are dépendent on that composition. Compared with conventlonal diesel, there are some négative factors of fatty acid profile should be optlmized by tradltional breeding or genetic engineering to optimlze biodiesel fuel characteristics. Various studies suggest that biodiesel with high levels of methyl oleate will hâve excellent, characteristics with regard to Ignition quality, NO_X émissions and fuel stability. For example, whlie unsaturation tends to reduce the cetane number of blodlesel, that of methyl oleate Is higher than the minimal blodlesel standard. Additlonally, It has been estimated that biodiesel fuels with an average of 1.5 double bonds per molécule will emlt an équivalent amount of NO, compared with conventions! diesel, thus a fuel high ln oleates should not resuit ln higher NO_X émissions. Finally, given that polyunsaturated fatty acids hâve a major effect on the auto-oxidation of biodiesel, high oleic acid with reduced polyunsaturated fatty acid content will Improve the stability of the fuel (Durrett et al., 2008).

Soybean Unes with high levels of oleic acid and low levels of saturated and polyunsaturated fatty acids hâve been developed using a transgenic strategy that results In down-regulation of one single gene fatty acid desaturase 2 (FAD2). Consistent with prédictions, biodiesel synthesized from these high-oleic soybeans demonstrated Improved fuel characteristics with regard to cold-temperature flow propertles and NO_X émissions (Tat et al., 2007; Graef et al., 2009).

During the last several years, many countries hâve begun to target biofuel research as a national priority and Implement compulsory blending of fossil fuel with biofuel. The Increasing demand for biofuel, however, Is exerting more pressure on food production because of the compétition between fuel crops and food crops for arable land. One way to ease this compétition Is to use marginal land for blo-energy production (Carroll and Somerville, 2008).

Jatropha curcas, a small woody plant beionglng to Euphorbiaceae, Is a non-food crop mainly grown ln the tropical and subtropical régions. This plant possesses several propertles rendering It suitable for biodiesel production, such as Its rapld growth, ease of propagation, short gestation period, low seed cost, high oil content, wide adaptability, and drought tolérance (Jones N, 1991; Falriess, 2007). Furthermore, Jatropha may yîeld more than four Urnes as much fuel per hectare as soybean, and more than ten times that of malze (com) (http7/en.wiklpedia.org/wiki/Jatropha_oil). Especlally Important Is that Jatropha can thrlve on degraded soil (Falriess, 2007) maklng It an attractive crop for blodiesel feedstock since It can be planted on a large-scale on marginal land unsultable forfood crops.

Plants syntheslze fatty adds via a common metabollc pathway known as the fatty acid synthase (FAS) pathway. Beta-ketoacyl-ACP (acyl carrier proteln molety) synthases are Important ratellmlting enzymes In the FAS of plant cells and exist in several versions. Beta-ketoacyl-ACP synthase I catalyzes chaln élongation to palmitoyl-ACP (C16:0), whereas Beta-ketoacyl-ACP synthase II catalyzes chaln elongaUon to stearoyl-ACP (C18:0). Beta-ketoacyl-ACP synthase IV Is a variant of Beta-ketoacyl-ACP synthase II, and can also catalyze chaln élongation to 18:0ACP. In soybeans, the major products of FAS are 16:0-ACP and 18:0-ACP. The desaturation of 18:0-ACP to form 18:1-ACP is catalyzed by a piastld-locallzed soluble delta-9 desaturase (also referred to as stearoyl-ACP desaturase).

The products of the piastidial FAS and delta-9 desaturase, 16:0-ACP, 18:0-ACP, and 18:1ACP, are hydrolyzed by spécifie thloesterases. Plant thloesterases can be classified Into two gene families based on sequence homology and substrate preference. The first famlly, FATA, Includes long chaln acyl-ACP thloesterases having activity primarily on 18:1-ACP. Enzymes of the second famlly, FATB, commonly utilize 16:0-ACP (palmitoyl-ACP), 18:0-ACP (stearoylACP), and 18:1-ACP (oleoyl-ACP). Such thloesterases hâve an Important rôle ln determining chaln length during de novo fatty add biosynthesis In plants, and thus these enzymes are useful In the provision of various modifications of fatty acyl compositions, particularty with respect to the relative proportions of various fatty acyl groups that are présent In seed storage oils.

The products of the FATA and FATB reactions, the free fatty acids, leave the plastlds and are converted to thelr respective acyl-CoA esters. Acyl-CoAs are substrates for the lipldblosynthesls pathway (Kennedy Pathway), which is located in the endoplasmlc réticulum (ER). This pathway Is responslble for membrane lipld formation as well as the biosynthesis of triacylglycerols, which constitute the seed oil. In the ER there are additional membrane-bound desaturases, which can further desaturate 18:1 to polyunsaturated fatty acids.

Various technologies for generating mld to high olelc add levels In soybean plants are known. For example, U.S. Patent Publication No. 2007/0214516 disdoses a method for obtaining soybean plants that hâve moderately Increased levels of olelc acid.

SUMMARY OF THE INVENTION

The présent invention relates to genes, coding sequences, other sequences, construis and vectors that can be used to provide a method to croate and select high oleic acid Unes containing around 80% oleic acid from the original level of around 40% In Jatropha seed oil. The genes, coding sequences, other sequences, constructs and vectors described herein, demonstrate the ability to effidently Incorporate an enhanced oil quality trait into elîte varieties of Jatropha plants without the expensive crosslng and évaluation used In traditional Jatropha broeding.

The Jatropha genome possesses two mlcrosomal ω6 oleate desaturase, deslgnated JcFAD2-1 and JcFAD2-2. Two cDNAs were Identified and they encoded proteins of 383 (SEQ ID NO:2) and 387 amino acids (SEQ ID NO:5) that were 74% Identical to each other and 77.3% and 72.1% identical to Arabidopsis FAD2, respectively. The cDNA with higher sequence Idenfrty to the FAD2 enzyme famlly was designated JcFAD2-1 and another one Is named as JcFAD2-2. FAD2-1 and FAD2-2 are found in the ER where they can further desaturate oleic add to poiyunsaturated fatty adds. The deita-12 desaturase catalyzes the insertion of a double bond Into oleic add (18:1), forming linolelc add (18:2) which results In a conséquent réduction of oleic add levels. A delta-15 desaturase (FADS) catalyzes the insertion of a double bond Into linolelc add (18:2), forming linolenic add (18:3).

To produce sélection marker free transgenic Jatropha, a chemically Indudbie Cre-loxPmediated slte-spedfic recombination System, which was first developed by Zuo J et al.(Zuo et a/., 2001) In Arabidopsis, was tested. JcFAD2-1 was silenced to make high oleic add and marker free transgenic Jatropha. Slmilar transformation procedure like above was taken to get hygromydn-reslstance régénération shoots (see WO 2010/071608, Incorporated herein in by référencé in Its entlrety). Once there are visible shoots cornes out, we transfer small shoots to marker free Induction medium without hygromydn. After two weeks Induction, these wellgrowing shoots were subsequently transferred Into régénération medium II but without hygromydn. The remaining procedures are same as above normal transformation procedure.

To Increase oleic add level and reduce the unexpected environmental adaptation risk, a seed spécifie promoter to produce a seed spedfic high oleic add In Jatropha was used. A soybean (Glycine max) seed storage protein 7S seed-spedfic promoter was chosen to drive hpRNA expression to downregulate JcFAD2-1 RNA. Two fines were found to contain 77.4% and 74.7% oleic add In T1 génération endosperm. The linolelc add were reduced to less than 5% of total fatty add In these fines.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and 1B show the amino acid composition of fatty acid desaturase enzymes In Jatropha (SEQID NO:2 and SEQID NO:5, respectively).

Figures 2A and 2B show a comparison between amino add sequences of fatty acid desaturease enzymes from plants (Fig. 2A) and expression pattern of various genes (Fig. 2B). The sequences are as follows: AtFAD2: SEQ ID NO:7; RcFAD2: SEQ ID NO:8; RcFAH12: SEQ ID NO:9; JcFAD2: SEQ ID NO:2; JcFAD2-2: SEQ ID NO:5; VfFAD2: SEQ ID NO:10; and VfFAX:SEQID NO:11.

Figures 3A-3C show β-estradiol mediated Cre-lox marker free system In transformation medium. Fig. 3A: Schematic diagram of the silencing cassette and β-estradiol-lnduced DNA exdsion for high oleic add. Size bar=1kb. Fig. 3B: Genotyping analysis for primary transgenic shoots #1*1 and #1-2. Fig. 3C: GC analysis for primary transgenic shoots #1-1 and #1-2.

Figures 4A and 4B show genotyping of X7-JcFAD2-1 RNAi (A) and X8-JcFAD2-1 RNAi (B). Fig. 4A: Upper DNA gel showed one of genotyping resuit of line 1-26 with hygromycin résistance gene primer pair (hpf) for X7-JcFAD2-1 RNAi. Lower DNA gel showed partial resuit of Unes 1-26 with marker free primer pair (P1+P4). Note: * indicated one example line with chimeras of marker free and marker together in one plant ** Indicated one example line with complété marker free. Fig. 4B: Upper DNA gel showed one of genotyping resuit of lines 25-49 with hygromycin résistance gene primer pair (hpt) for X8-JcFAD2-1 RNAi. Lower DNA gel showed partial resuit of lines 25-49 with marker free primer pair (P7S+P4). Note: * Indicated one example line (X8#34) with chimeras of marker free and marker together in one plant.

Figures 5A-5C show molecular and oil composition analysis of X7-FAD2-1 RNAi lines. Fig. 5A: RNA analysis in T1 endosperm of #79 and #170 lines. Fig. 5B: RNA analysis in T1 leaves of #79 and #170. Fig. 5C: GC analysis to show moderate fatty acid composition changes in X7FAD2-1 RNAI fines with T1 seeds of 35S.GFP as control.

Figures 6A-6F show higher oleic add transgenic lines with soybean 7S seed spedfic promoter. Fig. 6A: analysis In T1 endosperm of #34 and #291 lines. Fig. 6B: RNA analysis In T1 cotylédons of #34 and #291 lines. Fig. 6C: RNA analysis In T1 true leaves of #34 and #291 lines. Fig. 6D: Oil content analysis in endosperm of #34 and #291 lines. Fig. 6E: GC analysis to show fatty add composition changes in X8-FAD2-1 RNAi lines with T1 seeds of 35S:GFP as control. Fig. 6F: GC analysis to show no obvious fatty add profile change In T1 true leaves.

Figures 7A-7C show Southem blot analyses on primary and T1 plants from X8-FAD2-1 RNAI fines. Fig. 7A: Shows an EcoRV fragment containing JcFAD2-1. Fig. 7B: Total genomic DNA dlgested with Xhol, and probed with soybean 7S promoter. * indicates the positive genomic bands containing marker. ** indicates the positive genomic bands that are marker free. Fig. 7C: Total genomic DNA digested with EcoRV and Xbal, and probed with FAD2-1 open reading frame (ORF) In the left panel and the same membrane was stripped and reprobed with hpt ORF In rlght panel.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and sdentific terms used herein hâve the same meanlng as Is commonly understood by one of skill in the art to which the invention belongs.

As used herein, allele refers to any of one or more alternative forms of a gene locus, ali of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two aileles of a given gene occupy corresponding lod on a pair of homologous chromosomes.

As used herein, FAD2 refers to a gene or encoded protein capable of catalyzing the Insertion of a double bond Into a fatty acy! moiety at the twelfth position counted from the carboxyl terminus. FAD2 proteins are also referred to as delta-12 desaturase or “omega-6 desaturase. The term FAD2-1 Is used to refer to a FAD2 gene or protein defined as sequence In the FIG. 1A (SEQ ID NO:2), coding sequence shown in ORF sequence In SEQ ID NO:1 or whole genomic sequence SEQ ID NO:3 that Is naturally expressed In a multiple tlssues, Induding the seed préférable model. The term FAD2-2 Is used to refer a FAD2 gene or protein defined as Fig. 1B (SEQ ID NO:5), coding sequence shown In ORF sequence In SEQ ID NO:4 or whole genomic sequence SEQ ID NO:6 that Is (a) a different gene from a FAD2-1 gene or protein and (b) Is seed spedfic expression.

As used herein, gene refers to a nudelc add sequence that encompasses a 5' promoter région assodated with the expression of the gene product, any Intron and exon réglons and 3’ or 5’ untranslated régions assodated with the expression of the gene product.

As used herein, génotype’ refers to the genetic constitution of a cell or organism.

As used herein, phenotype refers to the détectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.

A fatty add Is a carboxylic acid that generally has a long unbranched aliphatic carbon chain. The désignations (18:2), (18:1), (18:3), etc., refer to the number of carbon atoms In the fatty add chain and the number of double bonds thereln, respectively. For example, olelc add (18:1) contains 18 carbon atoms and 1 double bond.

The présent Invention relates to the field of plant molecular biology, more partlcularly Jatropha mlcrosomal ω6 oleate desaturases. The présent Invention also relates to Jatropha plants or plants of other oil crops having seeds with altered ratios of monosaturated and poiyunsaturated fats. In particular, the présent invention relates to Jatropha plants or plants of other oil crops where the plants exhibit eievated levels of oleic acid.

Thus, in a first aspect, the présent Invention provides an Isolated nudeic acid encoding a JcFAD2-1 protein comprising the amino acid sequence set forth In SEQ ID NO:2. In one embodiment, the nucleic acid comprises the nucléotide sequence set forth In SEQ ID NO:1. In another embodiment, the nucleic acid comprises the nucleotlde sequence set forth in SEQ ID NO:3. In a further embodiment, the nucleic acid further comprises a plant opérable promoter operabiy linked to the coding sequence. In one embodiment, the promoter Is a seed spedfic promoter. In another embodiment, the seed spedfic promoter Is derived from an oil crop.

In a second aspect, the présent Invention provides an isolated nudelc add encoding a JcFAD22 protein comprising the amino add sequence set forth in SEQ ID NO:5. In one embodiment, the nudeic add comprises the nudeotide sequence set forth In SEQ ID NO;4. In another embodiment, the nudelc acid comprises the nudeotide sequence set forth In SEQ ID NO:6. In a further embodiment, the nudeic add further comprises a plant opérable promoter operabiy linked to the coding sequence. In one embodiment, the promoter Is a seed spedfic promoter. In another embodiment, the seed spedfic promoter Is derived from an oil crop.

In a third aspect, the présent invention provides a construct or vector comprising an isolated nucleic add as described herein. In one embodiment, the construct or vector Is an expression construct or vedor. In another embodiment, the construd or vector further comprises a selectable marker. In a further embodiment, the construct or vector comprises a Cre-lox recombination marker free system.

In a fourth aspect, the présent invention provides a transgenic plant comprising a nudeic add or vector described herein. In one embodiment, the transgenic plant may be any plant spedes. In another embodiment, the transgenic plant may be a plant of an oil crop. In a further embodiment, the transgenic plant may be a Jatropha plant.

In a fifth asped, the présent invention provides for the down régulation of a JcFAD2-1 and/or JcFAD2-2 gene using RNA interférence (RNAI), indudlng mlcroRNA and hairpln RNA. In one embodiment, a nudelc acid is provided which down régulâtes the JcFAD2-1 gene. In another embodiment, a nucleic acid Is provided which down régulâtes the JcFAD2-2 gene. In a further embodiment, a nudelc add Is provided which down régulâtes the JcFAD2-1 gene, and a nudeic add Is provided which down régulâtes the JcFAD2-2 gene. In one embodiment, the nudeic add further comprises a plant opérable promoter operabiy linked to the coding sequence. In one embodiment, the promoter Is a seed spedfic promoter. In another embodiment, the seed spedfic promoter Is derived from an oil crop. According to thls aspect, the présent Invention also provides a vector comprising an isolated nudelc add as described herein. In one embodiment, the vector Is an expression vector, In another embodiment, the vector further comprises a selectable marker. In a further embodiment, the vector comprises a Cre-lox recombination marker free system. According to thls aspect, the présent Invention further provides a transgenlc plant comprising a nudelc add or vector described herein. In one embodiment, the transgenlc plant may be any plant spedes. In another embodiment, the transgenlc plant may be a plant of an oil crop. In an additional embodiment, the transgenlc plant may be a castor bean plant. In a further embodiment, the transgenlc plant may be a Jatropha plant In one embodiment, seed of the transgenlc Jatrohpa plant has an olelc add content greater than 50%, preferably greater than 60%, more preferably greater than 70%, most preferably greater than 75%. In another embodiment, seed of the transgenlc Jatropha plant has a linolelc add content less than 5%.

According to thls fifth aspect, the nudelc add Is selected to Inhlbit expression of the native DNA sequence within a plant's tissues to achieve a deslred phenotype. In thls case, such Inhibition mlght be accomplished, for example, with transformation of a plant cell to comprise a promoter linked to an antlsense nudeotide sequence, hairpin, RNA Interfertng molécule, double stranded RNA, mlcroRNA or other nucleic acid molécule, such that tissue-preferred expression of the molécule Interfères with translation of the mRNA of the native DNA sequence or otherwise Inhiblts expression of the native DNA sequence In plant cells. For further description of RNAi techniques or mlcroRNA techniques, see, e.g., U.S. Patent Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also International Patent Publications WO 97/01952, WO 98/36083, WO 98/53083, WO 99/32619 and WO 01/75164; and U.S. Patent Publications 2003/0175965, 2003/0175783, 2003/0180945, 2004/0214330, 2005/0244858, 2005/0277610, 2006/0130176, 2007/0265220, 2008/0313773, 2009/0094711, 2009/0215860, 2009/0308041, 2010/0058498 and 2011/0091975. One example of an RNAI molécule Is described herein. However, the présent Invention Is not limited to this single example. Additional RNAI molecuies or mlcroRNA molécules can be prepared by the skilled artisan using techniques weii known in the art, Induding techniques for the sélection and testing of RNAI molecuies and mlcroRNA molecuies that are useful for down reguiating a JcFAD2-1 and/or JcFAD2-2 gene.

The constnict typically Indudes reguiatory réglons operatively linked to the 5* side of the nudelc add described herein (such a nudeic add encoding a JcFAD2 protein or a nudelc add encoding an RNAI molécule to down regulate a JcFAD2 gene) and/or to the 3* side of the nudelc add. A cassette containing ail of these éléments Is also referred to herein as an expression cassette. The expression cassettes may additionally contain 5* leader sequences In the expression cassette construct. The reguiatory réglons (I.e., promoters, transcriptionai regulatory régions, and translatlonal termlnatlon régions) and/or the polynucleotide encoding a signal anchor may be natlve/analogous to the host cell or to each other. The promoters and tlssue-specific promoters are particularly useful for preparing constructions for the transformation of Jatropha, as well as for the transformation of other oil crops. Altematively, the regulatory régions and/or the polynucleotide encoding a signal anchor may be heterologous to the host cell or to each other. See, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670, 2006/0248616 and 20090100536, and the référencés dted therein. The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known In the art and indude those described in international Publication No. WO 2008/094127 and the référencés dted therein.

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. That Is, the nudelc adds can be combined with constitutive, tissue-preferred, or other promoters for expression In the host cell of Interest. Such constitutive promoters Indude, for example, the core promoter of the Rsyn7 (WO 99/48338 and U.S. Pat. No. 6,072,050); the core CaMV 35S promoter (Odell et al., 1985); rice actln (McElroy et al., 1990); ublqultin (Christensen and Quai), 1989; Christensen et al., 1992); pEMU (Last et al., 1991); MAS (Velten et al., 1984); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters Include, for example, those disdosed In U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Other promoters indude Indudble promoters, particularly from a pathogen-lndudble promoter. Such promoters Indude those from pathogenesls-related proteins (PR proteins), which are Induced followlng infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitlnase, etc. Other promoters indude those that are expressed locally at or near the site of pathogen Infection. In further embodiments, the promoter may be a wound-lndudble promoter. In other embodiments, chemlcai-regulated promoters can be used to modulate the expression of a gene In a plant through the application of an exogenous chemical regulator. The promoter may be a chemlcal-indudble promoter, where application of the chemical induces gene expression, or a chemlcal-repressible promoter, where application of the chemical represses gene expression. In addition, tissue-preferred promoters can be utilized to target enhanced expression of a polynucleotide of Interest within a particular plant tissue. Each of these promoters are described In U.S. Pat. Nos. 6,506,962, 6,575,814, 6,972,349 and 7,301,069 and in U.S. Patent Application Publication Nos. 2007/0061917 and 2007/0143880.

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Generally, the expression cassette may additionally comprise a selectable marker gene for the sélection of transformed cells. Selectable marker genes are utilized for the sélection of transformed cells or tissues. Usually, the plant selectable marker gene will encode antiblotic résistance, with suitable genes Including at least one set of genes coding for résistance to the antiblotic spectinomydn, the streptomycln phosphotransferase (spt) gene coding for streptomydn résistance, the neomydn phosphotransferase (nptll) gene encoding kanamycln or genetldn résistance, the hygromydn phosphotransferase (hpt or aphlv) gene encoding résistance to hygromydn, acetolactate synthase (als) genes. Altemativeiy, the plant selectable marker gene will encode herbldde résistance such as résistance to the sulfonylurea-type herblddes, giufosinate, glyphosate, ammonium, bromoxynil, Imldazolinones, and 2,4dichlorophenoxyacetate (2,4-D), Indudlng genes coding for résistance to herblddes which ad to Inhlblt the adlon of glutamine synthase such as phosphlnothricln or basta (e.g., the bar gene). See generally, International Publication No. WO 02/36782, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670, 2006/0248616,2007/0143880 and 2009/0100536, and the référencés dted thereln. See also, Jefferson et al. (1991),* De Wet et al. (1987); Goff et al. (1990); Kaln et al. (1995) and Chiu et al. (1996). This list of seledable marker genes ls not meant to be iimiting. Any selectable marker gene can be used. The seledable marker gene ls also under control of a promoter opérable in the plant spedes to be transformed. Such promoters Indude those described In international Publication No. WO 2008/094127 and the référencés dted thereln.

Altemativeiy, the expression cassette may additionally comprise a Cre-lox recombination marker free system, such as described herein. Such a system ls useful for produdng sélection marker free transgenlc Jatropha plants or plants of other oil crops.

tn preparing the expression cassette, the various DNA fragments may be manlpulated, so as to provide for the DNA sequences In the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adaptera or linkera may be employed to join the DNA fragments or other manipulations may be Involved to provide for convenant restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, In vitro mutagenesls, primer repalr, restriction, anneallng, resubstitutions, e.g. transitions and transveralons may be Involved.

Once a nucleic add has been doned Into an expression vector, It may be Introduced Into a plant cell using conventional transformation procedures. The term plant cell” ls Intended to encompass any cell derived from a plant Including undifferentiated tissues such as callus and suspension cultures, as well as plant seeds, pollen or plant embryos. Plant tissues suitable for transformation Indude ieaf tissues, root tissues, meristems, protoplasts, hypocotyis, cotylédons, scutellum, shoot apex, root, Immature embryo, pollen, and anther, Transformation* means the directed modification of the genome of a cell by the externat application of recombinant DNA from another cell of different génotype, leading to Its uptake and Intégration Into the subject cell's genome. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained.

DNA constructs containing the promoters of the présent Invention can be used to transform any plant and particulariy oil paim plants. The constructs may be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described In the technical and scientific literature. Transformation protocols may vary dependlng on the type of plant or plant ceil, l.e., monocot or dlcot, targeted for transformation, as is well known to the skilled artisan. For example, the DNA construct may be introduced directly Into the genomlc DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using balllstic methods, such as DNA particle bombardment. Altematively, the DNA constructs may be combined with suitable T· DNA flanking régions and introduced into a conventional Agrobacterium tumefaclens host vector. The virulence fonctions of the Agrobacterium tumefaclens host will direct the insertion of the construct and adjacent marker Into the plant ceil DNA when the cell is infected by the bacteria. Thus, any method, which provides for effective transformatlon/transfection may be employed. See, for example, U.S. Patent Nos. 7,241,937, 7,273,966 and 7,291,765 and U.S. Patent Application Publication Nos. 2007/0231905 and 2008/0010704 and référencés cited thereln. See aiso, International Published Application Nos. WO 2005/103271 and WO 2008/094127 and référencés cited thereln. Techniques which hâve been used to transform oil paim include bioiistic-mediated transformation and Agrobacterium-mediated transformation. See, for example, Masii et al. (2009); Omidvar et al. (2008); Parveez et al. (2008); Abduliah et al. (2005); Parveez et ai. (2000); Chowdhury, et al. (1997); and U.S. Patent Application Publication No. 2009/0038032. In addition, transformation of Jatropha has been described In International Publication No. 2010/071608.

Transformed plant celis which are derived by any of the above transformation techniques can be cultured to regenerate a whoie plant which possesses the transformed génotype and thus the desired phenotype, e.g., a transgenlc plant. A transgenlc plant’ Is a plant into which foreign DNA has been introduced. A transgenlc plant encompasses ail descendants, hybrids, and crosses thereof, whether reproduced sexually or asexually, and which continue to harbor the foreign DNA. Régénération techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biodde and/or herbicide marker which has been Introduced together with the desired nucléotide sequences. See for example, International Published Application No. WO 2008/094127 and référencés dted therein.

The foregoing methods for transformation are typically used for producing a transgenic variety in which the expression cassette is stably incorporated. After the expression cassette Is stabiy 5 Incorporated In transgenic plants, it can be transferred to other plants by sexual crossing. in one embodiment, the transgenic variety could then be crossed, with another (non-transformed or transformed) variety, In order to produce a new transgenic variety. Altematively, a genetic trait which has been engineered into a particular cotton line using the foregoing transformation techniques could be moved Into another fine using traditional backcrossing techniques that are 10 weli known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite variety into an elite variety, or from a variety containing a forelgn gene In Its genome Into a variety or varieties which do not contain that gene. As used herein, crossing can refer to a simple X by Y cross, or the process of backcrossing, depending on the context. Any of a number of standard breeding techniques can 15 be used, depending upon the species to be crossed.

Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedures. Transgenic seeds can, of course, be recovered from the transgenic plants. These seeds can then be planted in the soi! and cultivated using conventional procedures to produce transgenic plants. The culitvated transgenic plants will 20 express the DNA of interest In a tissue-preferred or tissue-specific manner as described herein.

The practice of the présent invention employs, unless otherwise Indicated, conventional techniques of chemistry, molecular bioiogy, microbiology, recombinant DNA, genetics, Immunology, cell bioiogy, cell culture and transgenic blology, which are within the skiil of the art.

See, e.g., Maniatis et al., 1982, Molecular Clonlng (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook et al., 1989, Molecular Clonlng, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook and Russell, 2001, Molecular Clonlng, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Ausubel et al., 1992), Current Protocols In Molecular Bioiogy (John Wiley & Sons, 30 including periodic updates); Glover, 1985, DNA Clonlng (IRL Press, Oxford); Russell, 1984,

Molecular blology of plants: a laboratory course manua! (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Anand, Techniques for the Analysis of Complex Genomes, (Academie Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Bioiogy (Academie Press, New York, 1991); Hariow and Lane, 1988, Antibodies. (Cold Spring 35 Harbor Laboratory Press, Cold Spring Harbor, New York); Nucleic Acid Hybridization (B. D.

Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Uss, Inc., 1987); Immobllized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Clonlng (1984); the treatlse, Methods In Enzymology (Academie Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academie Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Welr and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Sdentific Publications, Oxford, 1988; Fire et al., RNA Interférence Technology: From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interférence In Practice, Wiley-VCH, 2005; Engelke, RNA Interférence (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interférence, Edtting, and Modification: Methods and Protocols (Methods In Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silendng byRNA Interférence: Technology and Application, CRC, 2004.

EXAMPLES

The présent Invention Is described by référencé to the foliowing Examples, which are offered by way of illustration and are not intended to lîmît the Invention In any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE 1

Matériels and Methods

Expiant material for transformation·. Seeds were collected from Jatropha curcas (Je-MD) plants, which were selected by Drs. Yan Hong and Chenxin Yi (Yï et al., 2010) and served as starting materials. Cotylédons from 5-7 day old seedlings, germinating from ’/ί Murashige and Skoog sait medium, were eut Into small pièces (5X5 mm).

Jatropha transformation procedure: For detailed procedure, please refer to Mao et al. (2009). Slmply, there are 4 steps as followed procedures. 1) Co-cultivatlon. Small cotylédons pièces incubated with Agrobacterium cells harboring the target expression cassette in 20ml of medium Il for 10-20 min at 25° C. Expiants were then transferred to the co-cultivatlon medium for 2-3 days at 22° C in the dark. Foliowing co-cultivation, expiants were rinsed several times with stérile water, foliowing one wash with 300mg Γ¹ cefotaxine. Cotylédon tissues were blotted dry by puttlng them on a pad of sterilized paper to remove excess surface water. Expiants on the callus formation medium plate were transferred to darkness at 25±1° C for three weeks. Under this condition, un-transformed expiants normally will tum brown.

2) Shoot régénération. Expiants with newly emerged hygromycln-resistant callus were transferred onto the shoot régénération medium I for 3 weeks at 25° C with 16h light (100 pmol m'²S‘¹)/ 8h dark cycles. During this period, any shoots regenerated from callus (about 40-50%) were transferred to the shoot régénération medium II. Callus with no regenerated shoots were transferred to the shoot régénération medium III for further régénération.

3) Shoot élongation. After 4 weeks, régénération shoots were transferred onto shoot élongation medium for élongation and bud multiplication.

4A) Rooting. The elongated shoots about 2.5cm were rooted In rooting medium. Normally it will take more than one month to get roots. Or

4B) graftlng can be used to increase survival rate. Elongated shoots also can be used as scions for grafting onto non-transgenic root stocks. Healthy and vigorously growlng Jatropha plants were chosen to be rootstocks. Both scions and rootstocks were eut into the cambium région so that phloem tissues from both will connect after joinlng. The graft joint was wrapped with parafilm and secured by a tape. Grafted Jatropha plants were malntained under low light intensity and 85% humility for 7 days.

Transgenic plasmids construction and materials: To generate the β-estradiot chemicalregulated inducible RNAi lines under-expressing JcFAD2-1_t a gene-speclfic 862-bp fragment corresponding to the coding région of nt 85 to 946 of the JcFAD2-1 cDNA was PCR-amplifled with forward primer 5'-ATCACTCGAGCCACCATTCACACTTGGTCAG-3' (SEQ ID NO:12) and reverse primer 5'-GTATAAGCTTCATGAGTGTCTGTAATGTTATG-3' (SEQ ID NO: 13). This fragment was Inserted In sense orientation into the Xho\IHind\\\] sites of pSK-Int vector as described previously (Guo et al., 2003). The same fragment, ampllfied with forward primer 5'CAATATCTAGACCATGGGTGCCGGTGGCAGAATG-3' (SEQ ID NO:14) and reverse primer S'-TATTGGATCCGGAAACTTGTTTTTGTACCAGAACAC-S* (SEQ ID NO:15), was subsequently placed in antisense orientation Into the BamHI/Xbal sites of pSK-Int already carrylng the sense fragment to form pSK-int-FAD2-1 RNAI. Finally, the entire RNAI cassette comprising the sense and antisense fragments Interspersed by the actin II Intron was excised from pSK-Int using the flanklng XhoVXba\ sites and Inserted into the Xho\IXba\ site of pX7-GFP vector yieldlng the construct pX7-FAD2-1 RNAI, whose sequence Is set forth In SEQ ID NO:33. To generate the β-estradiol chemlcal-regulated Inducible and seed-specific RNAI lines underexpressing JcFAD2~1, soybean 7S seed promoter was amplified by overlapplng PCR and used to substitute for the G10-90 constitutive promoter In pX7-GFP to yield a seed-specific promoter marker free vector deslgnated pX8-GFP. The entire FAD2-1 RNAI cassette above In pSK-Int vector was Inserted Into pX8-GFP to substitute for the GFP coding région to form the pX8-FAD2-1 RNAI vector, whose sequence Is set forth In SEQ ID NO:32.

Transformants were selected and events (X7#79, X7#170 from pX7-FAD2-1 RNAI; X8#34, X8#291 from pX8-FAD2-1 RNAI) were establlshed using gene markers, fatty acid compositional analysis of endosperm of Individuai seeds. Plants were grown In a greenhouse under naturel photoperiods and température condition (ranged from 25°-35° C).

Fatty add analysis: For leaf lipld profile analysis, total lipld, extracted from 100 mg fresh Jatropha leaves with the similar method described In (Ye et al., 2009b). Dried Jatropha seeds were collected and after removing the outer seed coat, seeds were surface sterilized for 60 seconds with 75% (v/v) éthanol, followed by Immersion In 10% (v/v) H₂O₂ for 1 h, then rinsed with stérile water for two times, finally immersed In stérile water ovemight at 28° C In darkness for 24 hrs. Seed endosperm was separated carefully from the embryo. The dry endosperm part was then ground to a fine powder, and the iipids were triple extracted with hexane. The supematant was transferred In a glass vial, and the hexane was evaporated with a flow of dry nitrogen gas at 50° C. The raw oll was welghted and the oil content was recorded as the ratio of raw oll to endosperm amount.

About 10-mg of the oll was transmethylated with 3N methanolic-HCi (SIGMA, USA) plus 400 μι. 2,2, Dimethoxypropane (SIGMA, USA). The résultant FAMEs were separated by GC and detected by using GC Agilent 6890 (Palo Alto, CA, USA) employlng hélium as the carrier gas and DB-23 columns for components séparation. The GC analyticai method was performed at 140* C for 50 sec and a 30* C min'¹ ramp to 240* C, and the final température was maintained for 50 sec. Peaks were Identified based on their rétention times compared with a FAME référencé mixture (SIGMA, USA). Fatty acid composition value induded In the analyses was calculated based on peak area percentage of total fatty acids In three biological replaçâtes and presented as mean 1 standard déviation.

RNA extraction and analysis: 100 mg leaf or endosperm tissues were ground In liquid N₂ and extracted with plant RNA purification reagent (Invltrogen, USA). RNA concentration was measured by Nanodrop (Thermo, USA). M-MLV reverse transcriptase (Promega, USA) was used for reverse transcription reactions. Real-time PCR was performed with Power SYBR® Green PCR Master (Applied Biosystems, USA) and run In ABI7900HT. Ail samples were run in triplicates and data was analyzed with RQ manager at a pre-set Ct value (Applied Biosystems, USA). The Jatropha rbcL mRNA served as an Internai control for leaf and Jatropha a-tubulin mRNA served as an internai control for seed samples. Ct values Induded in the analyses were based on 3 blological replicates, with three technicai replicates for each blological sample. Standard déviation was calculated based on 3 blological replicates. Real-time PCR primer sequences are shown In Table 1.

TABLE 1

Real-time PCR Primer Sequences

Primer	Sequence	SEQ ID NO:
FAD2- 1-R	GGTTGAGGAAGGAGGTGGAAG	17
FAD2- 1-F	CCACCATTCACACTTGGTCAG	18
FAD2- 2-F	AGCAATCAAGCCTATATTGGGC	19
FAD2- 2-R	CCAGAGAACTCCTCGGTTGG	20
FAD6-F	TGGTGCATCATACGGCTC	21
FAD6R	ATGTGAACATTGATATCATG	22
rbd-R	CTTCTCCAGCAACGGGCTC	23
rbcl-F	GGAGTTCCGCCTGAGGAAG	24
a-tub-F	GAGGCTGGATCTGGCAAACACGTT	25
a-tub-R	TGTGTAATGACCTCTAGCAAAATTA	26
P7S	TCAATCCATGATGAGCCCACA	27
P4	GTATAAGCTTCATGAGTGTCTGTAATGTTATG	28
P1	GCCGCCACGTGCCGCCACGTGCCGCC	29
hpt-R	TACTTCTACACAGCCATCGGTCCA	30
hpt-F	AAAAAGCCTGAACTCACCGCGACGTCT	31

Southem blot analysis: Total genomic DNA was Isolated from glasshouse-grown material representing the indicated transgenic lines, together with control Jc-MD DNA, by Cetyltrimethyl ammonium bromlde (CTAB) method. Genomic DNA was digested with restriction enzymes EcoFN and Xba\ and separated on 0.8% agarose gels. The gels were processed and transferred to a nylon Hybond-N* membrane (GE Biosclences, USA) following standard procedures (Sambrook et al., 1989). Membranes were hybridized with HPT and FAD2-1 ORF probes. The probes were labelled with [a-“P]-deoxycytidine triphosphate ([a-“P]-dCTP) by random prime synthesis using Amersham Redlprimer II Random Prime Labelllng System (GE Biosciences, USA), following the manufacturées protocol. Hybridization was performed ovemight at 42* C using the ULTRAHyb-Ollgo hybridization buffer (Ambion, USA) and signais were detected by autoradiography.

EXAMPLE 2

Isolation and Characterization of ω6 Oleate Desaturases Genes from J.crucas

The first step to generate high oleic add Jatropha Is to Isolate gene(s) encoding putative microsomal ω6 oleate desaturase. To this end, two cDNAs possessing extensive similarity to extant FAD2 enzymes were isolated from a J. curcas seed cDNA library (Yin ZC et al. unpublished data). The two cDNAs encoded proteins of 383 and 387 amino acids that were 74% identical to each other and 77.3% and 72.1% Identical to Arabldopsls FAD2, respectively. The cDNA with higher sequence identlty to the FAD2 enzyme famlly was deslgnated JcFAD2-1 and the other one was deslgnated JcFAD2-2. JcFAD2-1 has Identical amino add sequences with AtFAD2 at Its enzyme active centre In three conserved His-rich boxes (Fig. 2A), whiie JcFAD2-2 has a variation on a key residue Ala In active site His-rich box 3 (Thr In JcFAD2-2, Fig. 2A). The change of small hydrophobie Ala substituted with polar Thr could potentiaily alter FAD2-2 substrate specifidty and enzyme activity due to the hydrophobie core environment crudal for Its activity.

To Investigate gene expression patterns of FAD2-1 and FAD2-2, RNA was extracted from ail sets of seed development stages (3 weeks, 5 weeks, 7 weeks and 8 weeks after fertilization, correspondîng to the eariy, middle, later and mature stages of Jatropha seed development stages) and used In reverse transcriptase (RT)-PCR reactions containing primera spécifie for each cDNA As shown in Fig. 2B, the FAD2-1 gene Is expressed In both seeds and végétative tissues, while the FAD2-2 gene is expressed highly in seeds and not détectable in leaf. The expression pattern of these two FAD2 genes in Jatropha 1s very slmilar with those In the same Euphorbiaceae: FAD2 and FAH12 in castor bean (Ricinus commuais}, FAD2 and FAX In tung tree (Aleurites fordii). Ail the data above suggests that the JcFAD2-2 may fonction more like an unusual enzyme other than desaturase such as those of FADX and FAH12. Therefore, we chose FAD2-1 as our target for downregulation to produce a high oleic acid composition.

EXAMPLE 3 β-estradiol Induced Cre-lox Recombination Marker Free System in Jatropha

Increasing biosafety concems for genetically modified crops will definitely hinder its commerdalization and hâve led to greater demands for applying technologies aliowing the production of transgenic plants without selectable (e.g., antibiotic résistance) markers.

To produce selectable marker free transgenic Jatropha, we tested a chemically Indudble Cre//oxP-mediated site-specific recombination System, which was first developed by Zuo et al. (2001) in Arabidopsis. Instead of using GFP as a reporter, we selected to silence JcFAD2-1 to make high oleic add and marker free transgenic Jatropha.

A transformation procedure similar to that described above was used to get hygromycinresistance régénération shoots. Once visible shoots came out, we transfered small shoots to marker free induction medium without hygromydn. After two weeks induction, these wellgrowing shoots were subsequently transferred into régénération medium li but without hygromydn. The remalning procedures are same as the transformation procedure described above.

When chemically indudble Cre-lox mediated recombination and DNA exdslon happens, RNAI structure then can be directly driven by the foremost G10-90 In pX7 vector (see the diagram of Fig 3A). As a resuit of the down régulation of JcFAD2-1, there will be a change of fatty add profile. Therefore, we randomly selected 10 putative marker-free small shoots from the plates to extract the genomlc DNA for genotyplng analysis. Using one pair of primera consisting of a forward primer spedfic for the G10-90 promoter and a reverse primer spedfic for the FAD2-1, PCR analysis revealed the small fragment of expected size In 2 out of 10 regenerated shoots. Meanwhlle, there is an amplification band of hygromydn-resistant gene (hpf) in #1-1, suggesting it’s a chimera. On contrast, there is not any hpt gene PCR amplification bands In #12, suggesting it mlght be a pure marker-free transgenic Jatropha. In Arabidopsis, FAD2 encoded desaturase Is responsible for the desaturation of 18:1-ACP to 18:2-ACP. We hypothesized that a réduction of expression of FAD2-1 after Induction should biock the conversion of 18:1-ACP to 1&2-ACP fatty adds. We further used fatty add methyl ester (FAME)-Gas chromatographie (GC) to check thelr fatty add profile in leaf. As predicated, there is higher oleic add content in #1-1 and much higher levei of oieic add In #1-2 compared with regenerated shoots from WT Jatropha cotylédon (Fig. 3C) meanwhile the linoleic add level was slgniflcantly reduced In the marker-free Unes. Thus, we hâve shown that β-estradiol Induced Cre-lox recombination System can be used to generate marker-free transgenic Jatropha. We confirmed the function of JcFAD2-1 on controiling the conversion of oielc add to linoleic acid In Jatropha leaf by stable transformation.

EXAMPLE 4

Molecular Analysis and Oil Composition of X7-FAD2-1 RNAi Unes

Using PCR-based genotyplng, we identified 20 putative pure marker free X7-FAD2-1 RNAI Unes (Fig. 4A) and transferred them to blg pots In a greenhouse for further genetic and chemical analysis on seeds. We collected T1 seeds of these putative marker free events. Endosperm was separated carefully from embryo that we further germlnated at hormone-free medium for T1 plants. Oielc add of the two best Unes, #79 and #170 was found to increase to 50%-60% on contrast of 36.7% in 35S:GFP endosperm (Fig. 4A). Meanwhile the linoleic add was reduced to less than 25% from an original 41% In the control (Fig. 5C). But the change of oielc add composition Is moderate and not as dramatic as that found In medium and TRV-induced FAD21 RNAi Jatropha leaves (Fig. 3C and Ye et al., 2009a). We reasoned that the sllendng effect Is not as good In seeds due to the low Intenslty of G10-90 promoter activity. Our RNA data based on quantitative PCR further showed that there is still 20% JcFAD2-1 RNA in these two events (Fig. 5A). Further quantitative RT-PCR data proved this J cE4 D2-1 knock down effect was genespedfic for there is no affect on the FAD2 homologue FAD2-2 expression in endosperm of these two Unes.

We further germlnated T1 embryo on % MS medium to generate T1 plants to check the JcFAD2~1 RNAI effect on Its végétative organs. We found a simllar considérable réduction on JcFAD2~1 expression level (Fig. 5B).

EXAMPLE 5

Higher Oielc Add Transgenic Unes with Seed Spedfic Promoter

Because leaf oleic add content plays a rôle in envlronmental adaptation of plants It Is more désirable to spedfically change seed oieic add content with minimal effect on the same in végétative organs. To this end, we replaced the G10-90 promoter In the pX7-FAD2-1 RNAI vector with the soybean {Glycine max) 7S seed storage protein promoter which displays seedspedfic expression. The new vector with the soybean 7S promoter was named pX8-FAD2-1 RNAi (Fig. 3A). We generated 20 X8-FAD2-1 RNAI fines which were confirmed to be marker17068 free. RNA analysis showed that line #34 and #291 contained only 0.7% and 1.1% FAD2-1 transcript compared to 35S:GFP control In the endosperm (Fig. 6A). We found that the soybean 7S promoter was also active In Jatropha cotylédons as Indicated by a much lower FAD2-1 RNA accumulation In T1 cotylédons (Fig. 6B). However, there was no signîficant change of FAD2-1 transcript levels In végétative organs such as leaves (Fig. 6C).

There was no obvious oll content différence between line #39 with control endosperm (Fig. 6D). GC analysis data further proved much higher olelc add phenotypes In T1 endosperm of #34 and #291 with 77.4% and 74.7% olelc add accumuiated (Fig. 6E). The linoleic add levels were reduced to less than 5% of total fatty acid In these lines. The total unsaturated fatty acids (olelc and linoleic) In control Jatropha endospems was estimated to be about 78-79%. In lines #34 and #291 almost ail of the unsaturated fatty adds were stored as oleic add. Moreover, the stearic add level is also slightly reduced from 7.7% to 5.4-5.7%. There was no marked différence In C16 fatty adds composition between pX8-FAD2-1RNAI lines and control plants. Consistent with no changes on gene expression level, there is no statistic différence on fatty add profile of true leaf (Fig. 6F). This data further confirmed seed spedfic high olelc add in these lines.

EXAMPLE 6

Southem Blot Analysis on Marker Free Unes X8#34

We performed a Southem blot analysis on line X8#34 to détermine the complexity of the transgenic locus. There Is only one X/joI site in pX8-FAD2-1RNAI vector (Fig. 3A). As we knew X8#34 T0 plant Is a chimera partial marker-free (Fig. 4B). If there is only one copy of T-DNA Insertion In Jatropha genome of #34, there will be two bands with around 5.7kb size différence due to Cre-lox recombination event. Therefore total genomlc DNA of T0 and T1 plants were digested with Xhol and probed with soybean 7S promoter. Southem blot data in Fig. 4A showed two bands with size différence around 5-6 kb In #34 T0 plants and segregated In T1 plants (1-

4). It also suggested #34-2 and #34-4 are single copy and pure marker free, while #34-1 Is a chimera and #34-3 without marker free. To analysis whether #34-2 and #34-4 are marker free and single copy, we further treated total genomic DNA of these two T1 plants with EcoRV and Xbal, which was expected to release a 5K-band from the JcFAD2-1 genomic locus as suggested by Its genomic DNA sequence. An extra band was found in ail of 4 plants of X8#34 and X8#291 (Fig. 4B) but was absent in Jc-MD WT control plant. We stripped the membrane and hybridlzed It with an hpt ORF probe and no signal was detected In any of the transgenic plants. These results confirmed that ail these T1 plants were marker free.

The use of the terms a and “an and “the and similar referents in the context of describlng the invention (espedaliy in the context of the following daims) are to be construed to cover both the slngular and the plural, unless otherwise indicated herein or dearly contradicted by context. The terms comprising, having, induding, and containing are to be construed as openended terms (I.e., meaning induding, but not limited to,’) unless otherwise noted. Redtation of ranges of values herein are merely intended to serve as a shorthand method of referring indivldually to each separate value falling within the range, unless otherwise Indicated herein, and each separate value is incorporated into the spedfication as If It were Indivldually redted herein. Ail methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ail exemples, or exemplary ianguage (e.g., such as) provided herein, Is intended merely to better lllumlnate the invention and does not pose a limitation on the scope of the Invention unless otherwise claimed. No ianguage in the spécification should be construed as indicatlng any non-claimed element as essentiai to the practice of the invention.

Embodiments of thls Invention are described herein, Induding the best mode known to the Inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary sklii In the art upon reading the foregolng description. The inventors expect skilled artisans to empioy such variations as appropriate, and the inventors Intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this Invention indudes ail modifications and équivalents of the subject matter reclted in the daims appended hereto as permltted by applicable iaw. Moreover, any combination of the above-described éléments in ali possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise dearly contradided by context.

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Claims (19)

1. Claims:

   I. An isolated nucleic acid encoding a protein comprising the amlno acid sequence set forth In SEQ ID NO:5.
2. 2. The Isolated nucleic acid of daim 1 which comprises the nudeotide sequence set forth In SEQ ID NO:4 or SEQ ID NO:6.
3. 3. The isolated nudeic add of daim 1 or 2 which further comprises a plant opérable promoter operably linked to the nudeic add.
4. 4. The isolated nudeic add of daim 3, wherein the promoter 1s a seed spedfic promoter.
5. 5. A transgenlc plant cell, plant or plant seed comprising the Isolated nudeic add of any one of claims 1-4 stably integrated Into Its genome.
6. 6. The transgenlc plant cell, plant or plant seed of daim 5, wherein the plant Is Jatropha.
7. 7. The transgenlc plant cell, plant or plant seed of daim 5, wherein the plant Is castor bean or a plant of an oll crop.
8. 8. An Isolated nudeic add encoding an RNAI nucleic add that down régulâtes a native JcFAD2-2 gene.
9. 9. The Isolated nudeic add of daim 8, wherein the native JcFAD2-2 gene encodes a protein comprising the amlno add sequence set forth in SEQ ID NO:5.
10. 10. The Isolated nudeic add of daim 8 or 9 which further comprises a plant opérable promoter operably linked to the RNAI nudeic add.

    II. The isolated nudeic add of daim 10, wherein the promoter Is a seed spedfic promoter.
11. 12. A transgenic plant cell, plant or plant seed comprising the Isolated nuclelc acid of any one of clalms 8-11 stably Integrated Into its genome.
12. 13. The transgenic plant cell, plant or plant seed of daim 12 which further comprises a second nudelc add stably Integrated into its genome wherein the second nucleic add encodes a second RNAi nucleic add that down régulâtes a native JcFAD2~1 gene.
13. 14. The transgenic plant cell, plant or plant seed of daim 13, wherein the native JcFAD2~1 gene encodes a protein comprising the amino add sequence set forth in SEQ ID NO:2.
14. 15. The transgenic plant cell, plant or plant seed of daim 13 or 14, wherein the second nudelc add further comprises a plant opérable promoter operably linked to the second RNAI nudelc add.
15. 16. The transgenic plant ceil, plant or plant seed of daim 15, wherein the promoter Is a seed spedfic promoter.
16. 17. The transgenic plant cell, plant or plant seed of any one of daims 8-16, wherein the plant is Jatropha.
17. 18. The transgenic plant seed of daim 17, wherein, the seed has an oleic acid content greater than 50%, preferably greater than 60%, more preferably greater than 70%, most preferably greater than 75%.
18. 19. The transgenic seed of daim 18, wherein the seed has a linoleic add content less than 5%.
19. 20. The transgenic plant cell, plant or plant seed of any one of daims 8-16, wherein the plant is castor bean or a plant of an oll crop.

    ABSTRACT

    The présent Invention relates to the field of plant molecular blology, more particularty Jatropha mlcrosomal ω6 oleate desaturases. The présent Invention also relates to Jatropha plants 5 or plants of other oil crops having seeds with altered ratios of monosaturated and polyunsaturated fats, ln particular, the présent invention relates to Jatropha plants or plants of other oil crops where the plants exhlblt elevated levels of olelc add.

    2577208PCT2seqeunceLlsti ng.txt

    SEQUENCE LISTING <110> Temasek Life sciences Laboratory Limited