WO2009070872A1 - Procédé d'augmentation de la production d'acide érucique par silençage du gène fad2 de l'arn en épingle à cheveux et surexpression du gène fae - Google Patents

Procédé d'augmentation de la production d'acide érucique par silençage du gène fad2 de l'arn en épingle à cheveux et surexpression du gène fae Download PDF

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WO2009070872A1
WO2009070872A1 PCT/CA2008/002084 CA2008002084W WO2009070872A1 WO 2009070872 A1 WO2009070872 A1 WO 2009070872A1 CA 2008002084 W CA2008002084 W CA 2008002084W WO 2009070872 A1 WO2009070872 A1 WO 2009070872A1
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construct
erucic acid
plant
fad2
gene
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PCT/CA2008/002084
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Elzbieta Mietkiewska
David C. Taylor
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National Research Council Of Canada
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • 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
    • C12N15/8242—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
    • C12N15/8247—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 involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • This invention relates generally to biotechnology and, more particularly to methods for producing bio-products in plants.
  • erucic acid high erucic acid
  • Brassicaceae Modification of high erucic acid (HEA) germplasm of the Brassicaceae to increase the content of erucic acid (22:1 ⁇ 13) in the seed oil for industrial niche markets (Jadhav et al., 2005) is an important goal in the industry.
  • HEA cultivars are of high interest for industrial purposes because 22:1 is a valuable feedstock with more than 1000 potential or patented industrial applications (Scarth and Tang, 2006).
  • erucamide which is used as a surface-active additive in coatings and in the production of plastic films as an anti-block or slip-promoting agent.
  • Many other applications are foreseen for erucic acid and its hydrogenated derivative behenic acid, e.g.
  • the fatty acid desaturase (oleate desaturase), FAD2 is one of the crucial enzymes for the production of polyunsaturated fatty acids in plants (Okuley et al., 1994).
  • FAD2 fatty acid desaturase
  • antisense and cosuppression approaches it was possible to increase the pool of 18:1 available for elongation to enhance production of erucic acid in B. ca ⁇ nata seeds (Jadhav et al., 2005).
  • the antisense and cosuppression strategies have variable and unpredictable effectiveness and require the production of large populations of transgenic plants to obtain a reasonable number of lines showing sufficient levels of target gene suppression (Liu et al., 2002).
  • RNA interference in plants is mediated by sequence-specific degradation of dsRNA has led to the development of highly efficient methods of post transcriptional gene silencing (PTGS).
  • Constructs specially designed to express dsRNA in plants in the form of self-complementary hairpin RNA (hpRNA) elicit a high degree and frequency of PTGS of endogenous genes (Smith et al., 2000; Stoutjesdijk et al., 2002).
  • hpRNA constructs have great potential for genetic manipulation to improve crop traits (Wang et al., 2000).
  • B. carinata holds considerable promise as an alternative crop platform for industrial oil production and high-erucic oils in particular.
  • S. carinata is easily transformed at very high efficiency (Babic et al., 1998), is highly disease (e.g. blackleg)-resistant, and is drought-resistant, amenable to growth in hotter, drier regions such as the brown soil areas of southern Saskatchewan. It also has negligible out-crossing to canola, and therefore poses little or no risk of contaminating oils destined for the food and feed chain.
  • nucleic acid construct for increasing production of erucic acid in Brassica carinata plants, the construct comprising: an intron-spliced hairpin RNA nucleotide sequence for silencing fatty acid desaturase (FAD2) gene expression, the hairpin RNA nucleotide sequence comprising intron-interrupted inverted repeats of a nucleotide sequence cloned from the 3' untranslated region of a fatty acid desaturase (FAD2) gene of Brassica carinata; a heterologous nucleotide sequence from Crambe abyssinica coding for a fatty acid elongase (FAE); and, a seed-specific promoter.
  • FAD2 intron-spliced hairpin RNA nucleotide sequence for silencing fatty acid desaturase
  • a partial 3'-UTR of the B. carinata FAD2 gene was used to prepare an intron-spliced hpRNA construct to silence the FAD2 gene and consequently, to increase the pool of oleic acid available for elongation.
  • the increased pool of oleic acid contributes to a dramatic increase in the content of erucic acid in Brassica seeds when combined with heterologous C. abyssinica FAE expression.
  • Any suitable intron for example the intron from pyruvate orthophosphate dikinase (pdk) gene, may be used to separate the inverted repeats of the hairpin RNA.
  • the level of erucic acid (22:1) in the triacylglycerols (TAGs) of cells, seeds and plants of the present invention is elevated by at least 10% by weight, based on weight of total TAGs, over the level of erucic acid in wild type cells, seeds and plants.
  • the seed-specific promoter allows for over-expression in seeds without affecting expression in other tissues.
  • preferred promoters used in over- expression of enzymes in seed tissue are the seed-specific napin promoter, and an ACP promoter as described in PCT International Publication WO 92/18634, published October 29, 1992, the disclosure of which is herein incorporated by reference.
  • the promoter and termination regulatory regions will be functional in the host plant cell and may be heterologous (that is, not naturally occurring) or homologous (derived from the plant host species) to the plant cell and the gene.
  • the seed-specific napin promoter is particularly preferred.
  • the termination regulatory region may be derived from the 3' region of the gene from which the promoter was obtained or from another gene. Suitable termination regions which may be used are well known in the art and include Agrobacterium tumefaciens nopaline synthase terminator (Nos T), A. tumefaciens mannopine synthase terminator (Mas T) and the CaMV 35S terminator (T35S). Particularly preferred termination regions for use herein include the Nos T termination region.
  • a construct for use herein is comprised within a vector, most suitably an expression vector adapted for expression in an appropriate host (plant) cell.
  • a vector most suitably an expression vector adapted for expression in an appropriate host (plant) cell.
  • any vector which is capable of producing a plant comprising the introduced nucleic acid sequence will be sufficient.
  • Suitable vectors are well known to those skilled in the art and are described in general technical references such as Pouwels et al., (1986).
  • Particularly suitable vectors include the pCR2.1 vector, the pRD400 vector and the Ti plasmid vector.
  • Transformation techniques for introducing the nucleic acid constructs into host cells are well known in the art and include such methods as micro-injection, using polyethylene glycol, electroporation, or high velocity ballistic penetration.
  • a preferred method relies on /4gro ⁇ actem//77-mediated transformation.
  • those plant cells or plants into which the desired nucleic acid has been incorporated may be selected by such methods as antibiotic resistance, herbicide resistance, tolerance to amino-acid analogues or using phenotypic markers.
  • Various assays may be used to determine whether the plant cell shows an increase in gene expression, for example, Northern blotting or quantitative reverse transcriptase PCR (qRT-PCR).
  • Whole transgenic plants may be regenerated from the transformed cell by conventional methods. Such plants produce seeds containing the genes for the introduced trait and can be grown to produce plants that will produce the selected phenotype.
  • Plants transformed with a construct of the instant invention may be grown. Seeds of the transgenic plants are harvested and the product of interest is extracted. The extracted product is used for subsequent incorporation into a composition, for example a pharmaceutical composition, a nutraceutical composition or a food composition.
  • Fig. 1 is a schematic diagram (not to scale) of XD and XS constructs used to transform Brassica carinata plants. Both constructs, driven by napin promoter (Napin P), have an inverted repeat of a 142 bp fragment (3'-UTR) corresponding to the 3'-UTR of the B. carinata FAD2 gene (GenBank accession no: DQ250814) separated by the intron of pdk (Wesley et al. 2001).
  • the XS construct also contains the coding region of the C. abyssinica FAE gene (CaFAE).
  • NPTW neomycin phosphotransferase gene
  • NosP NOS promoter
  • LB T-DNA left border
  • RB right border
  • the positions of the restriction enzyme sites used for the cloning are as indicated.
  • Fig. 2 depicts proportion of unsaturated fatty acids in seed oils from nontransformed B. carinata (WT) and B. carinata T 1 independent lines transformed with XD (A) and XS (B) constructs. The values are the average ⁇ SD of three determinations.
  • Fig. 3 depicts correlation of FAD2 gene silencing activity expressed as an oleic acid desaturation proportion (ODP) value (gray bars) with erucic acid content ( ⁇ ) in seed oils from nontransformed B. carinata (WT) and selected B. carinata T 1 independent lines transformed with the XD and the XS constructs.
  • ODP oleic acid desaturation proportion
  • Fig. 4 depicts northern blot analysis of FAD2 gene expression in nontransformed ⁇ . carinata (WT) and in T1 transgenic seeds carrying the XD and XS constructs.
  • Fig. 5 depicts fatty acid composition of individual lipids from mature seeds of wild type S. carinata (WT) and XS-18A transgenic line. The values are the average ⁇ SD of three determinations.
  • Brassica carinata plants were grown under sterile conditions on MS medium (Murashige and Skoog, 1962) during transformation and tissue culture.
  • Transgenic B. carinata plants were grown in the greenhouse at the Kristjanson Biotechnology Complex greenhouses, Saskatoon, SK, under natural light conditions supplemented with high- pressure sodium lamps with a 16 h photoperiod (16 h of light and 8 h of darkness) at 22 0 C and a relative humidity of 25 to 30%.
  • GTCTGCTACGGTCTCTACCG-3' was performed using the SMARTTM RACE kit (CLONTECH).
  • a cDNA prepared from S. carinata developing seeds was used as a template for PCR amplification during 30 cycles of the following program: 94 0 C for 30 sec,
  • a 142 bp region of the FAD2 3'-UTR (SEQ ID NO: 3) was amplified by PCR with primers: 5'-ctcgag GGATGATGATGGTTTAAGA-3' (SEQ ID NO: 4) (lower case shows restriction site for Xho ⁇ ) and 5'-ggtaccCCATATCACATAATTTAAAGCC-3' (SEQ ID NO: 5) (lower case shows restriction site for Kpn ⁇ ) and cloned in the sense orientation into Xho ⁇ and Kpn ⁇ sites of pKannibal resulting in pKannibal/A plasmid.
  • the 3' UTR was amplified with primers 5'-tctagaGGATGATGATGGTTAAGA-3' (SEQ ID NO: 6) (lower case shows restriction site for Xho ⁇ ) and 5'-aagcttCCATATCACATAATTTAAAGCC-3' (SEQ ID NO: 7) (lower case shows restriction site for Hind ⁇ ) and then cloned in the antisense orientation in Hind ⁇ and Xba ⁇ sites of pKannibal/A giving pKannibal/A-B.
  • the napin promoter (Josefsson et al., 1987) was ligated into pCR2.1 as an Xho ⁇ -Sac ⁇ fragment.
  • Xho ⁇ -Xba ⁇ cassette carrying intron-interrupted inverted repeats of the FAD2 3'-UTR was excised from pKANNIBAL/A-B and subsequently cloned into the respective sites of pCR2.1 vector (Invitrogen) behind the napin promoter.
  • the resulting plasmid was named XC.
  • a NOS terminator (Bevan, 1983) amplified by PCR with primers 5'-tctagaGATCGTTCAAACATTTGGCAA-3' (SEQ ID NO: 8) (lower case shows restriction site for Xba ⁇ ) and 5'-ggtcgacCGATCTAGTAACATAGATGAC-3' (SEQ ID NO: 9) (lower case shows restriction site for Sa/I) and subsequently as Xba ⁇ -Sal ⁇ fragment, was ligated with the Xba ⁇ -Sac ⁇ fragment from the XC plasmid into the respective sites of pRD400 (CLONTECH). The resulting plasmid was named XD (Fig. 1).
  • a C. abyssinica ORF (SEQ ID NO: 10) was amplified by PCR with the primers: 5'-cccgggATGACGTTCCATTAACGTAAAG-3' (SEQ ID NO: 11) (lower case restriction site for Sma ⁇ ) and 5'-ggatccTTAGGACCGACCGTTTTGG-3' (SEQ ID NO: 12) (lower case restriction site for BamH ⁇ ).
  • the napin promoter was amplified with primers 5'-gaattcAAGCTTTCTTCATCGGTG-3' (SEQ ID NO: 13) (lower case restriction site for EcoRI) and 5'-cccgggGTCCGTGTATGTTTTTAATC-3' (SEQ ID NO: 14) (lower case restriction site for Smal).
  • the NOS terminator was generated by PCR with the primers 5'-ggatccGATCGTTCAAACATTTGGCAA-3' (SEQ ID NO: 15) (lower case restriction site for BamH ⁇ ) and 5'-gagctcCGATCTAGTAACATAGATGAC-3' (SEQ ID NO: 16) (lower case restriction site for Sacl).
  • the napin promoter as an EcoR ⁇ -Sma ⁇ fragment, the C. abyssinica FAE as an Sma ⁇ -BamH ⁇ fragment and the Nos terminator as a BamH ⁇ -Sac ⁇ fragment were ligated into the EcoRI-Sacl sites of pBluescript Il (SK+), resulting in plasmid ZB. Subsequently the EcoRI-Sacl cassette was excited from the ZB plasmid and cloned into the respective sites of the XD plasmid resulting in plasmid XS (Fig. 1).
  • the final binary vectors XD and XS were electroporated into Agrobacterium tumefaciens cells strain GV3101 containing helper plasmid pMP90 (Koncz and Schell, 1986). Plasmid integrity was verified by DNA sequencing following its re-isolation from A. tumefaciens and transformation into E. coli.
  • Brassica carinata plants were transformed by the method of Babic et al. (1998). Transgenic plants were selected and analyzed as described by Montgomerykiewska et al. (2007).
  • RNA from B. carinata plant material was isolated as described by Lindstom and Vodkin (1991). 20 micrograms of RNA was fractionated on a 1.4% (w/v) formaldehyde-agarose gel and the gels were then stained with ethidium bromide to ensure that all lanes had been loaded equally (Sambrook et al., 1989). The RNA was subsequently transferred to Hybond N+ membrane (Amersham Biosciences, Baie d ' Urfe, Canada).
  • a 0.5-kb probe containing the 3' part of FAD2 gene was generated by PCR using primers ⁇ '-GTCTGCTACGGTCTCTACCG-S' (SEQ ID NO: 17) and ⁇ '-TCATAACTTATTGTTGTACCAG-S' (SEQ ID NO: 18) and subsequently radioactively labeled with 32 P using a Random Primers Labeling kit (Invitrogen).
  • Membranes were hybridized at 60 0 C overnight. The filters were washed once in 1x SSPE, 0.1% SDS for 15 min and in 0.1x SSPE, 0.1% SDS for 5-10 min at the temperature of hybridization. The blots were exposed to X-OMAT-AR film (Kodak, Rochester, NY, USA).
  • abyssinica FAE was generated by PCR using primers ⁇ '-ATGACGTCCATTAACGTAAAG-S' (SEQ ID NO: 21) and ⁇ '-GGACCGACCGTTTTGGGC-S' (SEQ ID NO: 22) and was used for the analysis of plants transformed with the XS construct.
  • the labeling and hydridization were as described above.
  • the total fatty acid content and acyl composition of B. carinata seed oils were determined by gas chromatography of the FAMEs with 17:0 FAME as an internal standard as described (Katavic et al., 2001 , Taylor et al., 2002, Ricokiewska et al., 2007).
  • the lipid class separation was carried out according to the method of Christie (1982). Polar and neutral lipids species were separated by TLC on Silica Gel 60 H plates developed 4 cm in diethyl ether, air dried and then developed in hexane:diethyl ethe ⁇ acetic acid (70:30:1 , v/v/v).
  • Brassica carinata plants were transformed with two constructs: XD, targeted at the endogenous FAD2 gene utilizing intron-spliced hpRNA-mediated gene silencing, and XS targeted at silencing the FAD2 gene along with heterologous expression of the Crambe abyssinica FAE gene (Fig. 1). Eleven T 0 plants carrying the XD construct arising from 7 independent transgenic lines, were identified that were both resistant to kanamycin and PCR-positive for the presence of the transgene. Thirty six plants were transformed with the XS construct representing 20 independent lines (data not shown).
  • the fatty acid composition of T 1 seeds from individual plants was determined for all transformants.
  • the best independent transgenic lines are shown in Fig. 2A.
  • Seed specific silencing of the FAD2 gene resulted in a significant reduction of the level of 18:2 from 19.0% in the wild type background to as low as 4.5 % in line XD-4D.
  • the strong reduction of the level of 18:2 was directly correlated with a significant increase in the proportion of oleic acid up to 21.2% in the best transgenic line carrying the XD construct, compared to 5.7% in the wild type background.
  • the increased pool of 18:1 was utilized by the endogenous microsomal elongation complex (FAE) and this led to the increase in the proportion of erucic acid to as high as 50.6% in line XD-5A, compared with 40.0% in WT seeds, a 26.5% proportional increase over wild-type levels.
  • the high increase in erucic acid achieved in the current study through ihpRNA-mediated silencing of the FAD2 gene is considerably greater than that reported by Jadhav et al. (2005), where transformation with FAD2 sequence in an antisense orientation resulted in a net maximum of 17% increase in the proportion of erucic acid in the best line, compared to the WT B. carinata seeds.
  • Oleate desaturase is highly active in developing seeds of wild type ⁇ . carinata, with 85% of 18:1 being converted to 18:2 and 18:3, for an oleic acid desaturation proportion (ODP) value of 0.85 (Table 1).
  • ODP oleic acid desaturation proportion
  • Many transgenic T1 plants carrying XD and XS constructs showed a considerable reduction in the ODP value, to as low as 0.39, indicating a profound (55%) down-regulation of oleate desaturation.
  • Most transgenic plants have ODP values from 0.4-0.7 meaning that only 40-70% of oleic acid produced in developing seeds carrying the silencing constructs was converted to polyunsaturated 18- carbon fatty acids compared with 85% in nontransformed B. carinata seeds. As shown in Fig.
  • thaliana the highest efficiency of FAD2 gene silencing was achieved using an iHP construct utilizing a 120-bp fragment of FAD2 3'- UTR (Stoutjesdijk et al., 2002).
  • the presence of the intron in an iHP construct may result in increased or more stable transcript levels than in the nonintron-containing hpRNA constructs (Tanaka et al., 1990; Stoutjesdijk et al., 2002).
  • TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes by a novel type of Agrobacterium binary vector. MoI. Gen. Genet. 204, 383-396.
  • Arabidopsis FAD2 gene encodes the enzymes that is essential for polyunsaturated lipid synthesis. Plant Cell 6, 147-158.

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Abstract

La 3'-UTR du gène FAD2 de Brassica carinata a été clonée par PCR et utilisée pour préparer un produit de construction d'ARN en épingle à cheveux à intron-épissé (ihpRNA). Ce produit de construction, lorsqu'il est exprimé dans B. carinata, a conduit à un degré élevé de silençage du gène FAD2 accompagné par une forte augmentation de la teneur en acides oléique et érucique jusqu'à respectivement 267 % et 27 %, par comparaison avec celle du fond de type sauvage (WT). Un second produit de construction contenant de l'ihpRNA ciblé sur le gène FAD2 endogène en plus du gène FAE hétérologue de Crambe abyssinica sous le contrôle du promoteur de la napine spécifique des semences a été utilisé pour transformer B. carinata. Cette démarche a conduit à une augmentation plus spectaculaire de la teneur en acide érucique, jusqu'à 41 % dans les semences de disjonction de T1 par comparaison avec celle du témoin WT.
PCT/CA2008/002084 2007-12-03 2008-11-26 Procédé d'augmentation de la production d'acide érucique par silençage du gène fad2 de l'arn en épingle à cheveux et surexpression du gène fae WO2009070872A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052162A1 (fr) * 2003-11-25 2005-06-09 National Research Council Of Canada Genes elongase d'acide gras (fae) et leur utilite dans l'augmentation de l'acide erucique et autres proportions d'acide gras a tres longue chaine dans l'huile de graines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052162A1 (fr) * 2003-11-25 2005-06-09 National Research Council Of Canada Genes elongase d'acide gras (fae) et leur utilite dans l'augmentation de l'acide erucique et autres proportions d'acide gras a tres longue chaine dans l'huile de graines

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KANRAR, S. ET AL.: "Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea fatty acid elongationl (BjFAEl) gene.", PLANT CELL REPORTS, vol. 25, May 2005 (2005-05-01), pages 148 - 155 *
MIETKIEWSKA, E. ET AL.: "Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet.", PLANT BIOTECHNOLOGY JOURNAL, vol. 5, September 2007 (2007-09-01), pages 636 - 645 *
MIETKIEWSKA, E. ET AL.: "Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds.", MOLECULAR BREEDING, vol. 22, 4 July 2008 (2008-07-04), pages 619 - 627 *
MIETKIEWSKA, E. ET AL.: "Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid.", PLANT PHYSIOLOGY, vol. 136, September 2004 (2004-09-01), pages 2265 - 2675 *
SMITH, N.A. ET AL.: "Total silencing by intron-spliced hairpin RNAs.", NATURE, vol. 407, September 2000 (2000-09-01), pages 319 - 320 *
STOUTJESDIJK, P.A. ET AL.: "hpRNA-mediated targeting of the arabidopsis FAD2 gene gives highly efficient and stable silencing.", PLANT PHYSIOLOGY, vol. 129, August 2002 (2002-08-01), pages 1723 - 1731 *

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