WO2007113608A1 - Production d'acides gras polyinsatures dans des brassica en utilisant une delta-6-desaturase innovante - Google Patents

Production d'acides gras polyinsatures dans des brassica en utilisant une delta-6-desaturase innovante Download PDF

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Publication number
WO2007113608A1
WO2007113608A1 PCT/IB2006/001023 IB2006001023W WO2007113608A1 WO 2007113608 A1 WO2007113608 A1 WO 2007113608A1 IB 2006001023 W IB2006001023 W IB 2006001023W WO 2007113608 A1 WO2007113608 A1 WO 2007113608A1
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WIPO (PCT)
Prior art keywords
nucleic acid
polyunsaturated fatty
desaturase
fatty acids
plant
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PCT/IB2006/001023
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English (en)
Inventor
Patell Villoo Morawala
K. R. Rajzashri
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Avestha Gengraine Technolgies Pvt Ltd.
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Application filed by Avestha Gengraine Technolgies Pvt Ltd. filed Critical Avestha Gengraine Technolgies Pvt Ltd.
Priority to PCT/IB2006/001023 priority Critical patent/WO2007113608A1/fr
Publication of WO2007113608A1 publication Critical patent/WO2007113608A1/fr

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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

  • the invention relates to a method of producing polyunsaturated fatty acids in transgenic Brassica plants by the Agrobacterium mediated gene transfer of genes involved in the desaturation of the polyunsaturated fatty acids and thereby encoding for polypeptides involved in the biosynthesis of polyunsaturated fatty acids based on the expression of the said genes.
  • the invention additionally relates to the construction of expression cassettes comprising nucleic acid sequences encoding delta-6 desaturase enzyme obtained from Schizochytrium SC-I and the host cells into which the vectors have been introduced.
  • the further aspects relate to the selection and regeneration of the putative transformants based on a method of positive selection.
  • Delta-6 desaturases are the key enzymes required for the synthesis of highly unsaturated fatty acids such as Arachidonic acid, docosahexaenoic acid.
  • the major metabolite product of the n-6 pathway is arachidonic acid (20:4n-6), whilst the major end products of the n-3 pathway are eicosapentanoic acid (EPA) (20:5n-3) and docosahexaenoic acid (DHA) (22:6n-3).
  • EPA eicosapentanoic acid
  • DHA docosahexaenoic acid
  • Delta-6 desaturases catalyses the first and the rate limiting step of the PUFA synthesis. It acts as a gateway for the flow of fatty acids through the desaturation and the elongation pathway.
  • delta-6 desaturase deficiency Although it can act on any long chain fatty acid, the substrate binding affinity increases greatly with the number of double bonds already present. Recent identification of a human case of delta-6 desaturase deficiency underscores the importance of this pathway (Nakamura et al., 2003). A number of delta-6 have been identified. In plants such as the herb, borage (Borago officianalis), the delta-6 desaturase has been identified (Sayanova et al., 1997).
  • an isolated nucleic acid molecule comprising the DNA sequence encoding for the enzyme delta-6 desaturase isolated from the marine organism Schizochytrium.
  • the subject invention relates to the transformation of the said nucleotide sequence into Brassica.
  • Brassica is a genus within the Brassicaceae (Cruciferae), commonly known as the mustard family.
  • the family of about 375 genera and 3200 species includes oilseed crops, vegetables, ornamentals, and many weeds.
  • Brassica contains about 100 species, including rapeseed, cabbage, cauliflower, broccoli, brussels sprouts, turnip, various mustards and weeds. (Willis, 1973).
  • oilseeds have the highest economic value.
  • the oilseed Brassicas are found within Brassica jimcea, Brassica carinata, Brassica srapa (syn.
  • Brassica campestris and Brassica napus and are commonly called oilseed rape.
  • the center of origin of Brassica jimcea Indian mustard is believed to be in Central Asia-Himalayas, with migration to secondary centers in India, China and the Caucasus (mountainous region of south-central Russia).
  • Brassica juncea is a major oilseed crop of the Indian subcontinent and is cultivated as a winter crop in about 6 million hectares of land in rain-fed areas of northern India. Brassica jimcea commonly called, as Black mustard is more important as a spice and oil plant, especially in India. It is the second most widely used oil in several northern and eastern states next to sunflower oil.
  • LC-PUFAs Long Chain Polyunsaturated Fatty Acids
  • EPA eicosapentaenoic acid
  • DPA docosapentaenoic acid
  • DHA docosahexaenoic acid
  • the current invention would permit the production of the novel PUFA's in Brassica species by the incorporation of recombinant vectors comprising the nucleic acid sequences encoding polypeptides possessing the activity of the enzyme delta-6 desaturase. It is the purpose of the present invention to respond to the aforementioned need by providing a new variety of cash crop having a nutritional significance by virtue of the described technology that teaches the method of transformation of the delta-6 desaturase gene into the host plant satisfying the dietary provisions of significant polyunsaturated fatty acids. Also described is the method of selection and regeneration of the putative transformants.
  • This selection system is the use of a positive selectable marker for an efficient transformation and regeneration system.
  • This positive selectable marker enables the identification and selection of genetically modified cells through imparting to the transformed cells the ability to metabolise compounds that cannot be metabolised by the non-transformed cells.
  • the addition of this new compound in the culture medium, as nutrient source during the regeneration process, allows the normal growth and differentiation of transformed cells, while non-transformed are unable to grow and regenerate de novo plants.
  • the published Patent document WO2006028839 relates to the identification of the genes involved in delta-6 desaturase activity, more specifically enzymes which would be utilized for the conversion of linoleic acid to alpha-linolenic acid and in the conversion of alplia-linolenic acid to stearidonic acid. Also described is the use of the enzyme in pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
  • Our invention differs from the aforementioned prior art as the source obtained for encoding delta-6 desaturase is from schizochytrium as against this patent application that has obtained the invention from the Fungi Delacroixia coronata. Additionally the application does not refer to the method of selection and the regeneration of the transformants.
  • the patent application number US6635451 describes relates to the identification of genes involved in the desaturation of PUFA' s at carbon 5 and carbon 6 and the uses thereof.
  • This invention describes the transformation of the genes encoding delta-6 desaturase into Yeast.
  • the current invention significantly differs from the stated prior as the construction of the vectors comprising the delta-6 desaturase gene and the entire transformation protocol is standardised for transformation into Brassica sp.
  • FIG 1 Screening of BAC library with ⁇ -6 desaturase partial cDNA.
  • FIG 2 Strategy followed for the cloning of AGT-D6-XI into pCAMBIA 1390.
  • FIG 3 Map of Construct
  • FIG 4 Amplification of ⁇ -6 desaturase from pGEM-T clone with the restriction enzyme primers.
  • FIG 5 Amplification of genomic DNA from leaf samples of To putative transgenic shootlets (isolated at the time of transfer to rooting media) transformed with AGT-D D- XI construct with (A) D 6 and (B) XI primers
  • FIG 6 Stages of development. SEQUENCE LISTINGS:
  • SEQ ID 1 Full length nucleotide sequence of delta-6 desaturase
  • SEQ ID 2 Corresponding amino acid sequence of delta-6 desaturase.
  • the present invention is directed to the transformation of delta-6 desaturase gene into Brassica with Agrobacterium tumefaciens harboring the recombinant vector comprising the nucleic acid sequence encoding the enzyme delta-6 desaturase.
  • an isolated nucleic acid molecule comprising the delta-6 desaturase sequence.
  • a polypeptide encoded by the nucleic acid molecule according to the invention is provided.
  • a particular aspect of the invention is directed to the isolation of the delta-6 desaturase gene construct from Schizochytrium.
  • a the nucleic acid sequences depicted herein are incorporated in a vector and is operably linked to a promoter or other regulatory elements for the expression of the gene in the host cell.
  • the most desired aspect of the invention relates to the transformation of the constructed vector into the host plant, distinctly teaching a person skilled in art the method of explant preparation, infection, co-cultivation as well as the method of selection and regeneration of the transformed explants.
  • Brassica juncea is a major oilseed crop of the Indian subcontinent and is cultivated as a winter crop in about 6 million hectares of land in rain fed areas of northern India. Brassica juncea commonly called, as Black mustard is more important as a spice and oil plant, especially in India. It is the second most widely used oil in several northern and eastern states next to sunflower oil.
  • ⁇ 6 desaturase is the first enzyme in the ⁇ -3 pathway that converts ⁇ -linolenic acid (18:3) to stearidonic acid (18:4).
  • SC-I Schizochytrium
  • sequencing of the SC-I EST library led to the identification of a partial desaturase cDNA. Further, the screening of the cDNA library of SC-I with the partial desaturase gene led to the identification of a number of clones. One of these clones of 617bp was found to be homologous to ⁇ -6 desaturase of several organisms.
  • the sequence had an ORP running through till 273 bases.
  • the 3'UTR is 401 bases
  • a polyadenylation signal "AATAA' is seen towards the 3' end of the sequence.
  • the ⁇ -6 desaturase sequence was subjected to a motif search for confirmation of the presence of the desaturase domain.
  • the gene has the complete desaturase domain and the Cytochrome b5 domain characteristic of the functional desarurases.
  • the ⁇ -6 desaturase ORF has been cloned in pGEM (T) Easy vector (Promega) .
  • the ORF of SCl ⁇ 6 desaturase was amplified from the pGEM-T easy vector using primers carrying the Kpnl and Spel sites.
  • the ⁇ 6 desaturase containing amplicon was restricted with Kpnl and Spel and directionally cloned into the corresponding sites of the pCAMBIA-1380 vector under the ubiquitin promoter (AGT-D6).
  • the GUS reporter gene was replaced with ⁇ 6 desaturase gene under the ' ubiquitin promoter.
  • the entire cassette comprising of Ubiquitin promoter and ⁇ 6 desaturase gene was restricted with Bam HI and Spel and directionally cloned into the corresponding sites of pC AMB IA-XI to obtain AGT-D6-XI.
  • FIG 3 shows the results of the amplification.
  • the map of the construct AGT-D6-XI is represented in FIG 4.
  • the AGT-D6-XI construct has the following components:
  • the construct has been transformed into Agrobacterium tumefaciens strain GV3101.
  • the colonies obtained were verified for the presence of XI and ⁇ 6 desaturase and used for subsequent transformation experiments.
  • TabIe-2 Fatty acid profile of 12 different varieties of Brassicajuncea seeds.
  • Brassica jimcea variety 'variina' is an Indian national check variety released in 1964 used widely in North India for Mustard oil production; It is a medium duration variety (115-130 days) with an average yield of 1200 kg/ha and an oil content of 39.8 % and is moderately resistant to Altemaria blight. Of the 12 varieties studied, this variety had the minimum erucic acid content (35.5%) and the maximum amount of the unsaturated fatty acids - linoleic acid and linolenic acid and was hence selected for transformation of Brassicajuncea.
  • the Brassica jimcea 'varuna' cotyledonary petioles explants were transformed with the AGT-D6-XI construct following a protocol comprising of the following steps.
  • A) Growing of seedlings 100-150 seeds were sterilized in 10 ml of 70% alcohol by rinsing for 2 minutes. The alcohol was decanted and the seeds were rinsed in sterile milliQ water. The seeds were subsequently treated in 10 ml of 0.1% Mercuric Chloride for 5 minutes and decanted. The sterilized seeds were washed vigorously 4-5 times with 20 ml of sterile milliQ water and dried on sterile blotting paper.30-35 seeds were placed per bottle of 1/2 MS media and allowed to grow for two days at 25° C in BOD (dark condition) and three days in light/dark conditions of 16h light: 8h dark, at 25 0 C temperature and 60% humidity.
  • the cotyledonary petiole region was infected by dipping in agro suspension for 10-15 seconds and immediately placed back into the cocultivation medium with petiole region pierced into the medium.
  • the infected explants were co-cultivated on Co cultivation Media (CCM-BJX) (MS media containing 0.1 mg/1 NAA +2mg/l BAP +15g/l sucrose +150mg/l Cefataxime) in BOD at 28 0 C for three days.
  • CCM-BJX Co cultivation Media
  • the explants were placed in Selection Media I (SlM-BJX) (MS media containing 0.2 mg/1 NAA +2 mg/1 BAP +25 g/1 Xylose +5g/l sucrose +250 mg/1 Cefotaxime) and allowed to grow for 4 weeks in light/dark conditions of 16h light: 8h dark, at 25 0 C temperature and 60% humidity.
  • Selection Media I SlM-BJX
  • the selected explants were transferred to II Selection Media (S2M-BJX) (MS media containing 0.1 mg/1 NAA +3 mg/1 BAP +29 g/1 Xylose +1 g/1 sucrose +250 mg/1
  • S2M-BJX II Selection Media
  • Cefotaxime Cefotaxime
  • BJX (MS media containing 3 mg/1 BAP +30 g/1 sucrose +250mg/l Cefotaxime) and allowed to grow for 2 week in light/dark conditions of 16h light: 8h dark, at 25 0 C temperature and 60% humidity
  • RM-BJX Rooting Media
  • FIG 6 shows the different stages of development of transgenic Brassica juncea plants carrying the delta-6 desaturase gene. These transgenic plants show no significant differences in the phenotype compared to the untransformed plants. Reported is the successful regeneration of transgenic Brassica juncea plants carrying ⁇ 6 desaturase gene using Xylose Isomerase as the selection marker. A transformation efficiency of ca. 35 % is observed using this selection system.
  • delta-6 desaturase corresponds to the production of stearidonic acid which is the product of delta-6 desaturase in omega-3 pathway.
  • the transgenic Brassica juncea containing the transgene and the native elongase gene can also produce Docosahexaenoic acid as one of its end products that may be formed by further desaturation and elongation and is indicative of the fact that incorporation of delta-6 desaturase can be used to transform plant cells for altered polyunsaturated fatty acid compositions.

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Abstract

La présente invention concerne un procédé de production d'acides gras polyinsaturés dans des plants de Brassica transgéniques par le transfert sous la médiation d'Agrobacterium de gènes qui participent à la désaturation des acides gras polyinsaturés et qui codent ainsi pour des polypeptides qui participent à la biosynthèse des acides gras polyinsaturés à la suite de l'expression desdits gènes. L'invention concerne également la construction de cassettes d'expression comprenant des séquences d'acide nucléique codant pour l'enzyme delta-6 désaturase tirée de Schizochytrium SC-I et les cellules hôtes dans lesquelles les vecteurs ont été introduits. D'autres aspects concernent la sélection et la régénération des transformants putatifs par un procédé de sélection positive.
PCT/IB2006/001023 2006-04-06 2006-04-06 Production d'acides gras polyinsatures dans des brassica en utilisant une delta-6-desaturase innovante WO2007113608A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173277B (zh) * 2007-10-23 2010-12-08 北京市农林科学院 一种获得转基因玉米的方法
CN103167518A (zh) * 2011-12-15 2013-06-19 上海粱江通信系统股份有限公司 基于位置分析提高发现非法复制手机sim卡准确率的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003072784A1 (fr) * 2002-02-27 2003-09-04 Rothamsted Experimental Station Delta 6-desaturases de primulacees, plantes exprimant ces dernieres et huiles contenant des acides gras polyinsatures
WO2004071467A2 (fr) * 2003-02-12 2004-08-26 E. I. Du Pont De Nemours And Company Production d'acides gras polyinsatures a chaines tres longues dans des plantes a graines oleagineuses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003072784A1 (fr) * 2002-02-27 2003-09-04 Rothamsted Experimental Station Delta 6-desaturases de primulacees, plantes exprimant ces dernieres et huiles contenant des acides gras polyinsatures
WO2004071467A2 (fr) * 2003-02-12 2004-08-26 E. I. Du Pont De Nemours And Company Production d'acides gras polyinsatures a chaines tres longues dans des plantes a graines oleagineuses

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Title
ABBADI A ET AL: "Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: Constraints on their accumulation", PLANT CELL, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 16, no. 10, October 2004 (2004-10-01), pages 2734 - 2748, XP002334261, ISSN: 1040-4651 *
HALDRUP A ET AL: "Positive selection: A plant selection principle based on xylose isomerase, an enzyme used in the food industry", PLANT CELL REPORTS, SPRINGER VERLAG, DE, vol. 18, no. 1-2, November 1998 (1998-11-01), pages 76 - 81, XP002407645, ISSN: 0721-7714 *
KINNEY ET AL: "Metabolic engineering in plants for human health and nutrition", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 17, no. 2, April 2006 (2006-04-01), pages 130 - 138, XP005365879, ISSN: 0958-1669 *
WARD ET AL: "Omega-3/6 fatty acids: Alternative sources of production", PROCESS BIOCHEMISTRY, ELSEVIER, NL, vol. 40, no. 12, December 2005 (2005-12-01), pages 3627 - 3652, XP005637930, ISSN: 1359-5113 *
WU G ET AL: "STEPWISE ENGINEERING TO PRODUCE HIGH YIELDS OF VERY LONG-CHAIN POLYUNSATURATED FATTY ACIDS IN PLANTS", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 23, no. 8, August 2005 (2005-08-01), pages 1013 - 1017, XP007900180, ISSN: 1087-0156 *
ZHOU ET AL: "Heterologous production of GLA and SDA by expression of an Echium plantagineum DELTA6-desaturase gene", PLANT SCIENCE, LIMERICK, IE, vol. 170, no. 3, March 2006 (2006-03-01), pages 665 - 673, XP005254587, ISSN: 0168-9452 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173277B (zh) * 2007-10-23 2010-12-08 北京市农林科学院 一种获得转基因玉米的方法
CN103167518A (zh) * 2011-12-15 2013-06-19 上海粱江通信系统股份有限公司 基于位置分析提高发现非法复制手机sim卡准确率的方法

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