High γ-Linolenic Acid Gene Engineering Of Brassica Napus L. And Helianthus Annuus L.

Gamma-linolenic acid (GLA; C18:3 A -6,9,12) is a nutritionally important polyunsaturated fatty acid for human. However, GLA sources are not ideal for dietary supplementation due to large fluctuations in availability. As a result, there is a considerable interest in the large-scale production of GLA by transgenic oil crops. My research is focused on introducing â–³6- desaturase gene to conventional oil crops (rapeseed and sunflower) in order to produce r -linolenic fatty acid for functional foods and pharmaceutical products.(1) Cloning and identification of â–³6- fatty acid desaturase gene: The entire coding sequence of â–³6-desaturase was cloned by RT-PCR from filamentous fungus Mucor Circinelloides. The PCR product was subcloned into yeast expression vector pYES2 to generate a recombinant plasmid pYES412. Transformation of Saccharomyces cerevisiae strain INVScl was done by the lithium acetate method, and the recombinant yeast cells were selected on the uracil-deficient medium. Under appropriate medium and temperature, liloleic acid was provided as a substrate to yeast cultures, the level of Y -linolenic acid reached 50.07%. So far, the result we obtained is the best in terms of the level of expression of â–³6-desaturase gene in Saccharomyces cerevisiae , which has a high potential for industrial use.(2) Cloning and identification of seed-specific promoter from sunflower: 200bp , 500bp andl300bp fragment of sunflower Ha ds10 G1 gene promoter was amplified by PCR. The PCR products were ligated to plant expression vector pCAMBIA1301 harbouring the hpt gene as a selectable marker and the gus gene as a reporter gene. The plant expression vectors Ha ds10-GUS were transformed into Tobacco NC89 mediated by Agrobacterium tumefaciens EHA105. It was observed that 1300bp fragment before ATG is a seed-specific promoter by GUS staining the different tissue from transgenic tobacco.(3) Constructing constitutive and seed-specific plant expression vectors: â–³6-desaturase gene was ligated to plant expression vectors driving by 35Spromoter and seed-specific promoter, respectively. The characteristic of two sense plant expression vectors is carrying a â–³6-desaturase gene and a reporter gene at different expression boxes in order to identify transgenic plants by GUS staining.(4) rapeseed genetic transformation with â–³6-desaturase gene to produce GLA: Agrobacterium-mediated transformation had been used to introduce the â–³6- fatty acid desaturase gene into Brassica Napus L. zhongshuang 6. We obtained 10 transgenic rapeseeds which were identified by PCR and Southern hybridization.(5) Establishment of Agmbacterium-mediated transformation of Sunflower and obtaining transgenic sunflower to produce GLA: Sunflower is rich in linoleic acid (50-70%) which is substrate of GLA, so sunflower is a very ideal oil crop to product GLA. The transformation frequency was evaluated by explants with uniform GUS-positive shoot 7 d after co-culture. The results were indicated that hygromycin B concentration of 10mg/L was optimal for selection of transformed tissue;Agrobacterium suspension concentration was OD600= 0.8; Explants were treated with the enzyme solution (0.05% Pectinase and 0. l%cellulase) for 30min; The optimal co-culture temperature was 24C ; Acetosyringone (AS) concentration in the co-culture medium was 100 umol / L, the frequency of explants with uniform GUS expression reached 32.8%. We obtained 11 positive transgenic sunflowers which were identified by PCR, it is pity that fertile plants have not been obtained due to early flowering, We are studying the problem of early flowering leading to abort.