Cloning Of Genes Associated With PUFAs Synthesis Of Mortierella Alpina And Transformation Of Brassica Napus L.

Polyunsaturated fatty acids were one kind of important physiological activity substance to human and had high officinal value. It was a great potential method to produce polyunsaturated fatty acids through oil crops, and to produce long-chain polyunsaturated fatty acids by transferring the related genes in the biosythesis of polyunsaturated fatty acids into plants has turned out to be the hot spot of international lipids conferences recently. Rape is one plant of the Brassica genus of the family Cruiferae. Brassica napus L. has become the third important oil crop following soybean and palm in the world at present. The planting area and output of rape of our country accounted for about thirty percent of the world's total amount, which had an important strategic position in the agriculture production and grain safety. In our research, the related genes in the synthesis of AA and EPA obtained from Mortierella alpina was linked to the plant expression vector with seed-specific promoter, and were transformed to B. napus by Agobacterium-mediated transformation method to build biosynthesis pathway for AA and EPA, which provided one new source for AA and EPA production. The main contents and results of this research work are as follows:(1) A6 desaturase gene,â–³6 elongase gene andâ–³5 desaturase gene were cloned from M. alpina. Then the amino acid sequences encoded by the three genes were analyzed through bioinformatics methods. A domain of cytochrome b5 and three histidine conserved domains namely domainâ… (HX3H) domainâ…¡(HX2HH) and domainâ…¢(QIEHHLFP) were found in the amino acid sequences encoded by bothâ–³6 desaturase gene andâ–³5 desaturase gene. Meanwhile, in the amino acid sequence encoded byâ–³6 elongase gene, a histidine enrichment domain and a tyrosine enrichment domain were found in the amino acid residues position of 183-188 and 221-225, respectively. The evolution of the three genes was analyzed in the research as well.(2) Yeast expression vectors for the three genes were constructed and transferred into Pichia pastoris GS115 to build recombined engineering strains for the function identification of the three genes, respectively. Total protein of the recombined yeast containingâ–³6 desaturase gene was extracted. The target protein band with the size of 51.8 kDa was found after SDS-PAGE electrophoresis, demonstrating that theâ–³6 desaturase could be transcribed and translated normally in yeast. The fatty acid analysis showed thatâ–³6 desaturase could transform LA yielded by yeast itself into GLA, and the output of GLA accounted for 6.3% of the total fatty acids. There was no detection of OTA during the experiment which demonstrated that the A6 desaturase gene could not synthesize new product utilizing ALA or the amount of synthesized product could not be detected. In view of these results, we concluded thatâ–³6 desaturase gene had preference to the LA substrate. After the addition of GLA substrate,â–³6 elongase could catalyze the synthesis of DHGLA, which accounted for 0.31% of the total fatty acids in recombined yeast. While after the addition of DHGLA substrate,2.85% AA was detected in the yeast transformed withâ–³5 desaturase gene. According to the experimental results we suggested that the pathway ofâ–³6 elongase catalysis was the rate-limiting step in the synthesis of polyunsaturated fatty acid.(3) The napin promoter of seed-specific expression in B. napus was cloned. The DNA sequence analysis of the promoter showed that there were rich AT nucleotides and had much regulatory elements, including three TATA-box, seven CAAT-box, one GATA-box, two G-box, and three (CA)n-motif, which was the character of the higher plants promoter. Furthermore, the expression cassettes ofâ–³6 elongase gene andâ–³5 desaturase gene were constructed through the method of enzyme cleavage connection mediated PCR, and the trivalent plant expression vector pCAMBIA1303-D6ELD5 (napin promoter) was built with the napin promoter.(4) B. napus 7633 was transformed with Agrobacterium tumefaciens LBA4404 carrying the pCAMBIA1303-D6ELD5(napin promoter) vector through the methods of callus transformation, vacuum infiltration, and floral dip. The transformation efficiency of callus transformation achieved 1.2%, and the maximum transformation efficiency reached 1.2%,1.8%, and 2.5%, respectively. Hygromycin B was applied to screen the transformed explants and seeds, and more than 500 transgenetic plants were obtained during the reseach. PCR and Southern blot were utilized to analyze the screened positive transgenic plants. The results showed that the exogenous genes have been integrated into the genome of rape.