高山被孢霉多不饱和脂肪酸合成相关基因克隆及转化甘蓝型油菜研究-生物制药工程专业论文.docxVIP

华中科技大学博士学位论文 华 中 科 技 大 学 博 士 学 位 论 文 IV IV accounted for 6.3% of the total fatty acids. There was no detection of OTA during the experiment which demonstrated that the Δ6 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 that Δ6 desaturase gene had preference to the LA substrate. After the addition of GLA substrate, Δ6 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 with Δ5 desaturase gene. According to the experimental results we suggested that the pathway of Δ6 elongase catalysis was the rate-limiting step in the synthesis of polyunsaturated fatty acid. 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 of Δ6 elongase gene and Δ5 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. 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