Construction Of Transgenic Strains With Yield Of PUFAs

Polyunsaturated fatty acids (PUFAs), especially long-chain polyunsaturated fattyacids, were important in physiology and health. At present, the main resource ofω6polyunsaturated fatty acids was microbial fermentation while the main resource ofω3polyunsaturated fatty acids was fish oil. The source of fish oil was limited and theextraction of fish oil was complicated. Theω6 andω3 polyunsaturated fatty acids havetwo common disadvantages, expensive and cannot meet market demand. Using themethod of genetic engineering to improve the yield of long-chain polyunsaturated fattyacids of microbial and produce long-chain polyunsaturated fatty acids in oil crops isavailable solutions. In this study, we used the△6,△5 desaturase gene and△6 elongasegene from Phaeodactylum tricornutum and the Pichia pastoris expression vector pAO815to construct a vector contains△6,△5 desaturase gene and△6 elongase gene(pAO1×D6E6D5). Target genes were single copy in this vector pAO1×D6E6D5. Based onthe pAO1×D6E6D5, we constructed a vector pAO2×D6E6D5 in which target genes weredouble copy. Pichia pastoris were transformed with pAO1×D6E6D5 and pAO2×D6E6D5,we had obtained genetic engineering yeast (PpDED and Pp2D2E2D) producinglong-chain polyunsaturated fatty acids. In order to construct plant seed specific expressiontrivalent vector,△6,△5 desaturase gene and△6 elongase gene was respectively under thecontrol of napin promoter and target genes were ligated to vector pCAMBIA1303.Arabidopsis thaliana and Brassica napus were transformed with trivalent vector and wehad obtained transgenic Arabidopsis thaliana producing long-chain polyunsaturated fattyacids and transgenic Brassica napus seedlings. The detailed results are as follows:1. The results of Southern blot of transgenic Pichia indicate that△6,△5 desaturasegene and△6 elongase gene have integrated into the genome of Pichia pastoris. Targetgenes were single copy respectively in transgenic Pichia which transformed withpAO1×D6E6D5, and were double copy respectively in transgenic Pichia whichtransformed with pAO2×D6E6D. 2. The results of semi-quantitative RT-PCR of transgenic Pichia indicate that theexpression level of△6,△5 desaturase gene and△6 elongase gene in Pp2D2E2D were1.87,1.92,1.74 times of in PpDED. This shows that increasing the copy number of targetgene could significantly improve the expression level of mRNA.3. GC-MS was used to analyze the total fatty acids of induced transgenic Pichia.GLA, DGLA, AA, SDA, ETA, and EPA respectively account for 3.5%, 1.4%, 0.1%, 0.6%,0.1%, and 0.05% of total fatty acids in PpDED。In Pp2D2E2D, GLA, DGLA, AA, SDA,ETA, and EPA respectively account for 4.2%, 2.4%, 0.3%, 0.6%, 0.2%, and 0.1% of totalfatty acids in Pp2D2E2D. In Pp2D2E2D, the content of AA and EPA was 3 and 2 times ofin PpDED.4. The conversion efficiency of△6 desaturase gene was 22.7% (ω6) and 20.7% (ω3)in PpDED, in Pp2D2E2D the efficiency was 33.9% (ω6) and 33.3% (ω3). The conversionefficiency of△6 elongase gene was 28.6% (ω6) and 14.3% (ω3), in PpDED, inPp2D2E2D the efficiency was 36.4% (ω6) and 25% (ω3). The conversion efficiency of△5desaturase gene was same in the pathway ofω3 (33.3%) and in the pathway ofω6,increased from 9.7% to 11.1%.5. We had obtained the transgenic Arabidopsis thaliana seedlings producinglong-chain polyunsaturated fatty acids. The results of Southern blot indicated that△6,△5desaturase gene and△6 elongase gene had integrated into the genome of Arabidopsisthaliana. The target genes were especially expressed in seed. In the seeds of transgenic A.thaliana, the content of GLA, DGLA, AA, SDA, ETA and EPA respectively was 6.2%,1.6%, 0.5%,0.9%,0.5% and 0.05% of total fatty acids.6. Through the screening by hygromycin, we had obtained transgenic Brassica napusseedlings.