Manipulation Of Key Genes For Fatty-acid Synthesis To Increase Erucic Acid Content In Rapeseed Seeds

Erucic-acid(C22:1) in Brassica napus seeds which belongs to very long chain fatty acids(VLCFAs) is an important feedstock for industrial needs,and the main resource is high erucic acid brassica seeds.The fael gene encodes a condensing enzyme involved in the biosynthesis of VLCFAs,and is the ratelimiting factor for C22:1 synthesis.The content of oleic-acid(C18:1),the substrate of C22:1,is also closely related to 22:1.Therefore the change of C 18:1 through the regulation of fad2 and fad3 could influence the content of C22:1.Together,manipulation of above three genes could result in dramatic increase of C22:1 content in rapseeds.In the study,the fae1,fad2,fad3 and napin gene were cloned from high C22:1 Brassica napus,and 5 vectors were constructed including over-express vector of fae1; anti-sense express vector of fad2 and fad3;RNAi vector of fad2 and fad3. Agrobacterium turnefaciens-mediated transformation of Arabidopsis and rapeseed hypocotyls was conducted to generate transgenic plants with increased C22:1 content.For transgenic Arabidopsis,T₂ seeds were harvested and analysed for fatty acid composition,the major results are as follows.(1)In fad2 anti-sense express vector transformants,the average content of oleic-acid(C18:1),linolic-acid(C18:2) and linolenic-acid(C18:3) was 13.93%,32.97%, 19.69%,while 9.70%,33.44%,24.09%,respectively,in wild type arabidopsis.In fad2 RNAi vector transformants,the average content of 18:1,18:2 and 18:3 was 13.94%, 31.07%,19.61%.Compared to Wt,18:1 was ascended and 18:2,18:3 were descented. Meanwhile,C22:1 was also up to 2.31%and 2.37%,higher than Wt(2.30%).(2)In fad3 anti-sense express vector transformants,the average content of C18:1, C18:2 and C18:3 was 13.58%,35.25%,17.59%,while 9.70%,33.44%,24.09%, respectively,in wild type arabidopsis.In fad3 RNAi vector transformants,the average content of C18:1,C18:2 and C18:3 was 12.04%,33.48%,18.13%.Compared to Wt, 18:1 was ascended and 18:3 were descented.It can also accumulate 18:1.At the same time,the fael over-express vector and fad2 anti-sense express vector were introduced into high erucic rapesed.In total,34 plants(T₁:15,T₀:19) for fael transformants were regenerated and,42 plants(T0) for.fad2 transformants were regenerated.GC analysis was taken to 27 transformants of fae1 over-express vector. There were 17 individuals show a higher 22:1 than Wt(45.78%),and 9 of them were beyonded 55%in 22:1.The highest content of C22:1 in some individuals of transgenic was up to 61.53%,indicating that the transformants are capable of producing higher amounts of erucic acid due to fae1 over-express.