Double RNAi Vector Construction Of Fad2 And Fae1 Gene And The Transformation Of Rapeseed (Brassica Napus L.)

One of the main purposes of rapeseed breeding is to obtain the high quality varietieswhich contain high oleic acid, low erucic acid and low glucosinolates. Carbon chainextending and desaturating are determined by two key enzymes which are transcribedfrom Fael and Fad2 genes, respectively, in the down stream of oleic acid biosyntheticpathway. To obtain higher oleic acid content, we utilized RNA interference to silencethe two gene expression. Two conservative sequences, one was 349bp from Fae1gene and the other 426bp from Fad2 gene, were cloned, and the two fragments wereligated together by use of 3'-end-linking-to-5'-end way to form a longer complexfragment involving the two conservative sequences. Then the complex fragment wasinserted to the both sides of Chalcone sythase intron of pFGC5941 plasmid, with aseed specific expression NapinA promoter instead of CaMV35S, in forward andreverse direction, respectively, to construct a double-gene RNAi vector which wasused to interfere the two gene expression at the same time. The double-geneinterferefering cassette was transfered into hypocotyls and cotyledons of Xiang You15(B.napus) by Agrobacterium-mediated. Transformed explants were screened byPPT. PPT concentration for the two kind of explants were 4mg/L and 3mg/L,respectively. Finally we had obtained 268 anti-PPT seedlings in total, and amongthem 5 transgenic seedlings were tested by PCR amplification. This research openedup a new approach and obtained a new kind of germplasm resource for geneengineering breeding of oilseed crops. Meanwhile, it established the foundation for thefunction genome studies of rapeseed.