Clone And Expression Analysis Of Bnfad2 And BnaLCR78 Genes Of Brassica Napus And Construction Of Its Corresponding RNAi Vectors

High oleic acid rapeseed oil refers to the oil whose oleic acid contents are more than 75%.It has favorable health functions and endurable storage features.While cooking under high temperature,it doesn't produce lampblack easily.It is a superior edible fat and oil which can be on a par with olive oil and tea oil.As its production costs and cultivation difficulties are less than that for the above-mentioned high-grade oil crops,the development of rapeseed oil with high oleic acid has became an important direction for the supply to improve advanced edible oil.For a long time,studies on genetic engineering of oil-seed rape focused on the actual production.Previous studies on many rapeseed fatty acid desaturation enzyme genes found that even the silence of these genes cannot hinder the synthesis of polyunsaturated fatty acids,which restricted the constant increase of oleic acid contents.Current studies focused on fatty acid desaturase genes on known pathways and genes affecting oleic acid contents of rape seeds by other means.In this research,we cloned fad2 and BanLCR78 gene from low oleic acid content conventional rape cultivar zhongyou 82,medium oleic acid Male sterile maintainer line 100B and its high oleic acid mutant.This paper studied expression patterns of these two genes and constructed corresponding RNAi vector.Here are some results.Firstly,two kinds of fad2 genes were acquired from three types of rape above:Bnfad2-? and Bnfad2-?.Bnfad2-? could encode complete proteins while gene translation of Bnfad2-?terminated in advance.Secondly,expression results of Bnfad2-? and Bnfad2-? in different tissues suggested that the expression of Bnfad2-? and Bnfad2-? was different from previously proposed HO1 and H02 modes.Thirdly,BnaLCR78 complete genome sequence and CDS sequence were obtained respectively from three materials.The full length was 376bp and regional length of coding region was 237bp.cDNA of BnaLCR78 in the high oleic acid mutant had two forms.One was a common form in which introns were eradicated while the other one retained the introns.Results of phylogenetic tree suggested that this gene was close to LCR gene family of Arabidopsis thaliana.Protein prediction results found that BnaLCR78 has the structural domain to bind with SLR1(S locus-related glycoprotein 11)transcription inhibition proteins.Fourthly,quantitative PCR of BnaLCR78 showed that the expressions of BnaLCR78 in stems with high oleic acid were significantly lower than that of low oleic acid material and the control material.After 35d of bloom,the expressions of BnaLCR78 in capsules of three materials were higher than that in capsules after 27d of bloom.It was predicted that the expression of BnaLCR78 was closely related with fatty acid synthesis at later period of seed development.Fifthly,three RNAi vector were established by using three conserved domin of Bnfad2.Two RNAi vector containing two sectors of BnaLCR78 were constructed,which layed the foundation for further study on the influence of two genes on fatty acid synthesis pathways of cabbage type rape and breed cabbage type rape materials with high oleic acid.