Clonning And Analysis Of Transcription Factors WRI1 Gene In Lipid Synthesis From Brassica Napus.L

As one of the most important oil crops in the world,rapeseed is the main source of edible oil and the potential source of energy oil^[1].The synthesis route of the plant triglyceride has been elucidated.It has been carried out that the work of gene cloning and identification,transgenic breeding of the related.Transcription factors have become the hotpot because of the diversity of target genes.It is said that WRI1 played an important role in the accumulation of triglyceride（TAG）and carbon metabolism in fat synthesis.About the rape breeding,the character of high fat accumulation does not conflict with high oleic acid,however,there is very little research on whether the expression of WRI1 is related to oleic acid content.In this paper,we studied the experimental plot of higher oleic acid content by using Brassica napus（the cytoplasm is come from XinJiang wild rape species）to try to explain the positive effects of WRI1 on oleic acid content.The results are as follows.1.Three copies of the BnaWRI1 gene in Brassica napus L.were cloned by RT-PCR,named as BnaWRI1-1A（accession number is NM₀01315608.1,the length of CDS is1242bp）,BnaWRI1-N3（accession number is XM₀13848500.1,the length of CDS is1230bp）,BnaWRI1-N7（accession number is HM370542,the length of CDS is 1248 bp）,which are actually homologous with 7 mutation gene types founded by writer.the material of7 gene types come from the cDNA of 35 days after anthesis,and are name as BnaWRI1-1,BnaWRI1-2,BnaWRI1-3,BnaWRI1-4,BnaWRI1-5,BnaWRI1-6,BnaWRI1-7.And BnaWRI1-7 is completely homology with BnaWRI1-N3.The phylogenetic tree was constructed by MEGA5.1 software.The conserved protein of WRI1 gene was found to be highly conserved among different species.In some plants,such as Arabidopsis thaliana,there were many copies and gene polymorphism.2.There are four kinds of cDNAs,named BnaWRI1-2,BnaWRI1-3,BnaWRI1-4 and BnaWRI1-6,which are different from each others.These cDNAs were used to construct the plant expression vector.And we can obtain the first-generation of transgenic plants of of Arabidopsis thaliana by agro bacterium-mediated method..3 BnaWRI1-1,BnaWRI1-4,BnaWRI1-5 and BnaWRI1-7 were selected to construct the yeast eukaryotic expression vector,and were expressed in Saccharomyces cerevisiae H1246.The analysis of protein Activity shows that there were no significant differences in the expression of four genes in yeast.4.We studied the expression changes of PDAT-A02,DGAT1 and GPAT6 in oil synthesis,and WRI1 with its upstream named LEC1 and LEC2 by RT-qPCR in the process of seed development.We can draw conclusions,from DAF 15,DAF 20,DAF 25,DAF 30,DAF35,DAF 40,toDAF 45,by the result of RT-qPCR from these genes.The peak of LEC2gene expression was earlier than that of LEC1 gene.The expression patterns of LEC1 and LEC2 were similar to WRI1.PDAT-A02 had higher gene expression at the early stage of pod development,but the DGAT1 and GPAT6 are the opposite.