Mapping And Functional Analysis Of Yellow Seed Color Candidate Gene In Brassica Juncea L.

Rapeseed is one of the world's major oil crops,China is one of the world's largest producer of rapeseed,In 2014,the acreage of rapeseed is 7.6×10⁶ hm²,acreage of soybean is4.6×10⁶ hm²,but these cannot meet the oil need of our lives,therefore,improving the oil content of rapeseed is still an important subject for research of breeding.A large number of studies show that,in the same genetic background,the oil content of yellow-seed rapeseed is generally higher than that of brown-seed and black-seed rapeseed and yellow-seed rapeseed has thinner seed coat,transparent oil and higher protein content.However,Brassica napus is widely cultivated in China,which have no pure natural yellow-seed germplasm,Wuqi mustard has pure natural yellow-seed germplasm,genetic model is relatively simple,is an ideal material of yellow-seed gene.In this research,the BC₉ population of Wuqi mustard?yellow seed?and Guanzhong mustard?brown seed?was used for RNA-Seq,seeds of 30 days after pollination from 30yellow-seed plants and 30 brown-seed plants were combined to form Y and B bulks.We obtained significant different expression genes?DEGs?,than we analysised the correlation between the relative gene expression and the content of yellow-seed index in different seed developent stages.According to the BSR analysis results,we confired the candidate gene.In addition,we construct the over-expression vector of candidate gene and than transformed candidate gene into Arabidopsis thaliana to verify function of candidate gene.The research results provide important clues for revealing the molecular mechanism of yellow seed trait in Brassica juncea.The main results are as follows:1.In this study,the BC₉ population of Wuqi mustard?yellow seed?and Guanzhong mustard?brown seed?was used for RNA-Seq,at 30 days after pollination,seed from 30yellow-seed plants and 30 brown-seed plants were combined to form Y and B bulks.After filtering and ressembling the data,we got 42386 gene sequences,which were compared with Nr database,we noted 41018 gene sequences,among them 245 gene expressed differently significantly,209 genes up-regulated and 36 genes down-regulated.By KEGG annotation of these genes,we found most of these genes were annotated to the flavonoid biosynthesis pathway.The DEGs involved into flavonoid biosynthesis pathway is BjBAN,BjDFR,BjLDOX,BjTT5,BjC4H,BjTT8,BjTT4,BjPAL,BjF3H.2.By BSR analysis,Brassica rapa genome?Chiifu-401?is the reference genome,we got the 6825 SNPs between yellow-seed and brown-seed pool,we found they mainly located on chromosome A09,so we guess the yellow-seed candidate gene is located on chromosome A09.Among the DEGs we got before,BjDFR,BjTT5,BjF3H,BjTT4,BjTT8 located on chromosome A09.So we initially identified these 5 genes as a candidate gene of Wuqi yellow mustard yellow seed trait.3.In order to further determine the Wuqi mustard yellow-seed gene,the seeds pollinated after 9,16,23,30,38,45 days of Wuqi mustard and Wugong mustard were used to test the relative expression of these 5 genes,and the content of the flavonoid,anthocyanin,melanin and total phenol,then analysis the correlation between them,we found that BjTT5 and BjF3H genes have significant correlation.Therefore,we further define that BjTT5 and BjF3H genes may be candidate genes of Wuqi mustard yellow-seed trait.4.Combined with the previous results,candidate gene located on chromosome A0923.304²9.402Mb,BjF3H gene was found in the area.5.By homologous cloning method,CDS sequence of BjF3H gene was cloned in Wuqi mustard,its sequence was 1077bp,encoding 358 amino acids.Then we constructed over-expression vector of BjF3H gene,transformed it into wild type Arabidopsis?brown seed?,we have obtained two T₂ generation transgenic plants?yellow seed?.