The Function Of BnaC03.MYB56 In Regulating Seed Coat Pigment Synthesis In Brassica Napus L.

Brassica napus L.is the main oil crop in China and an important source of edible oil.The quality of rapeseed oil is closely related to the color of rapeseed.Compared with black-seeded rapeseed,yellow-seeded rapeseed has the characteristics of high protein content,thin seed coat and clear oil quality.Yellow-seeded rapeseed is the main breeding goal.The formation of yellow seeds is due to the synthesis of proanthocyanidin,a flavonoid substance in the endodermis of seed coat.The lack of pigment in the seed coat gives it the color of the embryo,that is yellow.B.napus has no yellow seed resources in natural germplasm,and its genetic background is complex,so it is very difficult to obtain stable genetic yellow seed rapeseed.At present,most of the studies on seed color of B.napus are based on the flavonoid pathway of Arabidopsis thaliana,and their homologous genes are found and verified in B.napus.However,B.napus is an allotetraploid and has a large number of homologous genes.Compared with A.thaliana,the mechanism of seed color regulation is more complex.In the previous studies,some important QTL loci and related candidate genes have been found by QTL mapping and genome-wide association analysis,but the function of candidate genes has not been verified.The regulation network of rapeseed seed color still needs to be improved.Therefore,it is important to identify the key genes regulating the seed color and analyze the molecular regulation mechanism of seed color of B.napus in the high quality rapeseed breeding.In this study,a hub gene BnaC03.MYB56 was found through co-expression analysis of seed coat at different developmental stages in ZS11 transcriptome,which may be involved in phenylpropane biosynthesis pathway,anthocyanin and proanthocyanidin biosynthesis pathway.Until now,it has not been reported whether BnaC03.MYB56 is involved in flavonoid pathway synthesis in B.napus.This study focused on the biological function of BnaC03.MYB56 in pigment synthesis in seed coat of B.napus.The main results are as follows:(1)Based on the analysis of transcriptome data and q RT-PCR results in different periods of ZS11,BnaC03.MYB56 and BnaA03.MYB56 were highly expressed in the seed coat 20-30 days after flowering,and the expression level of BnaC03.MYB56 was the highest in the seed.In addition,there was only a little expression in flowers and roots.The BnaC03.MYB56 gene promoter was fused with the expression vector with GUS protein and transformed into wild-type A.thaliana.The homozygous transgenic A.thaliana was stained with GUS.Strong GUS signals were detected in the early stage of seeds,roots in the seeding,trichomes and inflorescences,which was consistent with the q RT-PCR results.The results of subcellular localization showed that BnaC03.MYB56 was located in the nucleus,and transcriptional activity analysis showed that BnaC03.MYB56 had high transcriptional inhibitory activity.(2)We constructed a BnaC03.MYB56 overexpression vector to transform wild-type A.thaliana.Compared with WT,no significant phenotypic difference was found in the myb56 mutant,but the seed color of overexpressed A.thaliana was yellow and the accumulation of seed coat pigment decreased.In addition,staining of overexpressed seeds was lighter than WT when the seeds were stained with vanillin.The secondary metabolites of overexpressed and WT seeds were analyzed by UPLC-HESI-MS/MS,and the results showed that the contents of epicatechin and the three complexes proanthocyanidins decreased significantly.In addition,the overexpressed plants had small and curly leaves,short roots and abnormal lateral root development,indicating that BnaC03.MYB56 could affect the growth and development of A.thaliana.(3)BnaC03.MYB56 overexpressed rapeseed was obtained by genetic transformation and tissue culture.The overexpressed rapeseed seeds at 25 days after flowering were stained with vanillin,which was lighter than that in the WT.The differential metabolites were detected using UPLC-HESI-MS/MS,the results showed that epicatechin and five complexes of proanthocyanidins significantly decreased.The contents of kaempferol and erucic acid decreased.The results showed that BnaC03.MYB56 affected the synthesis of flavonoids and proanthocyanidins in the seeds of B.napus.(4)The knockout vectors of BnaMYB56 were constructed.And 25 plants were obtained by genetic transformation and tissue culture,and one knockout rapeseed plant was obtained by target sequence detection for further study.(5)We analyzed the transcriptome of WT and overexpressed rapeseed seeds.KEGG analysis showed that the differentially expressed genes were mainly enriched in phenylpropane biosynthesis pathway,starch sucrose metabolism pathway and flavonoid biosynthesis pathway.We selected the candidate target genes 4CL,CHIL2,FLS2,BAN2,LDOX and DFR1 for downstream regulation of BnaC03.MYB56.The results of promoter analysis showed that all of these genes had MYB binding sites.q RT-PCR showed that the expression levels of these candidate target genes in overexpressed rapeseed were significantly down-regulated compared with the WT.In addition,the expression levels of key genes of flavonoid pathway in WT and overexpressed A.thaliana were identified by q RT-PCR,we found that At PAL,At C4 H,At4CL,At CHI,At BAN,At LDOX,At DFR,At TTG1 and At TT8 were down-regulated.All together,BnaC03.MYB56 reduced the synthesis of proanthocyanidins and its derivatives by regulating the expression of genes related to flavonoid pathway.We also found that BnaC03.MYB56 interacted with flavonoid regulatory factor BnaMYB61,root development protein BnaMYB77,lignin-related protein BnaMYB54 and function unknown protein Bnab HLH96 through Y2 H and Bi FC.To sum up,BnaC03.MYB56 can reduce the content of flavonoids,proanthocyanidins and the accumulation of seed coat pigment through negatively regulate the transcriptional level of key genes in flavonoid pathway.BnaC03.MYB56 plays an important role in the formation of seed coat color in B.napus,and its molecular mechanism needs to be explored further.