Study On The Molecular Mechanism Of Transcription Factor BnbHLH92a Regulated Seed Coat Color Formation In Brassica Napus L.

Rapeseed(Brassica napus L.)is one of the most important oil crops in the world.The characteristics of yellow seed are related to the improvement of feed value of cake,oil and protein content.However,as the main harvesting organ of rapeseed,the pigments and polyphenols in seed coat not only make the seed coat show different colors(such as black,yellow,red,reddish-brown and black-brown),but also seriously affect the quality of rapeseed oil and feeding value of cake.Therefore,breeders hope to reduce the pigment content through the strategy of breeding yellow-seeded rapeseed,so as to improve the quality and commercial value of rapeseed.Flavonoids,as secondary metabolites,play an important role in plant growth,especially in color change.Although the downstream structural genes(DFR,LDOX and ANS),MYB and b HLH families and their MBW complexes in Brassica napus have been proved to be widely involved in a variety of primary and secondary metabolic processes of phenylpropane-flavonoid metabolic pathway in plants,and play an important role in the final determination of tissue and organ color formation.The molecular mechanism in the formation of seed color in B.napus is still unclear due to the relatively complex genome and multiple copies of the genes.Based on the analysis of the functional mechanism of the important seed color regulatory gene Bn MYB47,and possibly interacts with Bnb HLH92 by mediating the procyanidin metabolism.Overexpressing Bnb HLH92 a in Arabidopsis thaliana resulted in yellow seed coat color.We speculated that Bnb HLH92 a involved in the change of seed color in Brassica napus.Therefore,the molecular mechanism of Bnb HLH92 a participating in the regulation of seed color coat has been deeply studied.The main research results are as follows:(1)By analyzing the expression patterns of Bnb HLH92 a in different tissues and organs of B.napus and the expression level of seeds in different development stages of yellow and black seeds,it was found that Bnb HLH92 a was mainly highly expressed in yellow seeds.Through bioinformatics analysis,Bnb HLH92 a was found to have a typical helix-loop-helix b HLH conserved domain,which was closely related to the transcription factor Lcb HLH92 that was reported to be involved in flavonoid metabolism.Subcellular localization and transcriptional activity analysis indicated that Bnb HLH9 a was a transcription factor localized in the nucleus with strong inhibitory activity.Therefore,we speculate that Bnb HLH92 a negatively affects the formation of seed coat color.(2)Overexpression of Bnb HLH92 a in Arabidopsis thaliana results in yellow seed coat.Anthocyanins/proanthocyanins were significantly reduced using histochemical staining and chemical method.Further analysis of the types and contents of polyphenols and flavonoids in transgenic lines by UPLC-HESI-MS/MS showed that proanthocyanins derivatives were the main differential metabolites.q RT-PCR showed that transcription levels of flavonoid biosynthesis pathway genes(TT3,TT6,TT8,TTG1,TT18 and BAN)were significantly down-regulated in transgenic lines.In conclusion,Bnb HLH92 a results in lighter seed coat color by decreasing anthocyanin/proanthocyanin accumulation.(3)UPLC-HESI-MS/MS analysis of transgenic rapeseed lines overexpressing Bnb HLH92 a gene showed that,compared with the control,the differential metabolites were still significantly enriched in the anthocyanin/proanthocyanidins biosynthesis pathway.Yeast two-hybrid analysis(Y2H)and bimolecular fluorescence complementation(Bi FC)assays further confirmed that the interaction of Bnb HLH92 a with Bn TTG1,which may form a functional complex and then participate in the regulation of anthocyanin/proanthocyanidins metabolism.In addition,yeast one-hybrid assay(Y1H),chromatin immunoprecipitation quantitative PCR(Ch IP-q PCR)assay and luciferase reporter gene experiments demonstrated that Bn TT18 is the downstream target gene site of Bnb HLH92 a.Bnb HLH92 a mainly inhibits its expression by directly binding to the promoter sequence of Bn TT18,so as to participate in flavonoid metabolism,but the specific target site needs to be further identified.Taken together,Bnb HLH92 a is a trans-inhibitor regulating flavonoid metabolic pathway.First,Bnb HLH92 a may form a competitive MBW complex with Bn MYB47 and Bn TTG1,interfering with the normal regulation of the structural genes of the flavonoid pathway by the MBW complex;Second,Bnb HLH92 a directly targets and regulates the structural gene Bn TT18,inhibits the accumulation of flavonoids metabolites,and then affects the change of seed coat color of Brassica napus,but the specific mechanism needs to be further studied.