Molecular Mechanism Of BnMYB52a Involved In The Regulation Of Lignin Biosynthesis In Brassica Napus L.

Lignin is an important secondary metabolites in plants,with important biological functions such as mechanical support,protection and transport,and is closely related to plant disease resistance and lodging resistance.Recently,lodging is one of the important factors affecting the high quality and yield of rapeseed,and lignin contributes the most to the lodging index of rapeseed.Therefore,it is important to analyze the regulatory mechanism of lignin biosynthesis in B.napus for the studying the lodging resistance in rapeseed.In previous study,we identified a MYB transcription factor BnMYB52 a,which was assumed to involvethe regulation of lignin biosynthesis in B.napus.In this study,the molecular mechanism of BnMYB52 a involved in the regulation of lignin biosynthesis was preliminarily analyzed with bioinformatics analysis,q RT-PCR analysis,phenotype analysis of transgenic plants,identification of interacting proteins and identification of the target genes.The main results are as follows.(1)The results of q RT-PCR analysis showed that BnMYB52 a was specifically highly expressed in the stem and silique pericarp;the subcellular localization results showed that BnMYB52 a was localized in the nucleus,indicating that BnMYB52 a might exert its function in the nucleus.(2)Compared with the WT,the BnMYB52 a overexpression transgenic Arabidopsis(OE-BnMYB52a)had a weaker growth phenotype,characterized by smaller leaves andstems,delayed root development,and fewer branching number;In contrast to OEBnMYB52 a plants,the mutant myb52 had better development than WT.In addition,both OE-BnMYB52 a and myb52 plants had an earlier bolting stage than WT.q RT-PCR results showed that the expression of PAL,C4 H,HCT,C3 H,COMT,F5 H,4CL,CCR and CAD,the key enzyme genes related to lignin biosynthesis,were significantly downregulated in OE-BnMYB52 a compared with the WT,indicating that BnMYB52 a mayhave an impact on the plants normal growth and development by inhibiting lignin biosynthesis in Arabidopsis.(3)Unlike the OE-BnMYB52 a Arabidopsis plants,OE-BnMYB52 a plants of B.napus showed stronger growth phenotype than the WT,OE-BnMYB52 a transgenic B.napus had a robust stem which was not easy to be broken as the WT,and displayed a better lodging resistance in the field than the WT.In addition,the OE-BnMYB52 a transgenic B.napus had shorter siliques with bigger pedicel,and later flowering stage than the WT.Compared with the control,OE-BnMYB52 a plants showed better lignification of parenchymal cell wall in the pith of the stem,better development of xylem vascular bundles,with more vessels in the vascular bundles and shorter gap between the vascular bundles;and the lignification degree of the silique pericarps was also stronger than the WT,with more layers of lignified parenchymal cells.The results of lignin content detecting assay showed that the total lignin content and S-type lignin percentage in the stems of OE-BnMYB52 a plants were significantly increased compared with the WT.The key enzyme genes of the lignin pathway F5 H,4CL,COMT,PAL,CCo AOMT,CCo AOMT1,C3 H,C3’H,C4 H,HCT and CCR in OE-BnMYB52 a plants was significantly up-regulated.The above results suggest that BnMYB52 a may enhance the accumulation of total lignin and S-type lignin by promoting the expression of key genes of the lignin biosynthesis pathway in rapeseed,strengthening the xylem,and thus further improving the lodging resistance of B.napus.(4)as the combining results of DAP-Seq,yeast-one-hybrid and dual luciferase reporter system assays preliminarily demonstrated that BnMYB52 a could specifically bind the promoters of Bn4CL4,BnCCR1,BnMYB4,BnMYB43,BnMYB26,BnCYP81F1 and BnIAA11,which lay a foundation for the further elucidation of the molecular mechanism of BnMYB52 a involving the regulation of lignin biosynthesis.(5)The yeast-two-hybrid and Bi FC assays preliminarily demonstrated that BnMYB52 a had obvious interaction with BnMYB54,BnMYB85,BnMYB43 and BnMYB26,which are involved in the regulation of lignin synthesis,indicating that the regulatory network of lignin synthesis in B.napus is complex and needs further investigation.In summary,this study provides preliminary evidence that BnMYB52 a is an important transcription factor which involved in the regulation of lignin biosynthesis in B.napus,and new evidence for uncovering its regulatory mechanism,which also provide new insights in the genetic resources creation and resistance-breeding in B.napus.