Identification Of BnMYB46 Cis-acting Elements And Screening Of Trans-acting Factors In Brassica Napus

Brassica napus L.is one of the most important oilseed crops in China.However,environmental stresses negatively impact on its growth and development.Previous studies have showed that MYB46 is involved in the biosynthesis of plant secondary walls and plays a role in plant stress resistance.Our research group have isolated MYB46 gene from B.napus,and studied its function through overexpression and suppression of BnMYB46 in rapeseed.In this study,subcellular localization and transcriptional activation analysis of BnMYB46 were carried on.Further,cis-acting elements of BnMYB46 were identified by promoter activity assay,and the upstream regulatory factors of BnMYB46 were screened by yeast one-hybrid system.These results lay the foundation for further understanding the function of BnMYB46and improving the regulatory network of BnMYB46.The main results are as follows:1.Subcellular localization and transcriptional activation analysisTo examine the subcellular localization of BnMYB46,a binary vector construct harboring GFP:BnMYB46 driven by the Ca MV 35S promoter was transferred to onion epidermal cells.The results showed that GFP:BnMYB46 was localized in the nucleus.Furthermore,BnMYB46 possessed transcriptional activation activity in yeast.Therefore,BnMYB46 has the general characteristics of a transcription factor.2.activity analysis of BnMYB46 promoterThe upstream 1108 bp sequence of the start codon of BnMYB46 in B.napus was amplified as the promoter segment.Bioinformatics analysis showed that BnMYB46 promoter contained several potential cis-regulatory elements.On this basis,eleven vectors fusing GUS with 5’-or 3’-truncated flanking sequences of BnMYB46 promoter were constructed,and then transient expressed in tobacco and Arabidopsis protoplasts,respectively.GUS staining and GUS quantitative results showed that the activity of BnMYB46 promoter was significantly reduced without the upstream sequence between-615 bp to-789 bp of the start codon.This promoter sequence between-615 bp to-789 bp containing cis-elements such as ARE and circadian indicated that the expression of BnMYB46 could be regulated by various factors.3.Screening regulatory factors of BnMYB46The above-identified sequence（M67）and cis-acting elements were used to construct the yeast one-hybrid bait vector,named as p Ab Ai-M67bait,p Ab Ai-ARE,and p Ab Ai-Circadian,respectively.The Ab A screening concentrations of the three bait vectors were determined by the bait self-activation,and finally the Ab A of 650 ng·m L^-1was apply to all three bait vectors.The B.napus c DNA library plasmids were transformed into bait strains containing p Ab Ai-M67bait,p Ab Ai-ARE,and p Ab Ai-circadian for library screening,respectively.Six regulatory factors were initially obtained by library screening,namely Bna A02g01000D,Bna A03g23700D,Bna A05g33620D,Bna A07g18880D,Bna A10g01790D,and Bna C08g06800D.According to the functional annotation of homologous genes in Arabidopsis,these genes are involved in biological processes such as regulation of cell wall,pollen germination,secondary wall biosynthesis,lipid transport,and cuticle development.