中国农业科学2025，Vol.58Issue(5)：991-1003,13.DOI:10.3864/j.issn.0578-1752.2025.05.013
乌菜BcDET2的克隆及其抽薹开花调控功能
Cloning and Expression of BcDET2 Gene and Functional of Its Regulatory Effect on Bolting and Flowering in Wucai(Brassica campestris L.)
摘要
Abstract
[Objective]The steroid 5α-reductase gene BcDET2(DE-ETIOLATED 2),involved in the brassinosteroid(BR)biosynthesis pathway,was cloned and analyzed in Wucai(Brassica campestris L.ssp.chinensis var.rosularis Tsen).The functions of BcDET2 in regulating bolting and flowering were investigated through genetic transformation,thereby providing a molecular basis for the genetic breeding of Wucai.[Method]The BcDET2 was obtained via homologous cloning,based on the DET2(BraA10g023600.3C)sequence from the Chinese cabbage(B.rapa)genome.Bioinformatics analyses were performed using online tools such as Expasy,SOPMA,SWISS-MODEL,and TMHMM.The expression pattern of BcDET2 was analyzed using quantitative real-time PCR(qRT-PCR).Subcellular localization of BcDET2 was determined through Agrobacterium-mediated transient transformation in tobacco(Nicotiana tabacum)cells.The role of the BcDET2 in regulating bolting and flowering was studied through genetic transformation of Wucai.Yeast two-hybrid(Y2H)assay was utilized to screen and identify BcDET2-interacting proteins,followed by qRT-PCR analysis to examine the response of BrbHLH96 to vernalization and its expression levels in BcDET2 transgenic plants.[Result]The cDNA sequence of BcDET2(825 bp),encoding 274 amino acids,was successfully obtained through homologous cloning.Bioinformatics analysis indicated that the BcDET2 protein predominantly consists of α-helices,is predicted to be a weakly hydrophilic,non-secretory membrane protein,and contains 39 phosphorylation sites and 5 transmembrane domains.Phylogenetic analysis revealed that BcDET2 shares a close evolutionary relationship with DET2 from Brassica rapa and Brassica napus.Subcellular localization showed fluorescent signals of BcDET2 in both the nucleus and cell membrane.qRT-PCR analysis indicated the highest expression level of BcDET2 at 15 days of vernalization,suggesting its responsiveness to the vernalization process.Functional assay demonstrated that overexpression of BcDET2 significantly promoted early flowering in transgenic Wucai plants.Y2H analysis revealed that BcDET2 interacts with the transcription factor BcBHLH96,which exhibited high transcription levels in the late vernalization stage and significantly increased expression in BcDET2 transgenic plants.[Conclusion]In this study,the cDNA of BcDET2 from Wucai was cloned,and it is likely involved in the regulation of bolting and flowering in Wucai via the vernalization pathway.关键词
乌菜/BcDET2/基因克隆/酵母双杂交/抽薹开花Key words
Wucai/BcDET2/gene cloning/yeast two-hybrid/bolting and flowering引用本文复制引用
郑雅琴,陈国户,刘雪晴,吴思文,唐小燕,杨丹妮,汪永康,Ahmad Aftab,Khna Afrsyab,汪承刚..乌菜BcDET2的克隆及其抽薹开花调控功能[J].中国农业科学,2025,58(5):991-1003,13.基金项目
安徽省自然科学基金(2308085MC96)、合肥市自然科学基金(202343)、安徽省重大基础研究项目(2023z04020005) （2308085MC96）
