乌菜Bc DET2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.)

作　　者：郑雅琴 刘雪晴 吴思文 唐小燕 杨丹妮 汪永康 Ahmad Aftab Khna Afrsyab 汪承刚[1,2] 陈国户 ZHENG YaQin;LIU XueQing;WU SiWen;TANG XiaoYan;YANG DanNi;WANG YongKang;AHMAD Aftab;KHAN Afrsyab;WANG ChengGang;CHEN GuoHu(College of Horticulture,Anhui Agricultural University/Anhui Provincial Engineering Center of Horticultural Crop Breeding,Hefei 230036;Anhui Provincial Wanjiang Vegetable Industrial Technology Institute,Maanshan 238200,Anhui)

机构地区：[1]安徽农业大学园艺学院/安徽省园艺作物育种工程中心,合肥230036 [2]安徽省皖江蔬菜产业技术研究院,安徽马鞍山238200

出　　处：《中国农业科学》2025年第5期991-1003,共13页Scientia Agricultura Sinica

基　　金：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)。

摘　　要：【目的】通过对乌菜(Brassica campestris L.ssp.chinensis var.rosularis Tsen)油菜素内酯(brassinosteroid,BR)合成途径中类固醇5α-还原酶基因BcDET2(DE-ETIOLATED 2)进行克隆和表达分析,遗传转化乌菜植株,分析其在抽薹开花调控中的作用,为乌菜遗传育种提供分子依据。【方法】基于大白菜(Brassica rapa L.)基因组DET2(BraA10g023600.3C)基因序列,通过同源克隆获得乌菜BcDET2。利用Expasy、SOPMA、SWISS-MODEL、TMHMM等在线工具对BcDET2进行生物信息学分析。运用荧光定量PCR(qRT-PCR)等方法分析BcDET2的表达模式。采用农杆菌介导法瞬时侵染烟草(Nicotiana tabacum)细胞,进行BcDET2的亚细胞定位;遗传转化乌菜分析BcDET2在乌菜抽薹开花中的调控功能。利用酵母双杂交技术筛选和鉴定BcDET2的互作蛋白,并运用qRT-PCR分析该互作蛋白基因对春化的响应,以及在BcDET2过表达植株中的表达水平。【结果】成功获得乌菜BcDET2的cDNA序列(825 bp),编码274个氨基酸。生物信息学分析结果显示,BcDET2蛋白质结构以α-螺旋为主,预测为弱亲水性、不稳定碱性蛋白;含有39个磷酸化位点和5个跨膜结构域,无信号肽,表明其可能为非分泌型膜蛋白。进化树分析表明,BcDET2与大白菜(B.rapa)和油菜(B.napus)的DET2具有较近的亲缘关系。亚细胞定位显示,BcDET2在细胞核和细胞膜上均有荧光信号。qRT-PCR等分析表明,BcDET2在春化15 d时表达量最高,表明其对春化过程具有响应性。功能研究发现,过表达BcDET2显著促进乌菜转基因植株的早花现象。酵母双杂交试验表明,BcDET2可与转录因子BcBHLH96互作,且该转录因子在春化后期具有较高的转录水平,并且在过表达BcDET2植株中表达水平显著升高。【结论】成功克隆了乌菜BcDET2,该基因可能通过春化途径参与乌菜抽薹开花的调控。【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 ofα-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 t

关 键 词：乌菜 BcDET2 基因克隆 酵母双杂交 抽薹开花 

分 类 号：S63[农业科学—蔬菜学]