小黑杨PsnbHLH35转录因子基因功能分析  

The functional analysis of PsnbHLH35 transcription factor gene in Populus simonii

作　　者：董立本 王爽[1] 姜廷波[1] 周博如[1] Dong Liben;Wang Shuang;Jiang Tingbo;Zhou Boru(State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin 150040,China)

机构地区：[1]东北林业大学林木遗传育种国家重点实验室,黑龙江哈尔滨150040

出　　处：《山西农业大学学报（自然科学版）》2022年第4期56-64,共9页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：黑龙江省应用技术研究与开发计划(GA20B401)。

摘　　要：[目的]bHLH是一类响应环境胁迫和次生代谢调控的转录因子。以小黑杨为试材,探究bHLH35转录因子抗盐胁迫分子机制,对培育抗盐胁迫小黑杨具有现实意义。[方法]提取小黑杨RNA,反转录成cDNA后设计引物克隆PsnbHLH35基因;提取小黑杨DNA克隆PsnbHLH35基因启动子,进行启动子元件预测;使用在线软件对Psnb⁃HLH35基因进行生物信息学分析;构建pBI121⁃bHLH35⁃GFP载体,利用烟草注射的方式进行亚细胞定位分析;构建pGBKT7⁃bHLH35载体,使用酵母双杂交的方法进行自激活活性检测;取培养1个月的小黑杨水培15 d后,用0.15 mol·L^(-1) NaCl溶液分别处理0、3、6、12、24、48 h提取DNA进行荧光定量PCR,计算相对表达量并绘制时空表达模型。[结果]以小黑杨为试材,克隆出735 bp的PsnbHLH35基因的cDNA。启动子预测结果显示该基因包含AAGAA⁃mo⁃tif、CGTCA⁃motif、TGACG⁃motif等多种胁迫应答元件。PsnbHLH35蛋白编码244个氨基酸,编码的蛋白没有信号肽,属于亲水蛋白。蛋白二级结构预测显示该蛋白由54.51%的α⁃螺旋、2.05%的β⁃折叠、34.43%的无规则卷曲和9.02%的延伸链所构成。蛋白三级结构预测结果显示该蛋白由碱性区域和“α螺旋1⁃环⁃α螺旋2”组成;保守结构域分析证明PsnbHLH35具有bHLH家族的保守结构域;酵母双杂交实验显示,PsnbHLH35不具有自激活活性。亚细胞定位表明,PsnbHLH35为核定位蛋白。利用RT⁃qPCR发现在盐胁迫后,PsnbHLH35基因在根、叶中表达量有明显提高。[结论]PsnbHLH35是一个定位在细胞核里没有自激活活性的应答盐胁迫的bHLH家族的转录因子。[Objective]bHLH is a class of transcription factors that respond to environmental stress and secondary metabolism regulation.Using Populus simonii×P.nigra as the test material,this study was aimed to explore the molecular mechanism of bHLH35 transcription factor resistance to salt stress,which is of practical significance for breeding P.simonii×P.nigrawith resistance to salt stress.[Methods]RNA was extracted from P.simonii×P.nigra and reversely transcribed into cDNA,then the primers were designed to clone PsnbHLH35 gene.PsnbHLH35 gene promoter was extracted and cloned from P.simonii×P.nigra DNA,and the promoter element was predicted.Bioinformatics analysis of PsnbHLH35 gene was performed by online software.The pBI121-bHLH35-GFP vector was constructed,and the subcellular localization analysis was carried out by tobacco injection.The pGBKT7-bHLH35 vector was constructed and the self-activating activity was detected using yeast two-hybrid method.The one-month old P.simonii×P.nigra tissue were hydroponic cultured for 15 days,then treated with 0.15 mol·L^(-1) NaCl solution for 0h,3h,6h,12h,24h and 48h.DNA was extracted for RT-qPCR analysis.The relative expression was calculated,and the spatiotemporal expression model was drawn.[Results]A 735bp PsnbHLH35 gene cDNA was cloned from P.simonii×P.nigra.Promoter prediction showed that the gene contained AAGAA-motif,CGTCA-motif,TGACG-motif and other stress response elements.The protein encoded 244 amino acids,and the encoded protein was a hydrophilic protein without signal peptide.The protein secondary structure prediction showed that the protein was composed of 54.51%α-helix,2.05%β-sheet,34.43%random coil and 9.02%extended chain.The predicted tertiary structure of the protein showed that the protein consisted of a basic region and“α-helix 1-loop-α-helix 2”.Conservative domain analysis proved that PsnbHLH35 had a conservative domain of bHLH family.The result of yeast two-hybrid experiments showed that PsnbHLH35 had no self-activating activity.Subcellular locali

关 键 词：小黑杨 bHLH转录因子 盐胁迫应答 生物信息学 

分 类 号：S722.36[农业科学—林木遗传育种]