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

[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 localization analysis indicated that the PsnbHLH35 protein was localized in the nucleus.In addition,we found that the expression of PsnbHLH35 gene in roots and leaves was increased significantly under salt stress using RT-qPCR technology(P<0.05).[Conclusion]PsnbHLH35 is a transcription factor of the bHLH family which is located in the nucleus and has no self-activating activity in response to salt stress.