小麦蛋白激酶TaNPK启动子的克隆及活性分析

Cloning and Activity Analysis of Protein Kinase TaNPK Gene Promoter in Wheat

【目的】克隆小麦蛋白激酶基因TaNPK启动子,并分析5′非编码区对盐胁迫的响应,探索TaNPK的调控机制,为解析小麦抗盐机制提供理论依据。【方法】利用染色体步移技术从小麦中分离TaNPK读码框上游序列,构建启动子缺失突变体,在PEG4 000介导下将重组质粒转入拟南芥叶片原生质体进行瞬时表达分析,以gus为报告基因研究不同调控序列的活性及盐诱导性分析。【结果】获得了TaNPK读码框上游2 004 bp的启动子序列,PEG介导的拟南芥原生质体瞬时表达分析表明,5′端缺失片段都在不同程度上驱动了GUS的表达;经过NaCl处理后的原生质体,GUS的表达量比不经处理的对照组明显提高,这与NaCl处理下TaNPK的实时荧光定量分析结果一致。【结论】经克隆获得了受盐胁迫诱导的小麦TaNPK启动子,启动子缺失区段在不同程度上驱动了gus的表达,-1 083—-1 296 bp区段可能含有响应盐胁迫的负调控元件。

【Objective】 Cloning the promoter of a plant protein kinase gene,TaNPK,and analyzing its response to salt stress,could be helpful to investigate the regulatory mechanism of TaNPK gene and to provide a theoretical foundation for analyzing salt-tolerance mechanism of wheat.【Method】 Genome walking technology was used to amplify the upstream regulatory sequence of TaNPK gene,and six different 5′UTR deletion mutants of the TaNPK gene promoter were amplified by PCR,and then inserted into the vector pBI121 to replace CaMV 35S promoter respectively.The recombinant plasmids were transferred into Arabidopsis leaf protoplasts by PEG-mediated transient expression system and the promoter activities were quantitatively estimated using gus report gene.【Result】 A 2 004 bp 5′flanking sequence was obtained by genome walking technology.Deletion analysis was made by comparing with the control.The results coincided with that the GUS gene driven by six 5′-end deletion mutants could be highly effectively expressed in protoplasts,and GUS activity increased in varying degrees with the treatment of NaCl compared with real-time fluorescent quantitative PCR analysis.【Conclusion】 The TaNPK gene promoter was cloned,the activity analysis showed that NaCl up-regulates TaNPK gene in wheat,and a negative regulatory factor maybe exist between-1 083—-1 296 bp area of the 5′UTR of TaNPK gene.