中国农业科学2011，Vol.44Issue(19)：3930-3936,7.DOI:10.3864/j.issn.0578-1752.2011.19.002
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
摘要
Abstract
[ 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 5UTR deletion mutants of the TaNPK gene promoter were amplified by PCR, and then inserted into the vector Pbi 121 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 5UTR of TaANPK gene.关键词
小麦/启动子/染色体步移/原生质体/瞬时表达Key words
wheat/promoter/genome walking/protoplast/transient expression引用本文复制引用
翟朝增,徐兆师,陈耀锋,刘沛,李连城,陈明,马有志..小麦蛋白激酶TaNPK启动子的克隆及活性分析[J].中国农业科学,2011,44(19):3930-3936,7.基金项目
国家转基因生物新品种培育重大专项(2009ZX08009-083B) （2009ZX08009-083B）
国家自然科学基金项目(31171546) （31171546）
