小麦蛋白激酶TaNPK启动子的克隆及活性分析

doi:10.3864/j.issn.0578-1752.2011.19.002

Abstract

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 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.

Key words:

wheat, promoter, genome walking, protoplast, transient expression

DI Chao-Zeng, XU Zhao-Shi, CHEN Yao-Feng, LIU Pei, LI Lian-Cheng, CHEN Ming, MA You-Zhi. Cloning and Activity Analysis of Protein KinaseTaNPK Gene Promoter in Wheat[J].Scientia Agricultura Sinica, 2011, 44(19): 3930-3936.

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Cloning and Activity Analysis of Protein KinaseTaNPK Gene Promoter in Wheat

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