摘要
【目的】克隆强抗寒性牧草短芒大麦钙依赖蛋白激酶(Calcium-dependent protein kinase,CDPK)基因,分析其生理生化特性,为理想抗逆工程基因的筛选和利用奠定基础。【方法】采用RACE-PCR技术,获得短芒大麦CDPK基因全长cDNA序列;采用Northern杂交分析该基因在逆境条件下(低温(4℃)、干旱(200 g/L PEG6000)、盐(300 mmol/L NaCl)、激素(100μmol/L ABA))的表达模式;采用农杆菌介导的方法,对该基因编码蛋白进行亚细胞定位,并对转基因烟草的离子渗漏率和脯氨酸含量进行测定。【结果】获得了短芒大麦CDPK基因的编码序列,将其命名为HbCDPK;HbCDPK编码的蛋白是一个膜定位钙依赖蛋白激酶,该基因在短芒大麦根、幼苗叶片和成熟胚中均有表达;在低温、干旱、盐和ABA胁迫条件下处理不同时间,HbCDPK的表达丰度有差异,其在低温胁迫条件下的表达信号最强;转HbCDPK基因烟草相对离子渗漏率降低,脯氨酸含量升高。【结论】HbCDPK基因参与了低温胁迫信号转导,具有提高植物抗寒性的潜能。
【Objective】 Cloning,physiological and biochemical characteristics analysis of CDPK gene in Hordeum brevisubulatum provided a rational basis for screening and utilization of ideal resistant gene.【Method】 The cDNA sequence of CDPK gene in H.brevisubulatum was isolated by RACE-PCR and the expressions profiling of HbCDPK in different tissues and under various stresses and phytohormone treatments（4 ℃,200 g/L PEG6000,300 mmol/L NaCl,100 μmol/L ABA） were investigated by using RNA gel blot analysis.In addition,the subcellular location characteristics of HbCDPK were investigated by the expression of HbCDPK：GFP fusion protein,and the ion percolation rate and proline content in transgenic tobacco were analyzed.【Result】 The coded sequence of calcium-dependent protein kinase gene in H.brevisubulatum was obtained,and named HbCDPK.HbCDPK is a membrane-associated Serine/Threonine protein kinase.HbCDPK gene was detected in root,leaf blade and mature embryo.Expression of HbCDPK at transcription level was induced obviously under cold,drought,salt and ABA stress.The abundance of HbCDPK mRNA in transgenic tobacco treated by cold stress was the highest,and its ion percolation rate decreased and proline content increased.【Conclusion】 HbCDPK gene participates in signal transduction in abiotic stress condition and has a potential to enhance cold and drought resistance of plants.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2011年第2期111-119,共9页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(30471229)
关键词
短芒大麦
钙依赖蛋白激酶
非生物胁迫
亚细胞定位
RACE
Hordeum brevisubulatum
calcium-dependent protein kinase
abiotic stress
subcellular localization
RACE
