柠条锦鸡儿CkCDPK基因的克隆、表达分析及载体构建
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摘要
钙依赖蛋白激酶作为一个关键的信号传导器,通过调控和参与植物体内的代谢途径等方式在植物抵御逆境胁迫过程中发挥着重要作用。为了探索柠条锦鸡儿相关基因的功能,本研究利用同源克隆的方法,从柠条锦鸡儿中克隆了1个CDPK类基因的CDS,命名为CkCDPK。通过测序和生物信息学分析,结果显示,该基因包含1 710 bp的开放阅读框,编码570个氨基酸,分子质量为64.03 ku,蛋白质等电点(PI)为9.12。结构分析显示,CkCDPK含有N端可变区、蛋白激酶区、4个EF手型结构和类似钙调素等结构域。利用实时荧光定量PCR检测了CkCDPK基因在不同逆境胁迫下的表达量,结果显示,NaCl胁迫和干旱胁迫下该基因的表达量呈现出单峰趋势,其中,盐胁迫6 h、干旱胁迫4 h表达量最高,外源ABA诱导下该基因的表达量基本呈逐渐上升趋势。表明该基因可能参与了柠条锦鸡儿的抗逆性调控。构建植物超表达载体pCAMBIA3301-CkCDPK,可为进一步研究CkCDPK基因在柠条锦鸡儿抗逆性调控中的作用奠定基础。
Calcium dependent protein kinase is a key signal transductor,and plays an important role in plant resistance to envirenment stress by regulating and participating in metabolic pathways of plants. To explore the related function genes of caragana,we isolated an open reading frame of CDPK gene from caragana by homology cloning strategy,termed CkCDPK. Bioinformatic analysis showed that the gene had a 1 710 bp open reading frame(ORF),encoding 570 amino acids,with a molecular weight of 64.03 ku and PI of 9.12. Structural analysis showed that the CkCDPK gene contained the N-terminal variable region,a protein kinase domain,EF hand structure,calmodulin domains,etc. Quantitative real-time PCR technique was used to detect CkCDPK expression level under adverse situations. The results revealed that the CkCDPK presented a unimodal trend after treated with salinity(NaCl)and drought(PEG),of which the gene expression levels were higher at 6 h of salinity stress and 4 h of drought stress. Under ABA stress,the gene expression was up-regulated compared with the contrast. These results indicated that the CkCDPK gene might be involved in the regulation of environmental stress responses in Caragana korshinskii. The plant expression vector pCAMBIA3301-CkCDPK was also constructed. This study provided the theoretic and experimental basis for further study of the CkCDPK function in stress-tolerance regulation in Caragana korshinskii.
Calcium dependent protein kinase is a key signal transductor,and plays an important role in plant resistance to envirenment stress by regulating and participating in metabolic pathways of plants. To explore the related function genes of caragana,we isolated an open reading frame of CDPK gene from caragana by homology cloning strategy,termed CkCDPK. Bioinformatic analysis showed that the gene had a 1 710 bp open reading frame(ORF),encoding 570 amino acids,with a molecular weight of 64.03 ku and PI of 9.12. Structural analysis showed that the CkCDPK gene contained the N-terminal variable region,a protein kinase domain,EF hand structure,calmodulin domains,etc. Quantitative real-time PCR technique was used to detect CkCDPK expression level under adverse situations. The results revealed that the CkCDPK presented a unimodal trend after treated with salinity(NaCl)and drought(PEG),of which the gene expression levels were higher at 6 h of salinity stress and 4 h of drought stress. Under ABA stress,the gene expression was up-regulated compared with the contrast. These results indicated that the CkCDPK gene might be involved in the regulation of environmental stress responses in Caragana korshinskii. The plant expression vector pCAMBIA3301-CkCDPK was also constructed. This study provided the theoretic and experimental basis for further study of the CkCDPK function in stress-tolerance regulation in Caragana korshinskii.
引文
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