摘要
已有研究结果表明,植物自身能产生剧毒的氰化物(如HCN),而氰丙氨酸合酶(Cyanoalanine synthase,CAS)是植物解氰作用的关键酶,在植物生长、发育及逆境胁迫响应过程中起重要作用。本研究通过克隆拟南芥CAS合酶关键基因CYS-C1全长后,构建原核表达载体p EASY-CYS-C1并导入大肠杆菌E.coli受体细胞中进行表达。结果表明,在大肠杆菌E.coli细胞中成功诱导出可溶性的CYS-C1蛋白,其中IPTG最佳诱导时间为4 h,最佳诱导浓度为0.2 mmol/L。原核表达的CYS-C1蛋白经Ni柱纯化并免疫新西兰白兔制备多克隆抗体,Western blot检测结果表明,CYS-C1蛋白多克隆抗体的特异性较好。本试验结果对于进一步开展CAS合酶的功能研究奠定了基础。
Previous study results showed that plant contained variety of cyanides such as HCN,which was toxic to the plant. However,cyanoalanine synthase was the key enzyme of cyanide detoxication in plant,which played important roles in plant growth,plant development and stress tolerance. In the present study,the full-length of Arabidopsis CYS-C1 gene was cloned and linked to p EASY vector to construct the prokaryotic expression vector p EASY-CYS-C1,which was then transformed into E. coli. The results showed that the soluble CYS-C1 protein was induced in E. coli. The optimum-induced time was 4 h and the optimum-induced IPTG concentration was 0.2 mmol/L. The recombinant CYS-C1 protein was purified by nickel column. Then the purified CYS-C1 protein was injected into rabbit to prepare polyclonal antibody. Western blot detection results showed that the obtained polyclonal antibody with high specificity that could be used for further experiments.
出处
《江苏农业学报》
CSCD
北大核心
2017年第6期1235-1241,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31400242)
关键词
拟南芥
氰丙氨酸合酶
多克隆抗体
Arabidopsis thaliana
cyanoalanine synthase
polyclonal antibody
