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[目的]从丹参(Salvia miltiorrhiza Bunge)培养细胞中克隆水杨酸结合蛋白(salicylic acid binding protein 2,SABP2)基因Sm SABP2并对其进行表达分析。[方法]用Trizol法提取丹参细胞总RNA,反转录得到c DNA,通过PCR扩增Sm SABP2,将其连接到p MD-19T simple载体中,利用酶切连接的方法构建p ET28a-Sm SABP2原核表达载体,再转化至大肠杆菌BL21(DE3)中进行表达,检测蛋白酯酶活性。利用RT-q PCR测定水杨酸处理2 h的丹参悬浮细胞中Sm SABP2的表达量。[结果]PCR扩增获得了长度为780 bp的Sm SABP2,成功构建了原核表达载体p ET28aSm SABP2,IPTG诱导得到了Sm SABP2,SDS-PAGE电泳结果显示诱导8 h优于4 h,q PCR检测发现水杨酸处理2 h内Sm SABP2相对表达量持续升高。[结论]从丹参培养细胞中克隆了大小为780 bp的Sm SABP2,成功表达出了有活性的Sm SABP2,并且该基因能够强烈响应水杨酸诱导。
[Objective] The purpose of this study was to clone and analyze the gene Sm SABP2 of salicylic acid binding protein 2 (SABP2) in cultured Salvia miltiorrhiza Bunge cells. [Method] The total RNA of Salvia miltiorrhiza cells was extracted by Trizol method and the c DNA was reverse transcribed. Sm SABP2 was amplified by PCR and ligated into p MD-19T simple vector. The p ET28a-Sm SABP2 Prokaryotic expression vector, and then transformed into E. coli BL21 (DE3) for expression, detection of protein esterase activity. The expression of Sm SABP2 in Salvia miltiorrhiza cells after salicylic acid treatment for 2 h was determined by RT-q PCR. [Result] Sm SABP2 with a length of 780 bp was obtained by PCR amplification, and the prokaryotic expression vector p ET28aSm SABP2 was successfully constructed. Sm SABP2 was induced by IPTG. The result of SDS-PAGE showed that the expression of Sm SABP2 was better than that of 4 h after induced by q PCR It was found that the relative expression of Sm SABP2 in salicylic acid treatment continued to increase. [Conclusion] Sm SABP2 with a size of 780 bp was cloned from Salvia miltiorrhiza cultured cells, and active Sm SABP2 was successfully expressed, and the gene was able to respond strongly to salicylic acid induction.