拟南芥组蛋白甲基化酶SUVH6中SRA结构域基因的克隆及表达  

Cloning and Expression of SRA Domain Gene in Histone Methylation Enzyme SUVH6 from Arabidopsis thaliana L.

作　　者：谭玉霞[1] 王显国[2] 刘荣堂[1] 

机构地区：[1]甘肃农业大学草业学院,甘肃兰州730070 [2]中国农业大学动物科技学院草地所,北京100193

出　　处：《安徽农业科学》2009年第16期7348-7350,7352,共4页Journal of Anhui Agricultural Sciences

摘　　要：[目的]研究甲基化修饰对遗传基因调节的作用。[方法]利用拟南芥RNA反转录得到的cDNA序列为模板,克隆出SUVH6-SRA基因,并成功构建了含有SUVH6-SRA基因的表达载体pMAL-c2P-SUVH6-SRA,然后将pMAL-c2P-SUVH6-SRA质粒转化到大肠杆菌BL21(DE3)中,诱导表达带有MBP标签的SUVH6-SRA融和蛋白。[结果]pMAL-c2P-SUVH6-SRA克隆在转化到大肠杆菌BL21(DE3)后,在37℃条件下,经过终浓度为1.0 mmol/L的IPTG诱导,大肠杆菌BL21(DE3)能够获得大量可溶性MBP-SUVH6-SRA融和蛋白并顺利表达,SDS-PAGE显示诱导表达的融和蛋白相对分子质量为66.0 kDa左右,与预测的结果相吻合。[结论]在MBP与目的蛋白之间引入了PPE酶切位点,可使MBP和SUVH6蛋白分离,并在使用MBP亲和层析和PPE酶切后,获得了纯度较高的SUVH6蛋白,表明成功构建了SUVH6-SRA-MBP融和蛋白原核表达系统。[ Objective ] The study aimed to research the effect of methylation modification on the genetic gene regulation. [ Method ] The eDNA sequence obtained by reverse transcription of Arabidopsis thaliana L. was used as the templet to clone the gene of SUVHO-SRA and the pMAL-c2P-SUVH6-SRA of expression vector with SUVHO-SRA gene was constructed successfully. Then the pMAL-c2P-SUVH6-SRA plasmid was changed into the Eseherichiq coli BL21 （ DE3 ） and the SUVH6-SRA thaw and albumen with MBP label was induced and expressed. [ Resuit] After pMAL-c2P-SUVH6-SRA cloning was translated into the E. coil BI21 （DE3）, the E. coil BL21 （DE3） could get lots of soluble MBP-SUVH6-SRA fusion protein which was expressed successfully under the condition of 37℃ through the IPTG induction with terminal concn. of 1.0 mmol/L. The SDS-PAGE showed that the molecular weight of the induced and expressed fusion protein was about 66.0 kDa and it was anastomosod with the result of forecast. [ Conclusion] The restriction site of PPE was introduced between MBP and target protein to separate the MBP and SUVH6 and after using MBP affinity chromatography and PPE restriction enzyme the SUVH6 protein with high purity was taken, showing that the SUVH6-SRA-MBP fusion protein prokaryotic expression system was constructed successfully.

关 键 词：SRA 组蛋白 甲基化 拟南芥 MBP融和蛋白 

分 类 号：S188[农业科学—农业基础科学]