长叶红砂咖啡酸-O-甲基转移酶（RtCOMT）基因的克隆及蛋白纯化

长叶红砂(Reaumuria trigyna)是内蒙古东阿拉善-西鄂尔多斯地区特有双子叶盐生小灌木,有极强的耐盐抗旱性,入药可以治疗湿疹、皮炎等疾病,具有一定的药用价值。咖啡酸-O-甲基转移酶(RtCOMT)是一个重要的甲基化酶,主要调控木质素合成过程中中间产物及木质素的变化。基于高通量测序结果,利用RT-PCR技术克隆获得长叶红砂COMT(RtCOMT)基因,构建RtCOMT原核表达载pET32a-RtCOMT,并将其转化大肠杆菌,重组阳性菌经IPTG诱导和SDS-PAGE电泳检测。结果表明:RtCOMT基因开放阅读框长1 023bp,编码340个氨基酸,推测蛋白分子质量37.24ku,理论等电点(pI)6.71,发现该基因在大肠杆菌中正常表达得到与预期大小一致的融合蛋白。采用Ni-IDA His-Bind亲和层析方法对RtCOMT重组蛋白进行纯化,体外获得目的蛋白。

Cloning and Purification of Caffeic Acid O-methyltransferase(RtCOMT) from Reaumuria trigyna

Reaumuria trigyna is a small shrub grown in the Eastern Alxa-Western Ordos area of Inner Mongolia. R.trigyna has a vital ecological function for its remarkable tolerance to salt and drought. Additionally, R.trigyna is a traditional herb to cure eczema, dermatitis and other diseases. Caffeic acid O-methyltransferase (COMT) is an important methyltransferases which involved in synthesis of S-lignin, and plays significant roles in various physiological processes in plant life cycle. We isolated an open reading frame (ORF) of RtCOMT, constructed expression vectors of RtCOMT and transformed it into E.coli lines. The ORF of RtCOMT was 1 023 bp which encodes a protein with 340 amino acids and characterized predicted isoelectric point (pI) 6.71 and molecular mass 37.24 ku. The overexpression and purification recombinant protein were extracted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the result showed that size of the recombinant protein was consistent with the predicted . The His-tagged recombinant proteins of RtCOMT were purified by Ni-IDA His-Bind to obtain RtCOMT in vitro, it is necessary for further study in protein structure and catalytic function of RtCOMT.