茶树氧甲基转移酶基因的克隆及原核表达

摘要

A full length cDNA of O-methyltransferase gene was obtained from Camellia sinensis and the prokaryotic expression vector for this gene was constructed. Based on total RNA from tea leaves, a O-methyltransferase cDNA sequence of tea was obtained by RT-PCR and RACE. The whole cDNA sequence 1 280 bp which contains an ORF of 1 068 bp and encodes 355 amino acids. The putative protein of this gene had an isoelectric point of 5.68 and a calculated molecular weight of 39.1 kD. The amino acid sequence of tea O-methyltransferase showed 73%, 71%identity with that of Vitis vinifera and Ricinus communis respectively. The coding sequence had been cloned into pET-28a and transformed into the host BL21. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG.%获得了一类茶树氧甲基转移酶基因 cDNA 全长并构建了该基因的原核表达载体。以从茶树叶片中提取的总RNA为模板，结合 RT-PCR与RACE克隆技术获得氧甲基转移酶（O-methyltransferase）基因cDNA全长1280 bp，其开放阅读框为1068 bp，编码355个氨基酸，推测的蛋白分子量为39.1 kD，理论等电点为5.68。其氨基酸序列与葡萄和蓖麻氧甲基转移酶基因相似性分别为73%、71%。将该基因片段连接到原核表达载体pET-28a中，转化大肠杆菌 BL21后诱导重组蛋白的表达，经IPTG诱导，SDS-PAGE检测到1条与预测融合蛋白分子量相符的外源蛋白。