苜蓿夜蛾促前胸腺激素基因的克隆及原核表达.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 10 苜蓿夜蛾促前胸腺激素基因的克隆与原核表达 周夏  郭博智  高艳玲  赵奎军  樊东 （东北农业大学农学院，哈尔滨 150030） 摘 要 ： 促前胸腺激素 （Prothoracicotropic hormone，PTTH）是一类重要的昆虫激素，它对昆虫生长、蜕皮、变态、滞育具 有重要调控作用。以苜蓿夜蛾5 龄幼虫为材料提取总RNA，利用RT-PCR 和RACE 技术，扩增得到苜蓿夜蛾促前胸腺激素基因的 cDNA 序列，该序列含有823 个碱基，包括1 个681 个碱基的开放阅读框，编码1 个含226 个氨基酸的蛋白，分子量约为26.3 kD， 等电点为8.51。序列分析表明，苜蓿夜蛾PTTH 的氨基酸序列与其他昆虫，尤其是鳞翅目夜蛾科昆虫的PTTH 高度同源。获得的基 因已登录GenBank，登录号为JF731347。利用原核表达载体pET21b 在大肠杆菌中成功表达了苜蓿夜蛾PTTH 基因，并用Ni-NTA 亲和层析柱将带His-tag 的目的蛋白进行纯化。 关键词 ： 苜蓿夜蛾 促前胸腺激素 基因序列克隆 原核表达 Molecular Cloning and Prokaryotic Expression of Prothoracicotropic Hormone cDNA Sequence from Heliothis viriplaca Zhou Xia Guo Bozhi Gao Yanling Zhao Kuijun Fan Dong （College of Agriculture ，Northeast Agricultural University ，Harbin 150030 ） Abstract: Prothoracicotropic hormone（PTTH）is a group of important insect hormones. They have vital effect on regulation of growth, ecdysis, metamorphosis and diapause of insects. Total RNA was isolated from the fifth instar larvae of Heliothis viriplaca. The cDNA sequence was cloned by RT-PCR and rapid amplification of cDNA ends（RACE）. The cDNA sequence was 823 base pairs in length and contained an open reading frame of 681 base pairs, encoding for a polypeptide of 226 amino acid residues, with a predicted molecular weight of 26.3 kD and pI 8.51. Sequence analysis showed that the predicted amino acid shared extensive similarities with those from other insects, especially the lepidopteran noctuid insects. The cDNA sequence has been deposited in GenBank with accession No. JF731347. The cDNA sequence was successfully expressed by the recombinant prokaryotic expression vector pET2