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家蚕G蛋白α亚基基因BmGα12的克隆及序列分析与融合表达

ISSN号：0257-4799
期刊名称：《蚕业科学》
时间：0
分类：S881.2[农业科学—特种经济动物饲养;农业科学—畜牧兽医] Q78[生物学—分子生物学]
作者机构：[1]安徽省农业科学院蚕桑研究所,合肥230061, [2]中国科学院上海生命科学研究院植物生理生态研究所,上海200032, [3]芜湖职业技术学院,安徽芜湖241006
相关基金：安徽省农业科学院院长青年创新基金项目（No.201005-A01）; 现代农业产业技术体系建设专项（蚕桑）; 安徽省蚕桑产业技术体系（No.ahnycytx-16）

作者：章玉萍[1], 蒋剑豪[2], 赵云坡[2], 汤强[3], 代君君[1], 王储炎[1], 吴传华[1], 范涛[1], 苗雪霞[2]

关键词：家蚕, G蛋白α亚基, 基因克隆, 序列特征, 融合蛋白, Bombyx mori, G protein α subunit, Gene cloning, Sequence feature, Fusion protein

中文摘要：

异三聚体G蛋白是真核细胞感知外界信号后将信号传递到细胞内的重要分子,参与生物体内广泛的信号转导途径。采用生物信息学方法和RT-PCR、RACE技术克隆了家蚕G蛋白α亚基基因BmGα12的全长cDNA序列,该序列全长1374 bp,开放阅读框（ORF）1 128 bp,编码375个氨基酸。将构建的表达载体pET-41b（＋）-Gα12转化E.coli BL21宿主菌进行诱导表达,经SDS-PAGE和Western blot检测显示,BmGα12可在E.coli BL21中高效表达,GST-BmGα12融合蛋白主要以包涵体形式存在,其分子质量约71 kD。系统进化分析显示BmGα12与大鼠和人Gα12/Gα13的亲缘关系较近,初步推测BmGα12介导的信号传导同样在细胞生长、胚胎发育中起重要作用。

英文摘要：

The heterotrimeric GTP-binding proteins are vital molecules that transmit signals into the cell after the cell senses extracellular signals and function in a wide range of signal transduction pathways in eukaryotes.Based on bioinformatic analysis,a full-length cDNA sequence of G protein α subunit was cloned by means of RT-PCR and RACE from Bombyx mori and named as BmGα12.This sequence was 1 374 bp long with an open reading frame（ORF） of 1 128 bp which codes for 375 amino acid residues.After the constructed expression vector pET-41b（＋）-Gα12 was transformed into E.coli BL21 for induced expression,SDS-PAGE and Western blot analysis showed that BmGα12 could be expressed efficiently in E.coli BL21.The fusion protein GST-BmGα12 mainly existed in the form of inclusion body.Its molecular mass was around 71 kD.Phylogenetic analysis revealed that BmGα12 has close genetic relationship with Gα12/Gα13 of rat and human.These results suggest that BmGα12-mediated signal transduction plays important roles in cell growth and embryonic development as well.