<FONT face="Verdana">水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析</FONT>

刘连盟,王玲,黄雯雯,刘恩勇,黄世文

doi:10.3969/j.issn.1001-7216.2010.04.04

中国水稻科学 >

2010 , Vol. 24 >Issue 4: 353 - 359

DOI: https://doi.org/10.3969/j.issn.1001-7216.2010.04.04

研究报告

水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析

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中国水稻研究所, 浙江杭州 310006； *通讯联系人， E-mail:swhuang666@sohu.com

收稿日期: 1900-01-01

修回日期: 1900-01-01

网络出版日期: 2010-07-10

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Cloning， Expression and Sequence Analysis of G Protein β Subunit Gene of Rice False Smut Pathogen Ustilaginoidea virens

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China National Rice Research Institute， Hangzhou 310006, China； *Corresponding author, E-mail: swhuang666@sohu.com

Received date: 1900-01-01

Revised date: 1900-01-01

Online published: 2010-07-10

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摘要

水稻稻曲病是由稻绿核真菌\[Ustilaginoidea virens (Cooke) Tak\]引起的一种常见的水稻后期穂部病害。G蛋白可能参与了稻曲病菌的致病过程。为了研究G蛋白在病菌致病过程中的作用，分离并分析了稻曲菌的G蛋白β亚基编码基因。根据丝状真菌G蛋白β亚基编码基因的同源保守序列设计简并引物，采用同源克隆和热不对称交错PCR的方法，分离得到了稻曲菌的G蛋白β亚基全编码基因序列。该序列的长度为2037 bp，包含4个内含子，5个外显子和1个编码359个氨基酸的开放阅读框。根据克隆到的UvGβ1 设计引物，通过RTPCR克隆到包含整个开放阅读框的cDNA序列。该基因的DNA序列和cDNA序列在GenBank中注册的登记号分别为GU014921和GU065745。系统进化分析表明该片段与栗疫病菌(Cryphonectria parasitica)的G蛋白β亚基基因亲缘关系最近。将该基因的整个开放阅读框连接于pET30a构建原核表达载体，通过诱导获得了重组蛋白。

关键词： 水稻稻曲病菌; 异三聚体G蛋白; G蛋白β亚基基因; 热不对称交错聚合酶链式反应; 原核表达

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刘连盟,王玲,黄雯雯,刘恩勇,黄世文 . 水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析[J]. 中国水稻科学, 2010 , 24(4) : 353 -359 . DOI: 10.3969/j.issn.1001-7216.2010.04.04

Abstract

Ustilagionidea virens is a common rice pathogen, which causes rice false smut. G protein may involve in the pathogenicity of U. virens. To study the role of G protein of U. virens in the pathogenicity, a gene that encodes G protein βsubunit had been isolated and characterized. The degenerate primers based on conserved domains of other reported genes were designed to obtain a homologous fragment. Thermal asymmetric interlaced PCR (TAILPCR) was used to isolate flank sequence of the homologous fragment. The gene was obtained by jointing all the fragments and named UvGβ1. Full length of UvGβ1 was 2037 bp, which contained 4 introns, 5 extrons, and an open reading frame (ORF) encoding 359 amino acids. The cDNA sequence containing entire ORF of UvGβ1 was cloned through RTPCR by primers based on the sequence of UvGβ1. The DNA and cDNA sequences of UvGβ1 have been registered in GenBank with accession numbers GU014921 and GU065745. Phylogenetic analysis showed that UvGβ1 had the nearest relationship with the G protein βsubunit gene of Cryphonectria parasitica. The ORF of UvGβ1 was subcloned into pET30a and was expressed in E. coli BL21. The recombinant protein was obtained by IPTG inducing.

Key words： Ustilaginoidea virens; heterotrimeric G protein; G protein beta subunit gene; thermal asymmetric interlaced PCR; prokaryotic expression

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