大豆立枯丝核菌G蛋白β亚基基因的克隆与分析  

Cloning and Analyzing of G-protein β-Subunit Gene in Rhizoctonia solani Causing Soybean Sharp Eyespot

作　　者：马炳田[1,2] 曲广林[1] 黄文娟[1] 林瑜凡[1] 李仕贵[1,2] 

机构地区：[1]四川农业大学水稻研究所,成都温江611130 [2]四川农业大学/西南作物基因资源与遗传改良教育部重点实验室,四川雅安625014

出　　处：《作物学报》2009年第2期370-374,共5页Acta Agronomica Sinica

基　　金：教育部长江学者和创新团队发展计划项目(IRT0453)资助

摘　　要：由立枯丝核菌[Rhizoctonia solani Khn,有性世代:Thanatephorus cucumeris(Frank)Donk]引起的大豆纹枯病(soybean sharp eyespot)是一种重要病害。G蛋白β亚基(guanine nucleotide binding protein beta-subunit)作为重要的信号传导因子,在植物病原菌致病分子机制中起着重要作用。为了解G蛋白β亚基基因的结构与功能,根据同源物种G蛋白β亚基相关序列设计引物,利用PCR和RT-PCR技术克隆了大豆立枯丝核菌G蛋白β亚基的基因序列和开放阅读框(G-protein beta-subunit of soybean Rhizoctonia solani,简写为gbsrs1,GenBank登录号为EU663628)。该片段全长1864bp,含有4个内含子和5个外显子;开放阅读框(ORF)长1047bp,编码348氨基酸残基,与多种真菌G蛋白β亚基的氨基酸序列相似程度较高,达79.72%～99.43%;该蛋白质具有2个α-螺旋和7个β-折叠的二级结构,形成无规则卷曲连接的桶形三级结构。将gbsrs1的ORF连接于原核融合表达载体pGEX-4T-2中,经IPTG诱导,获得了相应蛋白的表达。gbsrs1的克隆和特性研究为了解大豆立枯丝核菌的致病机理、有效防治纹枯病奠定了基础。Soybean sharp eyespot is one of the most serious diseases in world. The protein encoded by G-protein β-subunit （gua- nine nucleotide binding protein beta-subunit） gene plays an important role in pathogenesis mechanism. In this paper, the G-protein β-subunit from Rhizoctonia solani （Teleomorph： Thanatephorus cucumeris） causing soybean sharp eyespot was identified. The genome of 1 864 and 1 047 bp open reading frame （ORF） were amplified by PCR and RT-PCR. The gene included 4 introns and 5 exons, lntrons ranged in size from 54 to 65 bp, and their sequences complied with the rule of ”5′-gt“ and “ag-3″” （GenBank Accession No. EU663628）. The ORF encoded 348-amino acid polypeptide with 38.24 kD of calculated molecular weight and 6.31 of pl. There were two alpha-helixes and seven beta-sheets including four beta-strands each in its amino acid secondary structure. Two alpha-helixes in its N-terminal and seven beta-sheets formed barrel structure by non-regular curl in the tertiary structure. The deduced amino acid sequence of β-subunit was identical to that from Rhizoctonia solani （GenBank Accession No. EU267677, AY884129）, Lentinula edodes （GenBank Accession No. AAT74567）, Coprinopsis cinerea （GenBank Accession No. EAU92269）, Ustilago maydis （GenBank Accession No. AAN33051） and Filobasidiella neoformans （GenBank Accession No. AAD03596） with 99.43%, 89.19%, 87.97%, 83.66%, 80.23%, and 79.72%, respectively. The amplified ORF was ligated into the prokaryotic fusion expression vector pGEX-4T-2. E. coli BL21 was transformed with this recombinant vector and induced by IPTG for expression. The result indicated that the protein size of ORF matched the prediction. This cloning of this gene provides the evidence for controlling hyphal growth, development and virulence in R. solani.

关 键 词：大豆 立枯丝核菌 致病性 G蛋白β亚基 信号传导 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]