玉米大斑病菌G蛋白β亚基编码基因的克隆与表达  

Cloning of G Protein β Subunit Gene of Setosphearia turcica and Its Prokaryotic Expression

作　　者：郝志敏[1] 李志勇[1] 董金皋[1] 周程[2] 

机构地区：[1]河北农业大学植物分子病理学实验室,保定071001 [2]河北大学附属医院,保定071000

出　　处：《农业生物技术学报》2008年第6期1025-1030,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30471126);河北省自然科学基金项目(No.C2008000335)资助

摘　　要：研究克隆了玉米大斑病菌(Setosphearia turcica)G蛋白β亚基编码基因Stgb-1。Stgb-1基因全长1296bp,由5个外显子和4个内含子组成;开放阅读框(ORF)为1056bp,编码351个氨基酸,蛋白质计算分子量为39.081kD。其推导氨基酸序列与Cochliobolus heterostrophus、Cryphonectria parasitica和Aspergillus fumigatus的G蛋白β亚基编码基因均具有较高的同源性,分别为100%、80%和81%。将该序列提交GenBank登录号为No.EF407555。利用RACE(rapid amplification of cDNA ends)和Genomic walking技术获得了Stgb-1基因3'末端cDNA和3'端DNA侧翼序列,BlastN结果表明其cDNA3'-UTR为136bp,poly(A)由9个A构成。构建了pET28a-Stgb-1原核表达载体,SDS-PAGE检测和Western blot鉴定结果表明,目的蛋白在大肠杆菌(Escherichia coli)BL21菌株中诱导表达。Stgb-1 gene from the phytopathogenic fungus of Setosphearia turcica, which encoded a protein of 351 amino acid residues, was 1 296 bp and interrupted by four introns. It shared a high degree of amino acid homology with Gβ protein from Cochliobolus heterostrophus, Cryphonectn＇a parasitica and Aspergillus fumigatus, which was 100%, 80% and 81%, respectively. The sequence has been deposited in the GenBank/EBI Database（accession No. EF407555）. Results of RACE （rapid amplification of eDNA ends） and Genomic walking showed a 136 bp 3′UTR in its 3′ end eDNA with a poly （A） tail containing nine adenylates. The pET vector system was used to express Stgb-1 in vitro. Results of SDS-PAGE and Western blot showed that the recombinant protein with the calculated molecular mass of 40 kD was expressed in Escherichia coll.

关 键 词：玉米大斑病菌 G蛋白 Β亚基 原核表达 

分 类 号：S188[农业科学—农业基础科学]