家蚕葡萄糖-甲醇-胆碱氧化还原酶家族基因BmGMCβ2的克隆及序列与表达分析  

Cloning,Sequence and Expression Analysis of BmGMCβ2 Gene in the Silkworm GMC Oxidoreductase Family

作　　者：杨文娟[1] 刘博[1] 肖丽蓉[1] 冯丹丹[2] 周良宵[1] 沈以红[1] 张泽[1,2] 

机构地区：[1]西南大学蚕学与系统生物学研究所,重庆400716 [2]重庆大学农学及生命科学研究院,重庆400044

出　　处：《蚕业科学》2012年第4期634-642,共9页ACTA SERICOLOGICA SINICA

基　　金：西南大学博士创新项目(No.Kb2009019)

摘　　要：葡萄糖-甲醇-胆碱氧化还原酶(glucose-methanol-choline oxidoreductases,GMC)家族是昆虫体内一大类以黄素腺嘌呤二核苷酸(FAD)为辅酶的氧化还原酶类,家蚕基因组中含有43个GMC家族基因。以家蚕5龄幼虫cDNA为模板克隆到一个家蚕GMC家族基因,命名为BmGMCβ2(GenBank登录号:JQ965926)。该基因cDNA全长1 875 bp,编码624个氨基酸,预测蛋白质分子质量为69.3 kD,pI为6.21,定位于家蚕16号染色体的nscaf3058上,编码蛋白质含GMC家族共有的ADP-bindingβ-α-β折叠结构域。系统发育分析表明该基因是家蚕GMC家族β亚家族基因成员。RT-PCR检测家蚕不同发育时期和5龄第3天幼虫不同组织中BmGMCβ2基因mRNA转录水平,以5龄盛食期最高,并且主要在表皮、脂肪体和气管中转录表达;Westernblotting分析BmGMCβ2蛋白主要在5龄第3天幼虫的脂肪体中表达。将BmGMCβ2克隆到原核表达载体pET-28a(+)中,转化E.coli BL21表达菌株,IPTG诱导表达BmGMCβ2融合蛋白,通过His-亲和层析得到纯化的融合蛋白并制备多克隆抗体,为后续研究该蛋白在家蚕生长发育过程中的功能奠定了基础。Glucose-methanol-choline oxidoreductases （GMC） in insects are a large family of oxidoreductases using flavin adenine dinucleotide （FAD） as coenzyme, There are 43 members of GMC family in the genome of silkworm （Bombyx mori）. One GMC family gene in the silkworm was cloned by using cDNA from the 5th instar larvae as template and was designated as BmGMCβ2 （GenBank accession No, JQ965926）. Full-length cDNA of this gene is 1 875 bp long and encodes for 624 amino acids with a predicted protein molecular mass of 69.3 kD and an isoelectric point of 6.21. BmGMCf32 is located on scaf3058 of silkworm chromosome 16, and its encoded protein contains the ADP-binding β-a-β fold domain shared by GMC family. Phylogenetic analysis indicated that the gene is a member ofβ-subfamily in GMCfamily. The transcription levels of BmGMCβ2 mRNA in different developmental stages of silkworm and different tissues of day 3 larvae of the 5th instar were detected by RT-PCR. The results demonstrated that the transcription level was the highest at full appetite stage of the 5th instar, and BmGMCβ2 was expressed predominantly in integument, fat body and trachea. Western blotting analy-sis indicated that BmGMCβ2 protein was mainly expressed in the fat body of day 3 larvae of the 5th instar. We cloned BmGMCβ2 gene into the prokaryotic expression vector pET-28a（＋） and transformed the recombinant plasmid into Escherichia coli expression strain BL21. After induction with IPTG and purification by His-affinity chromatography, the recombinant BmGMC#2 fusion protein was used to prepare polyclonal antibody. The obtained results establish a good basis for further studies on function of this protein during growth and development of silkworm.

关 键 词：家蚕 葡萄糖-甲醇-胆碱氧化还原酶家族基因GMCβ2 基因克隆 序列特征 表达谱 原核表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]