家蚕普通气味结合蛋白Ⅰ和Ⅱ的cDNA克隆序列分析及在大肠杆菌中的表达.docVIP

PAGE 第 PAGE 15 页 (共 NUMPAGES 16 页) 家蚕普通气味结合蛋白Ⅰ和Ⅱ的cDNA克隆、序列分析及在大肠杆菌中的表达 作 者:宋志冰 指导老师:阚云超 (南阳师范学院生命科学与技术学院2003级4班) 摘要: 利用RT-PCR 技术扩增了编码家蚕雌、雄虫触角普通气味结合蛋白Ⅰ和Ⅱ的cDNA 片段,将其克隆至pGEM-T载体,获得了普通气味结合蛋白Ⅰ和Ⅱ基因成熟蛋白阅读框序列。将该基因重组到表达型质粒pET30a ( + ) 中,并转化入原核细胞中表达。序列测定结果表明,家蚕触角普通气味结合蛋白基因的成熟蛋白阅读框全长分别为495 bp 和483 bp,各自编码164和160 个氨基酸残基。推导的氨基酸序列与已报道的10 种昆虫普通气味结合蛋白Ⅰ和Ⅱ高度同源(73 %～98 %) ,并具有气味结合蛋白的典型特征。经IPTG诱导,普通气味结合蛋白Ⅰ和Ⅱ基因能在大肠杆菌BL21(DE3) 中表达。 关键词： 普通气味结合蛋白; 触角; 基因克隆; 原核表达 Cloning and sequencing of cDNA encoding general odorant binding protein Ⅰand Ⅱin the Bombyx mori Author: Song Zhibing Guider: Kan Yunchao (The college of life science and technology of Nan Yang Normal University Class4 Grade2003) Abstract : The cDNA encoding the general odorant binding protein ⅠandⅡ (named as GOBP1andGOBP2) was isolated from themale and female antennae of Bombyx mori by reverse transcription polymerase chain reaction (RT-PCR) . The cDNA fragment wasfurther cloned into Pgem-T vector , and then constructed into expression vector pET-30a ( + ) for overexpression in prokaryotic cells. Structural analysis showed that the full length of mature GOBP1 and GOBP2 open reading frame (ORF) were 495 bp and 483bp, encoding 164 and 160 amino acid residues . The deduced amino acid sequence showed a high identity to the reported sequences ofGOBP1 and GOBP2 from other insects and shared the typical structural features of odorant binding proteins from other insects.Induced by IPTG, the full length of GOBP1 and GOBP2 were expressed in Escherichia coli BL21 (DE3) . Key words ： general odorant binding protein ; antenna ; gene cloning ; prokaryotic expression 1 引言 昆虫气味结合蛋白(odorant binding protein ,OBP) 是一类低分子的水溶性酸性蛋白(Pelosi andMaida ,1995) ,在昆虫识别外界气味物质中起重要作用(Li and Prestwich , 1997 ;王睿等,1999) ,多在昆虫嗅觉感器中表达。对于鳞翅目昆虫依据其生理功能将OBP 分为信息素结合蛋白( pheromone bindingprotein , PBP) 、普通气味结合蛋白Ⅰ(general odorantbinding protein Ⅰ, GOBP1) 、普通气味结合蛋白Ⅱ(GOBP2) 和PBP 相关蛋白( PBP2like protein) (Li andPrestwich ,1997) 。实验证明PBP 可与特定的信息素结合,它们主要存在于毛形感器中,在信息素