德国小蠊几丁质酶基因的克隆及表达模式分析  

Cloning and expression patterns analysis of a chitinase gene from Blattella germanica(Blattodea: Blattellidae)

作　　者：张道伟[1] 陈静[2] 袁媛[1] 杨静[1] 钱正敏[1] 

机构地区：[1]遵义师范学院生命科学学院贵州省赤水河流域动物资源保护与应用研究重点实验室,贵州遵义563002 [2]遵义医学院生化与分子生物学教研室,贵州遵义563003

出　　处：《环境昆虫学报》2015年第2期293-301,共9页Journal of Environmental Entomology

基　　金：贵州省联合基金项目(黔科合J字LKZ[2013]23号);遵义医学院博士启动基金(F-565)

摘　　要：几丁质酶广泛存在于自然界多个物种,其主要参与蜕皮、消化、防御与免疫等多项功能。本研究采用同源克隆结合RACE-PCR的方法,从德国小蠊中克隆到几丁质酶基因(Bg Chi)的c DNA全长序列(Gen Bank No:KJ789158)。Bg Chi全长为1881 bp,包含192 bp的3'非翻译区(UTR)和69 bp的5'UTR,其开放阅读框(ORF)为1620 bp,编码539个氨基酸。预测的相对分子量为59.7 k Da,理论等电点为5.0。Bg Chi具有几丁质酶典型的结构域:N-端信号肽,几丁质催化区,C-端几丁质结合区。系统进化树分析表明:Bg Chi与同翅目禾沫蝉Poophilus costalis的亲缘关系最近,为81%。组织分布表达显示,Bg Chi主要在表皮、中肠和气管组织有表达。定量PCR结果表明,Bg Chi在6龄蜕皮前表达量最高,随之下降到较低水平。本研究结果有助于提高我们对德国小蠊蜕皮发育机制的理解,也为从几丁质降解途径获取防治德国小蠊新方法奠定基础。Chitinases are widely distributed in nature as they are found in many species with different function including digestion,molting and defense / immunity. In the present study,Blattella germanica chitinase gene（ Bg Chi） was first obtained by using techniques of homological cloning and RACE- PCR.The full length of c DNA sequence is 1881 bp（ Gen Bank accession number： KJ789158）,containing a3’UTR（ untranslated region） of 192 bp,and a 5’UTR of 69 bp. The ORF（ Open Reading Frame） of B.germanica chitinase consisted of 1620 bp which encoding 539 amino acids residues. The deduced protein composition has a calculated molecular weight of 59. 7 k Da and theoretical PI of 5. 0. Bg Chi protein was composed of N- terminal leading signal peptide,catalytic domain,linker region and C- terminal chitin- binding domain showing a typical insect chitinase domain structure. Phylogenetic tree analysis showed that Bg Chi had the closest genetic relationship with Poophilus costalis（ up to 81%）. Tissue distribution showed that Bg Chi was mainly expressed in the epidermis,midgut and tracheal. The transcription level of Bg Chi mRNA increased before molting in 6thinstar larval,and decreased after molting by quantitativereal time PCR（ qRT- PCR）. These results help us to improve understanding the mechanisms of molting of B. germanica,and also provide a good foundation for obtaining new methods to control German cockroach from chitin degradation pathway.

关 键 词：德国小蠊 几丁质酶 基因克隆 序列分析 表达模式 

分 类 号：Q965[生物学—昆虫学] S433.89[农业科学—农业昆虫与害虫防治]