意大利蜜蜂AmCht11基因的克隆鉴定和发育时期表达分析  

Cloning,Identification,and Expression Analysis of AmCht11 Gene in Developing Apis mellifera

作　　者：王李[1] 王林玲[1] 马荣琴 衡惠 李治[1] WANG Li;WANG Linling;MA Rongqin;HENG Hui;LI Zhi(College of Life Science, Chongqing Normal University, Chongqing 401331, China)

机构地区：[1]重庆师范大学生命科学学院,重庆401331

出　　处：《重庆师范大学学报（自然科学版）》2018年第3期45-51,F0002,共8页Journal of Chongqing Normal University:Natural Science

基　　金：现代农业产业技术体系建设专项资金(No.CARS-44-KXJ21);国家高技术研究发展计划"863"项目(No.2013AA102507);重庆师范大学博士科研启动基金(No.13XLB009);重庆市教育委员会科学技术研究项目(No.KJ1703051);重庆市自然科学基金(No.31302037);重庆市前沿与应用基础研究计划项目(No.cstc2014jcyjA80039;No.cstc2015jcyjA80014)

摘　　要：【目的】克隆鉴定和定量PCR分析不同发育时期意大利蜜蜂(Apis mellifera)几丁质酶AmCht11基因。【方法】克隆测序鉴定AmCht11基因序列,通过多重序列比对分析该基因编码的氨基酸序列特征和结构域组成;在线预测AmCht11蛋白的二级结构、三级结构以及相互作用靶蛋白,构建系统进化树分析该蛋白在几丁质酶家族中的分类和进化关系;实时定量PCR分析AmCht11基因的组织表达特征。【结果】AmCht11基因c DNA序列全长1 404 bp,编码468个氨基酸;预测AmCht11蛋白质相对分子质量为53.5 k Da,等电点为7.21。多重序列比对和系统进化分析表明,AmCht11蛋白为几丁质酶GH18家族Group-Ⅷ亚家族成员,该蛋白的氨基酸序列N-端含有跨膜域,具典型的几丁质酶催化结构域,无结合结构域,也不具信号肽。进一步实时定量PCR分析表明,AmCht11基因表达量在意大利蜜蜂幼虫发育第2至第5日龄无明显波动,至幼虫后期第6日龄表达量明显上调。【结论】推测AmCht11基因与意大利蜜蜂幼虫发育后期的蜕皮有关。[Purposes]To clone,identify,and q PCR analysis of chitinase gene Am Cht11 in honeybees Apis mellifera. [Methods]Cloning,sequencing,and identification of the Am Cht11 gene sequence. Multiple alignment analysis of the Am Cht11 amino acid sequence features and domain composition. Prediction online and analysis of the Am Cht11 secondary structure,3 D structure model,and the interaction target protein. Construct phylogenetic tree to analyze the classification and evolution relationship of Am Cht11 in chitinase family. Real-time quantitative PCR analysis of Am Cht11 gene expression pattern in different tissues of A. mellifera. [Findings]The full length of Am Cht11 c DNA sequence was 1 404 bp,and encodes 468 aminoacids residues. Am Cht11 has a calculated molecular mass of 53. 5 k Da and a theoretical p I of7. 21. Multiple sequence alignment and phylogenetic analysis showed that Am Cht11 can be classified into the Group-Ⅷ of the GH18 family of glycoside hydrolase. Am Cht11 has the typical Glyco_18 catalytic domain but without the N-terminal signal peptide and the chitin binding domains as in most chitinase family members. Further real-time quantitative PCR analysis showed that the expression level of Am Cht11 genedid not significantly change from the 2 nd to 5 th day old,but up-regulated start at the 6 th day old of larval development. [Conclusions]Am Cht11 may have an important role in the molted of A. mellifera at the end of larval stage.

关 键 词：几丁质酶 GH18家族 克隆 表达谱 意大利蜜蜂 

分 类 号：Q966[生物学—昆虫学]