家蝇几丁质酶MDCht2基因的序列分析、克隆与表达模式分析  

Sequencing, cloning, and analysis of the patterns of expression of the chitinase 2 gene in Musca domestica

作　　者：苏佩佩 赵欣宇 李妍 马慧玲 杨隆兵 刘鉴 付萍 国果[1,2] SU Pei-pei;ZHAO Xin-yu;LI Yan;MA Hui-ling;YANG Long-bing;GUO Guo(The Key Laboratory of Modern Pathogen Biology and Characteristics, College of Basic Medicine, Guizhou Medical University,Guiyang, China,550025;Key Laboratory of Environmental Pollution and Disease Surveillance, Ministry of Education, Guizhou Medical University,Guiyang, 550000)

机构地区：[1]贵州医科大学基础医学院现代病原生物学特色重点实验室,贵州贵阳550025 [2]贵州医科大学环境污染与疾病监控教育部重点实验室

出　　处：《中国病原生物学杂志》2019年第6期679-685,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81760647,81560337);贵阳市科技局基金项目:筑科合同[2017] 5-30

摘　　要：目的从家蝇的基因组库中筛选几丁质酶2(MDCht2)基因,进行cDNA克隆及分子特性分析,对其时空表达模式进行初步探索。方法从家蝇基因组数据库中筛选MDCht2基因,以该基因的cDNA为模板进行PCR扩增,采用生物信息学相关软件对MDCht2基因及其编码蛋白质的基本理化性质、信号肽、蛋白质结构等进行分析,预测蛋白质功能。取家蝇不同生活史时期(卵,1龄幼虫,2龄幼虫,3龄幼虫,蛹,雌雄成虫)及3龄幼虫不同组织(体壁,气管,马氏管,唾液腺,脂肪体,肠道)标本,采用实时荧光定量PCR检测MDCht2基因表达情况。结果 MDCht2基因cDNA全长1 932 bp,ORF框全长1 530 bp,编码509个氨基酸,理论相对分子质量为57.8×10^3,pI 5.67,属于亲水性的酸性蛋白,有信号肽,功能结构域位于第37~385位氨基酸间,无几丁质结合域。二级结构分析显示存在无规则卷曲(Cc),α螺旋(Hh),β折叠(Ee)3种类型。PCR扩增得到MDCht2基因长为1 530 bp的序列片段。实时荧光定量PCR检测家蝇MDCht2基因在蛹期的表达量较卵期上调6.477 01倍(P<0.01),3龄幼虫表达量上调2.655 25倍(P<0.01),1龄幼虫的表达量上调2.475 01倍(P<0.05),表达水平排列顺序为蛹>3龄幼虫>1龄幼虫>2龄幼虫>卵>雄成虫>雌成虫。以体壁为参照,MDCht2基因在家蝇气管中的表达量较高,气管中的表达量较体壁上调1.816 51倍(P<0.01),表达水平为气管>体壁>唾液腺>脂肪体>肠道>马氏管。结论成功克隆了家蝇的MDCht2基因并初步探索了其时空表达模式,即MDCht2基因在蛹期及气管组织高表达,为MDCht2的功能研究奠定了基础。Objectives To screen for the chitinase 2(MDCht2) gene from the genomic library of Musca domestica and to preliminarily examine the temporal and spatial patterns of its expression using cDNA cloning and molecular characterization. Methods The M. domestica genome database was screened for the MDCht2 gene, and the cDNA of the gene was used as a template for amplification with PCR. The basic physicochemical properties, signal peptides, and protein structure of the MDCht2 gene and its encoded protein were analyzed using bioinformatic software to predict the protein’s function. Expression of the MDCht2 gene in different tissues and stages of M. domestica development(eggs, first instar larvae, second instar larvae, third instar larvae, pupae, and male and female adults) and different tissues of the third instar larvae(body wall, trachea, Malpighian tubules, salivary glands, fat body, and gut) was detected using real-time PCR. Results The total length of MDCht2 cDNA is 1 932 bp and the total length of the ORF is 1 530 bp. The gene encodes 509 amino acids. The protein it encodes has a theoretical relative molecular mass of 57.8×10^3 and a pI of 5.67. It is a hydrophilic acidic protein. Amino acids 1-29 of the MDCht2 gene are signal peptides, a cleavage site is located at amino acids 29 and 30, a functional domain is located between amino acids 37 and 385, and there is no chitin binding domain. The MDCht2 protein has 17 serines, 11 threonines, and 7 tryptophans that may be sites of protein kinase phosphorylation. Analysis of the protein’s secondary structure revealed the presence of three types of random coils(CC), alpha helices(HH), and beta sheets(EE). Amplification with PCR yielded a fragment of the MDCht2 gene 1,530 bp in length. The level of expression of the MDCht2 gene in M. domestica was up-regulated 6.47701-fold in the pupal stage compared to that in the egg stage(P<0.01), up-regulated 2.655-fold in third instar larvae(P<0.01), and up-regulated 2.47501-fold in first instar larvae(P<0.05) according to real-tim

关 键 词：家蝇 几丁质酶2 基因克隆 生物信息学分析 时空表达 

分 类 号：R384.2[医药卫生—医学寄生虫学]