摘要
旨在对家蝇伴侣蛋白CCTδ基因进行c DNA克隆、序列分析,并对其时空表达模式进行初步探索。采用EST测序技术从已构建的家蝇幼虫c DNA质粒文库中筛选到家蝇CCTδ基因,以该基因的c DNA文库质粒为模板,通过PCR的方法进行扩增。运用生物信息学方法对该基因及其编码蛋白进行结构与功能分析,构建系统进化树。取家蝇不同生活史时期虫体(卵、各龄幼虫、蛹、雄雌成虫),3龄幼虫不同组织部位(体壁、气管、唾液腺、脂肪体、马氏管及中肠),采用实时荧光定量PCR分析CCTδ基因在家蝇不同发育阶段和组织部位的表达情况。结果显示,经PCR扩增后,得到了1 600 bp左右的特异性家蝇CCTδ基因片段;CCTδ基因ORF全长1 602 bp,编码533个氨基酸,理论分子量57.16 k D,等电点为7.50;二级结构主要以α-螺旋和不规则卷曲为主;进化树分析显示其具有较好的保守性,与中华按蚊的遗传距离较近。时空表达谱显示:该基因在家蝇不同发育时期均有表达,以蛹期表达量最高;在3龄幼虫不同组织中,以气管表达量最高。成功克隆了家蝇CCTδ基因并初步探索了其时空表达模式。
This work aims to clone,have bioinformatic analysis and explore the expression patterns of chaperonin gene CCTδ from Musca domestica. We screened and isolated the gene CCTδ from the built c DNA plasmid library of M. domestica larva by EST sequencing,and having the plasmid as template,the amplification was conducted by PCR. Further,with bioinformatics we analyzed the structures and functions of gene CCTδ and the encoded protein,and constructed phylogenetic tree. We collected the bodies at different developmental stages of M. domestica life history（eggs,varied-instar larvae,pupae,female and male adults）,and chose 6 tissues of 3-instar larvae（body wall,trachea,salivary glands,fat body,markov tube,and midgut）,thus the expression patterns of gene CCTδ at different developmental stages and tissues of M. domestica were detected by real-time quantitative PCR. An about 1 600 bp specific fragment of housefly＇s gene CCTδ was acquired by PCR amplification. The open reading frame of gene CCTδ was 1 602 bp that encoded a putative protein with 533 amino acids. The predicted molecular weight of the protein was 57.16 k D and p I was 7.50. The secondary structures were mainly composed of α-helix and random coil. Comparative analysis of the phylogenetic tree showed that it was conservative,and the genetic distance was closer with Anopheles darlingi. The spatial and temporal expression patterns of gene CCTδ revealed that gene CCTδ expressed in each developmental stage of M. domestica,while the highest at pupa stage;and it expressed the highest in trachea among various tissues of 3-instar larvae. Conclusively,in this study,the gene CCTδ of M. domestica was successfully cloned,and the spatial and temporal expression patterns of gene CCTδ were preliminarily explored.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第3期115-121,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(81160204
81360254)
贵州省卫生厅基金项目(gzwjkj2014-2-100)
国家科技部支撑计划课题子课题(2011BAC06B12)
贵州省科学技术基金项目(黔科合J字[2012]2038号)
作者简介
陶如玉，女，硕士研究生，研究方向：昆虫分子免疫；E-mail：494858079@qq．com
通讯作者：国果，女，博士，研究方向：昆虫分子免疫；E-mail：guoguojsc@163．com
