埃及伊蚊腺苷脱氨酶相关基因的鉴定和表达分析  

IDENTIFICATION AND EXPRESSION ANALYSIS OF ADENOSINE DEAMINASE GENE FAMILY OF GENES IN AEDES AEGYPTI

作　　者：吕清巧 翟慧[1] 颜凤[2] 程金芝[1] 吴家红[1] 商正玲[1] 

机构地区：[1]贵州医科大学环境污染与疾病监控省部共建教育部重点实验室,贵州医科大学现代病原生物学实验室,贵州医科大学基础医学院,贵阳550025 [2]遵义市第一人民医院检验科,贵州遵义563002

出　　处：《寄生虫与医学昆虫学报》2017年第1期12-18,共7页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金项目(Nos:30600515,81060318,81260260);贵州省省长基金项目(黔省专合字(2007)48号)

摘　　要：为了筛选和鉴定埃及伊蚊全基因组中腺苷脱氨酶相关基因，并分析其在蚊虫不同发育时期、雌蚊不同组织中的表达差异，本文运用模式昆虫黑腹果蝇Dm-ADA基因的ADA结构域序列在NCBI埃及伊蚊全基因组数据库中筛选并鉴定埃及伊蚊ADA相关基因，采用ExPASy在线相关工具包分析其理化性质和结构域，运用SignalP4．0预测信号肽，结合使用GeneDoc和Mega6．0软件包进行同源性比对和构建系统进化树构建。首先提取埃及伊蚊不同发育时期（卵、I～Ⅳ龄幼虫、蛹、未吸血雌蚊、雄蚊）、雌蚊不同组织（唾液腺、中肠、脂肪体和卵巢）RNA并逆转录为cDNA，利用荧光实时定量PCR（qRT—PCR）方法对埃及伊蚊不同发育时期和雌蚊不同组织表达谱进行相对定量分析。结果显示，从埃及伊蚊全基因组中获得3条具有完整开放阅读码框的ADA相关基因（Aa-ADAL、Aa-ADGF-A、Aa-ADGF-B）；尽管不同物种ADA基因家族成员间相似性较低，但8个酶活性位点高度保守。Aa-ADAL在2龄幼虫期（7．88）、卵巢（8．05）高表达；Aa-ADGF-A在3龄幼虫期（7．25）高表达；Aa-ADGF-B在雌蚊唾液腺（10．07）显著高表达。经分析，2条属于ADA2／ADGF亚家族，1条属于ADAL亚家族。To identify the adenosine deaminase （ADA） -related genes in Ae. aegypti and their expression pattern at various developmental stages and in different tissues of female. The ADA-related genes were identified by an extensive search on the Ae. aegypti genome with the ADA domain of Drosophila melanogaster （GenBank No. NM_ 141609, NM_079406, AF384215, AF337552, AF337553） as clues. The domains were analyzed with the on-line tools, the signal peptides was predicted with Signal P4. 1 software. Sequence alignment and the phylogenetic tree were made by MEGA 6.0 and GeneDoc. Specific primers for each ADA member were disigned and quantitative analysis was conducted by real-time RT-PCR. Three ADA-related genes with complete ORF were finally identified from Ae. aegypti genome （ Aa-ADAL, Aa-ADGF-A, Aa-ADGF- B） , and high similarity was found only in the eight conserved ADA enzyme active sites. The three ADA-related genes were all expressed at different developmental stages and in various tissues of female. Among them, Aa-ADAL was expressed higher in the second instar （7.88） and the ovary （8.05）, Aa-ADGF-A was expressed higher in the third instar （7.25）, while the highest relative expression amount of Aa-ADGF-B transcripts was observed in salivary gland of female （ 10. 07）. Aa- ADGF-A and Aa-ADGF-B belonged to ADA2/ADGF subfamily, and Aa-ADAL was classified into ADAL subfamily.

关 键 词：埃及伊蚊 ADA基因家族 QRT-PCR 表达谱 

分 类 号：R384.1[医药卫生—医学寄生虫学]