微小牛蜱雌蜱唾液腺消减cDNA文库的构建与分析  

Construction and analysis of subtractive cDNA library of salivary gland of female Boophilus microplus

作　　者：杨朝霞[1] 关贵全[1] 马米玲[1] 刘军龙[1] 殷宏[1] 罗建勋[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2010年第5期441-445,共5页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2007BAD40B06);国家自然资源平台项目(2005DKA21100);国家自然科学基金项目(30571397);国家高技术研究发展计划(863)项目(2006AA10A207)

摘　　要：以我国微小牛蜱半饱血雌性成蜱唾液腺cDNA为检测子Tester）,饥饿雌性成蜱cDNA为驱动子（Driver）,采用抑制消减杂交技术和T/A克隆技术构建了微小牛蜱半饱血雌性成蜱唾液腺消减cDNA文库。对获得的158个阳性克隆进行PCR鉴定,显示插入片段主要集中在200-550bp之间,挑取60个阳性克隆进行测序,共获得了14个有效ESTs,生物信息学分析推测,它们所编码的功能性蛋白包括卵黄蛋白原、基质蛋白、富含脯氨酸蛋白质、OTM-3假定蛋白等。根据基因序列设计引物,以微小牛蜱DNA为模板,对所得ESTs进行鉴定,证明它们都是微小牛蜱所固有的。该项工作为进一步研究微小牛蜱唾液腺差异表达基因奠定了基础。With cDNA from salivary gland of semi-engorged adult female Boophilus microplus as tester and cDNA from starved adult female B.microplus as driver,a subtractive cDNA library of salivary gland of female B.microplus was constructed using suppression subtractive hybridization and T/A cloning.The inserts of the 158 positive clones were from 200 to 550bp in size according to PCR identification and fourteen valid EST sequences were obtained from sixty sequenced clones.Bioinformatics analysis revealed that proteins encoded by these ESTs had similarity to proteins such as vitellogenin,matrix protein,acidic proline-rich protein and OTM-3 hypothetical protein.PCR with primers derived from these ESTs was performed using genome DNA of B.microplus as templates and the results showed that all the ESTs belonged to B.microplus.This research provided foundation for the study of differential expression of genes in salivary gland of female B.microplus.

关 键 词：微小牛蜱 抑制消减杂交 表达序列标签 序列分析 

分 类 号：S852.746[农业科学—基础兽医学]