新疆荒漠昆虫光滑鳖甲cDNA文库的构建及功能基因筛选

Construction and Analysis of cDNA Library of Anatolica Polita Borealis

以过冬后早春的新疆荒漠拟步甲属昆虫光滑鳖甲(Anatolica Polita borealis)成虫为研究材料,应用SMARTTM cDNA Library Construction技术,通过总RNA提取,反转录合成cDNA,定向构建至噬菌体载体λTripEx2,经体外包装构建了光滑鳖甲的cDNA表达文库。测试结果表明库容量为2.2×106,重组率为84.8%。通过对cDNA文库克隆的序列测定和初步生物信息学分析,获得28个光滑鳖甲表达序列标签(ESTs),包括17个EST(60.7%)与NCBI中已注册的已知功能基因相似性较高,另外的11个EST(39.3%)则没有发现与之相似的序列,推测可能是功能未知的新基因。同时,利用PCR扩增文库技术从cDNA文库中克隆获得了光滑鳖甲的抗冻蛋白基因,初步表明光滑鳖甲cDNA表达文库构建的成功,为深入研究新疆荒漠昆虫的抗冻机理及发现新的昆虫功能基因奠定了基础。

Desert beetle Anatolica Polita borealis was collected in Zdhungar desert in early spring to construct cDNA library.Total KNA was extracted and cDNA was synthesized by LD-PCR （long-distance PCR） . cDNA library was constructed with SMARTTM cDNA Library Construction Kit from Clontech, The titer of the original library is 2.2 × 106. cDNA clones were randomly selected and partially sequenced. Partial sequence from 5＇end of each clone was obtained. Out of 28 expressed sequence tags （ESTs）, 17 ESTs （60.7%） had sequence homology to defined genes at gene and peptide level, while 11 ESTs （39.3%） were novel or unknown protein, Meanwhile antifreeze protein gene （AFP） was selected by PCR from this library to confirm the qualification of the library. The successful construction of cDNA library from Anatolica Polita borealis will lay a foundation for finding novel genes and for the study of how beetles survive cold winter at gene level.