小地老虎隐花色素基因cryptochrome1和cryptochrome2的克隆及mRNA表达分析
Molecular cloning and mRNA expression analysis of cryptochrome
程 鹏 徐蓬军 高希武 肖海军 付晓伟 等
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作者单位：1. 中国农业大学农学与生物技术学院北京100193; 2. 植物病虫害生物学国
中文关键词：小地老虎，隐花色素，基因表达
英文关键词：Agrotis ypsilon，cryptochrome，mRNA expression
中文摘要： 隐花色素( cryptochrome，cry)是一类广泛存在于生物体内的蓝光和近紫外光受体，介导生物对蓝光的一系
列反应并能导引生物钟。本研究利用RT-PCR 和RACE 方法获得了小地老虎Agrotis ypsilon cry1 和cry2 基因，分别命名为Aycry1 和Aycry2。Aycry1 基因(GenBank No． JQ616846)读码框1 587 bp，编码528 个氨基酸，预测分子量60. 5 ku，等电点6. 68。Aycry2 基因(GenBank No． JQ616847)读码框2 439 bp，编码812 个氨基酸，预测分子量92. 1ku，等电点8. 45。保守区分析表明:Aycry1 和Aycry2 均含有FAD 结合位点的PHR 区域和C 末端保守区域。氨基酸序列比对分析表明，小地老虎的AyCRY1 和AyCRY2 分别与其它鳞翅目昆虫的CRY1 和CRY2 有很高的一致性，其中与棉铃虫Helicoverpa armigera 的一致性最高，分别为89. 5% 和73. 8% 。NJ 聚类分析结果表明昆虫含有两类CRY，即CRY1 和CRY2，它们可分别以目为单位进行聚类，其中AyCRY1 和AyCRY2 分别与其它鳞翅目昆虫的CRY1 和CRY2 聚到一起。以室内饲养的小地老虎为材料，以3 h 为间隔检测了Aycry1 和Aycry2 的24 h 昼夜表达 变化，结果表明这2 个基因均表现出一定的昼夜节律性。Aycry1 和Aycry2 表达趋势白天高于晚上，表达峰值出现在ZT7( Zeitgeber time)。方差分析其昼夜波动差异不显著。
英文摘要： Cryptochrome ( cry) are blue and UV light photoreceptors and also act as an integral component of circadian clocks． In this study，two black cutworm ( Agrotis ypsilon ) cryptochrome genes，named Aycry1 ( GeneBank No．JQ616846) and Aycry2 ( GeneBank No． JQ616847 )，were cloned and characterized by RT-PCR and SMART RACE strategy． Their AUG-initiated ORFs ( open reading frames ) were 1 587 bp and 2 439 bp in length and encoded polypeptides were comprised of 528 and 812 amino acids respectively． Conserved domain analysis suggests that both Aycry1 and Aycry2 contain two basic conserved domains，the PHR region，which provides FAD binding sites，and the Cterminal conserved domain． Phylogenetic analysis indicates that insect CRYs are divided into two subgroups，CRY1 and CRY2，and AyCRY1 and AyCRY2 cluster with Lepidopteran CRY1 and CRY2 genes，respectively． Diel fluctuation of both Aycry1 and Aycry2 mRNA levels was determined by quantitative PCR analysis． The mRNA levels of both Aycry1 and Aycry2 were higher during the day than at night peaking at ZT7．