小地老虎隐花色素基因cryptochromel和cryptochrome2的克隆及mRNA表达分析

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Agrotisypsiloncryl和cry2基因，分别命名为Aycryl和Aycry2。Aycryl基因（GenBankNo．JQ616846）读码框1587bp，编码528个氨基酸，预测分子量60．5ku，等电点6．68。Aycry2基因（GenBankNo．JQ616847）读码框2439bp，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：Aycryl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HelicoverpaaFmigera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3h为间隔检测了Aycry1和Aycry2的24h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（Zeitgebertime）。方差分析其昼夜波动差异不显著。

Molecular cloning and mRNA expression analysis of cryptochrome genes of the black cutworm Agrotis ypsilon

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 Aycry! （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 Aycryl and Aycry2 contain two basic conserved domains, the PHR region, which provides FAD binding sites, and the C- terminal 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 Aycryl and Aycry2 mRNA levels was determined by quantitative PCR analysis. The mRNA levels of both Aycryl and Aycry2 were higher during the day than at night peaking at ZT7.