蜕皮激素受体和超气门蛋白基因在柞蚕发育过程及激素诱导后的表达模式

The Expression Patterns of Ecdysone Receptor and Ultraspiracle Genes in Antheraea pernyi During Development and Hormone-induced Process

昆虫蜕皮激素受体和超气门蛋白形成的异源二聚体介导蜕皮激素信号,起始包括转录因子表达在内的蜕皮级联反应,从而使昆虫进入蜕皮和变态等生命过程。克隆了柞蚕的2个蜕皮激素受体相关基因,命名为Ap EcRB1(Gen Bank登录号:KY411159)和Ap USP1(Gen Bank登录号:KY411160)。2个基因的ORF序列全长分别为1 758 bp、1 401 bp,编码585个、466个氨基酸。半定量RT-PCR检测这2个基因除在柞蚕幼虫血淋巴中不表达之外,在其他组织中均有表达,在丝腺和脂肪体中的表达量较高。实时荧光定量PCR(qRT-PCR)分析2个基因在幼虫蜕皮前及化蛹前后呈现大幅度上调表达的规律。注射20-羟基蜕皮甾酮(20E)诱导启动滞育蛹的发育,结果诱导组的滞育蛹经过17 d羽化,比对照组滞育蛹提早羽化。滞育蛹中2个基因的表达量在注射20E后均明显下降,其中Ap EcRB1在蛹发育后期的12 d内一直处于较低水平,但羽化前期其表达量急剧上升,蛹发育16 d(羽化前1 d)达到最高;Ap USP1基因的表达量从注射后8 d开始升高,至16 d时达到最高。以上研究结果丰富了柞蚕变态发育过程中蜕皮激素调控作用的分子信息。

In insect,ecdysone receptor and ultraspiracle protein form heterodimers to mediate ecdysone signaling and initiate molting cascade reactions including expression of transcription factors,so as to cause insect molting,metamorphosis and other life activities. Two ecdysone receptor related genes in Antheraea pernyi were cloned,and named as Ap EcRB1（ Gen Bank accession No. KY411159） and Ap USP1（ Gen Bank accession No. KY411160）. The open reading frames（ ORF） of Ap EcRB1 genes and Ap USP1 are 1 758 bp and 1 401 bp long,encoding 585 and 466 amino acids respectively. Semi-quantitative RT-PCR analysis showed that these two genes were expressed in all other tissues except the hemolymph of A. pernyi larvae,among which silk gland and fat body had higher expression levels over other tissues. The expression of these two genes was substantially up-regulated before larva ecdysis and around pupation as revealed by real-time fluorescent quantitative PCR（ qRT-PCR）. After injection with 20-hydroxyecdysone（20E）,pupae at diapause were induced to develop. The pupae of induced group appeared eclosion after 17 d,which was ahead of the contrast. The expression levels of both genes in pupae at diapause significantly decreased after 20 E injection. Ap EcRB1 gene maintained at a low level during 1 d to 12 d,but increased sharply before ecosion,and reached the highest level at 16 d（ the day before eclosion）. The expression level of Ap USP1 gene started to increase at 8 d,and reached the highest level at 16 d. The above results enriched information of molecular regulating effect of ecdysone on metamorphosis development in A. pernyi.