20E诱导家蚕抗菌肽CecropinB6的上调表达

Upregulation of CecropinB6 expression was triggered by 20E in silkworm,Bombyx mori

蜕皮激素(20-hydroxyecdysone,20E)是调控昆虫发育的重要激素,在昆虫的蜕皮和变态中起关键作用。近年来的研究表明,20E也调控昆虫抗菌肽的表达,揭示昆虫的发育和免疫之间具有重要的联系。家蚕是重要的经济昆虫,家蚕抗菌肽对蜕皮激素(20E)的应答及其调控机制仍有待研究。本文利用20E注射家蚕5龄第3天幼虫,qRT-PCR结果表明20E处理的脂肪体中抗菌肽CecropinB6基因(BmCecB6)的表达上调。通过对抗菌肽BmCecB6上游启动子的截短和双荧光素酶活性分析,结果显示BmCecB6响应20E的调控位点在启动子-448~-170区域,该区域内存在潜在的FoxO、E74A和BR-C等结合位点。本研究表明20E抑制了ILS通路水平,暗示ILS下游的转录因子FoxO被激活。进一步对BmCecB6的启动子进行FoxO结合位点的缺失突变,双荧光素酶检测结果表明20E对BmCecB6的诱导活性并没有丧失,推测BmCecB6对20E的应答不是通过转录因子FoxO结合BmCecB6启动子中的顺式调控元件直接调控的。20E激活BmCecB6表达的分子调控机制还需要进一步深入研究。

20-hydroxyecdysone(20E)is an important hormone involved in insect development and it plays a key role in insect molting and metamorphosis.Recent studies have shown that 20E also regulates the expression of antimicrobial peptides(AMPs),revealing the crosstalk between insect development and immunity.Silkworm is an important economic insect.The expression of AMPs in response to 20E in silkworm and the regulatory mechanism remain to be studied.In this study,we showed by qRT-PCR that the expression of CecropinB6(BmCecB6)gene in the fat body was upregulated when 20E was injected into the hemocoel of the 3 rd day of 5 th instar silkworm larvae.Then a series of BmCecB6 truncated promoters were constructed and analyzed for their transcriptional activities by Dual-luciferase reporter assays.The results showed that the 20E responsive elements were located in the promoter region between-448 and-170 bp,which contains potential DNA binding sites for FoxO,E74A and BR-C transcription factors.Our study showed that 20E inhibited the level of the ILS pathway,suggesting that FoxO,the transcription factor downstream of the ILS pathway,was activated.The Dual-luciferase activity results showed that the mutated BmCecB6 promoter,in which the potential FoxO binding sites were deleted,was still activated by 20E.Therefore,activation of BmCecB6 by 20E was not directly due to the binding of transcription factor FoxO to the cis-regulatory sites in the BmCecB6 promoter.The molecular mechanism of BmCecB6 expression in response to 20E needs to be further studied.