Expression and function of ecdysone receptor in tissue differentiation of insect

蜕皮激素，昆虫的蜕皮激素，渗透到靶细胞，并与细胞核中的蜕皮激素受体（EcR）结合。这种复合物（ecdysone-EcR/USP）调节一组特殊的基因表达，最终导致昆虫的蜕皮和变态。我研究了变态过程中组织分化的分子机制，主要研究EcR表达与蜕皮激素诱导的基因表达网络的关系。为此，我们首先利用5^1-RACE鉴定了家蚕EcR的两个同工异构体BmEcR-A和BmEcR-B1。用northern杂交方法分析了两种EcR亚型在变态过程中不同组织的表达模式。此外，我们确定了每个EcR异构体的转录起始位点及其侧翼序列。这些区域与假定的转录因子之间的相互作用通过凝胶转移试验进行了研究。极低剂量蜕皮激素（20羟基蜕皮激素）可诱导EcR mRNA的表达。在家蚕（Bombyx mori）中，BmEcR-B1主要在变态过程中表达，除前丝腺外，其余组织均表达。这一结果与果蝇的观察结果不一致；EcRB1主要在幼虫组织中表达，而EcRA主要在成虫组织中表达。因此，鳞翅目昆虫的组织发育命运与EcR亚型表达模式之间的关系是不同的。BmEcR A和B1的转录起始位点相距超过20kb，我们没有发现它们之间的同源结构。在两个起始位点的上游区域，都有启动子序列，而不是TATA盒。凝胶位移分析表明，许多因素参与了BmEcR异构体的转录调控和替代促进。

Ecdysone, the insect molting hormone, penetrates into the target cells and binds to the ecdysone receptor (EcR) in the nuclei. this complex (ecdysone-EcR/USP) regulates a special set of gene expression and finally causes molts and metamorphosis of insects. I studied the molecular mechanisms of tissue differentiation during metamorphosis, mainly focused on relationship between EcR expression and the network of gene expressions induced by ecdysone. For this purpose, we first identified two isoforms of the silkworm EcR,BmEcR-A and BmEcR-B1, using by 5^1-RACE.Expression patterns of two EcR isoforms in different tissues during metamorphosis were analyzed by northern hybridization. Further, we determined the transcriptional initiation site for each EcR isoform and their flanking sequences. Interaction between these regions and putative transcription factors were studied by gel shift assay.Expression of EcR mRNA was induced by a very low dose of ecdysone (20 hydroxyecdysone). In the silkworm, Bombyx mori, BmEcR-B1 was expressed mainly during metamorphosis in most tissues only except anterior silk glands. This result is not consistent with the observation in Drosophila ; EcRB1 is predominant in larval tissues while EcRA is major in adult tissues. Thus, the relationship between developmental fate of tissues and patterns of EcR isoform expression is different in a lepidopteran insect. The start sites of transcription for BmEcR A and B1 are located more than 20kb from each other and we could not find homologous structure between them. In upstream regions of both start sites, there are initiator sequences instead of TATA box. Gel shift analysis implied that many factors are involved in transcriptional regulation and alternative promoting of BmEcR isoforms.