Molecular Analysis of Eclosion Hormone in the Tobacco Hornworm

本项目将研究烟草角虫（Manduca sexta）的分子结构和羽化激素（EH）的合成调控。EH在大脑和腹侧中枢神经系统中合成，并在每次蜕皮前释放，引发一种预先编程的神经活动模式，最终导致蜕皮。该62个氨基酸的肽最近已被测序，我们打算利用基于蛋白质序列设计的合成寡核苷酸探针分离EH基因。EH基因的分离将为研究具有可识别神经元的昆虫相对简单的神经系统中神经肽的合成和作用提供机会。我们将通过核酸测序和转录图谱研究的结合来研究EH基因的结构，并将鉴定来自激素原的其他潜在肽。我们将确定EH mRNA合成的发育调控，并分析调节其表达的内分泌因素。EH转录本的细胞定位将通过脑和与腹侧中枢神经系统相关的细胞（EH的两个来源）的原位杂交来确定。最后，将通过与EH基因杂交来检测EH样序列在其他昆虫中的存在。

This project will examine the molecular structure and the regulation of synthesis of the neuropeptide, eclosion hormone (EH), in the tobacco hornworm, Manduca sexta. EH is synthesized both in the brain and in the ventral CNS and released before each molt to elicit a preprogrammed pattern of neural activity culminating in ecdysis. The 62 amino acid peptide has recently been sequenced and we intend to isolate the EH gene using synthetic oligonucleotide probes designed based on the protein sequence. The isolation of the EH gene will offer the opportunity to study the synthesis and action of a neuropeptide in the relatively simple nervous system of an insect with it's identifiable neurons. We will examine the structure of the EH gene by a combination of nucleic acid sequencing and transcript mapping studies and will identify other potential peptides derived from the prohormone. We will determine the developmental regulation of EH mRNA synthesis and will analyze the endocrine factors that regulate its expression. The cellular localization of the EH transcript will be determined by in situ hybridization both in the brain and in the cells associated with the ventral CNS, the two sources of EH. Finally, the presence of EH-like sequences in other insects will be examined by hybridization to the EH gene.