Synthesis and Action of Diapause Hormone of Bombyx mori.

滞育激素是一种神经肽，可诱导家蚕胚胎滞育。从成虫头部中提取出一种具有滞育诱导活性的新分子，经高效液相色谱(HPLC)纯化至均一状态，测得其表观分子质量为10,000，测定了N-端40个氨基酸序列，并利用编码滞育激素前体(DHP)的基因克隆了DHP基因，并对其组织结构和核苷酸序列进行了测定。该基因在末龄幼虫和蛹的食道下神经节(SG)和高温孵化的食道下神经节(SG)中均有明显表达。组织化学研究表明，在SG的12个神经分泌细胞中合成了水解酶，并通过心体-体释放到循环中。为了确定水解酶的作用点，我们首先克隆了一个编码卵巢海藻糖酶的基因，并建立了一种测定卵巢海藻糖酶mRNA含量的方法。将合成的水解物注射到卵巢发育进行的成虫体内，或添加到培养液中进行卵巢的体外培养。这两个实验都证明了水解酶能诱导卵巢海藻糖酶基因的表达，这种诱导作用可被放线菌素D抑制，而不能被放线菌亚胺抑制。因此，我们得出结论，水解酶在卵巢发育过程中直接诱导海藻糖酶基因的表达。

A neuropeptide, diapause hormone (DH) is responsible for induction of embryonic diapause of the silkworm, Bombyx mori. A new molecule having diapause inducing activity has been extracted from adult male heads and purified to the homogeneous state by HPLC.An apparent molecular weight of this peptide was 10,000, and the N-terminal 40 amino acid sequence was determined.Using the cDNA encoding diapause hormone precursor (DHP), the DHP gene was cloned, and its organization and nucleotide sequence were determined. The expression of this gene was clearly found in suboesophageal ganglion (SG) of the last instar larvae and pupae, and in pharate larvae which were incubated at high temperature. Histochemical studies using cDNA and anti-DH antibody clearly revealed that DH was synthesized in 12 neurosecretory cells of SG and released into circulation through corpus cardiacum-corpora allata.To estimate the action point of DH, we at first cloned a cDNA encoding ovary trehalase, and established a method for measurement of trehalase mRNA content in ovaries. The synthetic DH was injected into pharate adults in which ovarian development proceeds, or added to the medium for in vitro culture of ovaries. Both experiments have demonstrated that DH induces the expression of trehalase gene in ovaries, and this induction is inhibited by addition of actinomycin D, not cycloheximide. Thus, we concluded that DH expresses its function on developing ovaries to induce directly trehalase gene expression.