【作者】 宋霞；

【导师】 周开亚；

【作者基本信息】 南京师范大学 ， 动物学， 2001， 博士

【摘要】 甲壳类的生长包括蜕皮过程中外壳周期性的蜕壳。这个过程受两种相互拮抗的激素调控，即受到蜕皮激素（ecdysone）的刺激作用和蜕皮抑制激素（molt-inhibiting hormone，MIH）的抑制作用。MIH肽产生于端髓X器（MTXO），储存在眼柄的窦腺中，通过抑制Y器分泌蜕皮激素来调节蜕皮。尽管MIH详细的作用机制仍未清楚，但已经在虾类和蟹类显示MIH可能经环核苷依赖途径抑制蜕皮激素的产生。 通过重组DNA技术，已经从几种甲壳类动物分离、克隆和鉴定了几个编码MIH肽的cDNA。不同甲壳类MIH的cDNA和氨基酸的序列都显示高度的同源性，不仅如此，MIH与CHH家族中其它成员，如甲壳类高血糖激素（CHH）、颚器官抑制激素（MOIH）和性腺抑制激素（GIH），也显示了高度的同源性。这些激素的成熟肽通常由72-78个氨基酸残基组成，氨基酸的序列相似，均有6个保守的半胱氨酸残基。 中华绒螯蟹（Eriocheir japonicus sinensis）是我国重要的经济蟹类，俗称河蟹。近年来由于不同水系的河蟹种苗混杂，导致河蟹个体变小，严重影响了经济效益。蜕皮是河蟹生活史中重要的过程，但至今对河蟹蜕皮的激素调节尚未进行研究，蜕皮抑制激素的氨基酸序列及其基因的核苷酸序列尚未见报道。本文运用RT-PCR和反向PCR（inverse PCR，IPCR）技术，克隆了中华绒螯蟹蜕皮抑制激素基因的cDNA和基因，并分析了基因的结构组成。 1．用RT-PCR技术从中华绒螯蟹眼柄总RNA中分离编码蜕皮抑制激素的部分cDNA 根据美洲黄道蟹Cancer magister，可口美青蟹Callinectes sapidus，三叶真蟹Carcinus maenas和斑纹蟳Charybdis feriatus的MIH成熟肽氨基酸序列的密码子使用频率，设计2个简并引物（F1和R48）。以10只蜕皮间期的河蟹眼柄总RNA，经随机六核苷引物逆转录合成第一链cDNA，然后用简并引物经PCR扩增河蟹眼柄cDNA。 结果：经过RT-PCR扩增得到一个与预期大小一样的片段，长163bp。将获得的部分cDNA片段连接到PinPoint^TMXa-1质粒载体中，进行克隆测序。序列分析认为，获得的中华绒螯蟹蜕皮抑制激素（molt-inhibiting hormone for Eriocheir japonicus sinensis，Ers-MIH）基因的部分cDNA编码的蛋白与另4种蟹类的MIH肽同源。更多还原

【Abstract】 Growth in crustaceans involves periodic shedding of the exoskeleton during molting. This process is under the control of two antagonistic hormones. It is stimulated by the molting hormone (ecdysone) and suppressed by the molt-inhibiting hormone (MIH). MIH is produced in the medulla terminalis of the X-organ (MTXO) and stored in the sinus gland of the eyestalk. It regulates molting by inhibiting the production of ecdysone by the Y-organ. Although the detail mechanism of MIH actions is unknown, it has shown that MIH can suppress the production of ecdysone through cyclic nucleotide-dependent pathway in crab and crayfish.Several cDNAs encoding the MIH of crustaceans have been isolated, cloned and characterized by recombinant DNA technology. Both the cDNA and amino acid sequences of the MIH from different crustaceans show a high degree of homology. Moreover, MIH also shares a high degree of homology with other members of Crustacean Hyperglycemic Hormone peptides family (CHH family), such as the Crustacean Hyperglycemic Hormone (CHH), Mandibular Organ-Inhibiting Hormone (MOIH) and Gonad-Inhibiting Hormone (GIH). The mature peptides of these hormones usually consist of 72-78 amino acid residues and have a similar amino acid sequence with six conserved cysteine amino acid residues.The Chinese mitten crab (Eriocheir japonicus sinensis) is of important value in Chinese and overseas markets. The size of the crab was decreased in recent years because of the mixing of crab seedlings from different river systems. This in turn, affected the benefit of crab farms,. Molting is an important step during the growth of crabs. But the hormones regulating the molting of the mitten crabs have not been studied so far. There is no information on the MIH gene, not only its cDNA but also its structure and organization are unknown. Using RT-PCR and inverse PCR technology, complementary DNA and structure organization of MIH gene in the mitten crab were cloned and analyzed respectively.更多还原