中华绒螯蟹蜕皮抑制激素1(MIH1)基因的cDNA片段克隆和Northern印迹分析

Molecular cloning and Northern blot analysis of a cDNA fragment of molt-inhibiting hormone gene 1 from Eriocheir japonica sinensis

根据其他蟹类蜕皮抑制激素的氨基酸序列设计了简并引物,运用RT PCR和RACE技术,从中华绒螯蟹(E riocheirjaponicasinensis)成体的蜕皮间期眼柄中,克隆了1条长1145bp的cDNA。该cDNA编码60个氨基酸,包括942bp的3′端非翻译区。同源性分析结果表明,该cDNA编码的氨基酸序列和其他蟹类蜕皮抑制激素有较高的一致性,最高的一致性为64%,将获得的序列命名为中华绒螯蟹蜕皮抑制激素1基因(GenBank检索号:AY309062)。用Northern印迹分析的方法对处于各个发育阶段的胚胎及状幼体期该基因的表达进行了研究,结果表明,处于胚胎发育早期的卵裂期几乎没有检测到杂交信号,而胚胎发育的其他时期和状幼体期都检测到蜕皮抑制激素1基因的mRNA。中华绒螯蟹蜕皮抑制激素1基因部分cDNA序列的获得为进一步获得该基因的全长cDNA、研究其功能及阐明中华螯蟹蜕皮的分子机制奠定了基础。

Degenerate primers were designed according to the MIH amino acid sequences of Cancer magister,Callinectes sapidus,Carcinus maenas and Charybdis feriatus. Using reverse transcription PCR (RTPCR) and rapid amplification of cDNA end(RACE)techniques, a cDNA sequence of 1 145 bp was obtained from the eyestalks of Eriocheir japonica sinensis. The cDNA encodes a polypeptide of 60 amino acids and includes 3′ terminal untranslated region of 942 bp. The homology analysis revealed that the amino acid sequences coded by the cDNA obtained showed higher identities with the MIH of the four species above, and the highest identity was 64%. So it is named ErsMIH1 gene (moltinhibiting hormone 1 gene for Eriocheir japonica sinensis, i.e. ErsMIH1 gene) temporarily. The accession number of ErsMIH1 gene in GenBank is AY309062. Northern blot analysis was performed with the embryos at each developmental stage and the Chinese mitten crab zoaea Ⅲ to study the expression of this gene. The results showed that hybridization signal was hardly detected in the early developmental embryos at egg cleavage stage, while the mRNA of the ErsMIH1 gene was detected in the embryos at other developmental stages and in the Chinese mitten crab zoaea Ⅲ. By obtaining the partial cDNA sequence of ErsMIH1 gene, cloning of the fulllength cDNA sequence of this gene, a further study on its function and the elucidation of the molecular mechanism in molting of Chinese mitten crab will be possible.