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采用RT-PCR及RACE技术,从拟穴青蟹眼柄组织中克隆了两种Ⅰ型高血糖激素CHH基因(分别命名为C1与C2)cDNA全序列。序列分析结果表明:C1基因全长1859bp,开放阅读框长420bp,编码139个氨基酸,分子量为15.326kDa,等电点为5.540;C2基因全长1739bp,开放阅读框长426bp,编码141个氨基酸,分子量为15.621kDa,等电点为7.618。与其它物种CHH氨基酸序列进行同源性比较分析显示,拟穴青蟹CHH基因与榄绿青蟹CHH基因同源性最高(92%),依次为日本鲟(80%)、三疣梭子蟹(78%)。聚类分析表明,拟穴青蟹CHH氨基酸序列与榄绿青蟹和日本鲟紧密聚为一支。经荧光定量检测,拟穴青蟹CHH基因在眼柄、肝胰腺、心脏、肠中表达量较高,鳃、胃中表达量很少,肌肉中表达极少。盐度骤变试验结果表明:盐度胁迫24h后,C2的表达量是C1的30倍以上;C2基因盐度5时与对照组的表达量差异不显著(P>0.05),盐度15时差异显著(P<0.05),盐度22、25、30时差异极显著(P<0.01);盐度变化越大,C2的表达量越大。上述结果为进一步深入研究CHH基因的功能及调控机理奠定基础。
Using RT-PCR and RACE techniques, we cloned the full-length cDNAs of two types of hyperglycemic CHH genes (named C1 and C2, respectively) Sequence analysis showed that C1 gene was 1859 bp in length and 420 bp in open reading frame length, encoding 139 amino acids with a molecular weight of 15.326 kDa and an isoelectric point of 5.540. The C2 gene was 1739 bp in length and contained an open reading frame of 426 bp, encoding 141 amino acids. The molecular weight is 15.621 kDa and the isoelectric point is 7.618. Homology comparison of CHH amino acid sequences with other species showed that CHH gene of Quyang acupressure had the highest homology (92%) with CHH gene of Quercus acutissima, followed by Japanese sturgeon (80%), Portunus trituberculatus %). Cluster analysis showed that the amino acid sequence of CHH in the green crab acreage was closely related to the green crab and Japanese sturgeon. After fluorescent quantitative detection, the expression of CHH gene in the acupoints of Scylla serrata was higher in the eyelid, hepatopancreas, heart and intestine, but less in the gill and stomach, with little in the muscle. The results of salinity change test showed that the expression of C2 was over 30 times higher than that of C1 after salinity stress for 24 hours, while the expression of C2 gene was not significantly different from that of the control group at 5 (P> 0.05) (P <0.05). The differences of salinity at 22, 25 and 30 were significant (P <0.01). The greater the salinity, the higher the expression of C2. The above results lay a foundation for further study on the function and regulation mechanism of CHH gene.