| 摘要: |
| 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。 |
| 关键词: 拟穴青蟹, 高血糖激素CHH, 基因克隆, 表达分析 |
| DOI：10.11693/hyhz201204002002 |
| 分类号: |
| 基金项目:浙江省重大科技专项农业项目, 2008C12008号 |
附件 |
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| CLONING AND EXPRESSION OF TWO GENES ENCODING TYPE ⅠCRUSTACEAN HYPERGLYCEMIC HORMONE FAMILY FROM THE MUD CRAB SCYLLA PARAMAMOSAIN |
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SHU Miao-An, ZHANG Long-Tao, ZHOU Yu-Fang, HU Hang-Jiao, XU Bin-Peng, GUO Xiao-Ling
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College of Animal Science, Zhejiang University
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| Abstract: |
| The cDNAs of two crustacean hyperglycemic hormones, designated as C1 and C2, were isolated from the eyestalk of mud crab Scylla paramamosain through RT-PCR and RACE. Sequence analysis indicated that C1 had an open reading frame of 420bp encoding 139aa of 15.326kDa and pI at 5.540; C2 had an open reading frame of 426bp encoding 141aa of 15.621kDa and pI at 7.618. The amino acid sequences of C1 and C2 possessed 92%, 80%, 78% identity with the CHHs of S. olivacea, Charybdis japonica, Portunus trituberculatus respectively. C1 and C2 protein firstly clustered with CHHs of S. olivacea and Charybdis japonica in the phylogenetic analysis. The expression of C1 and C2 in tissues were analyzed by Real-Time PCR, the result showed that C1 and C2 were expressed in eyestalk, hepatopancreas, intestinal, heart, gill and stomach, but not in muscle. After 24h’s stress in salinity, the expression of C2 increased by a factor of thirty compared with C1. The expression of C2 in salinity 10 was not significant compared with salinity 5 (P>0.05), but significant with salinity 15 (P<0.05) and great significant with salinity 20, 25, 30 (P<0.01). The more the salinity changed, the more the C2 expressed. These results served further studies on functions and regulation mechanism of CHHs. |
| Key words: Scylla paramamosain, Crustacean hyperglycemic hormone (CHH), Gene cloning, expression analysis |
