| 摘要: |
| 为了研究3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methyl glutaryl coenzyme A reductase, HMGR)在甲壳动物蜕皮调控中的作用, 采用RT-PCR和cDNA 末端快速扩增技术(RACE), 克隆得到三疣梭子蟹(Portunus trituberculatus) HMGR基因的cDNA序列(GenBank登录号: KF280756)。该序列全长2575bp, 包括一个53bp的5′端非编码区, 一个686bp的3′端非编码区和一个长度为1836bp的开放阅读框, 编码611个氨基酸。该氨基酸序列与已公布的美洲海鳌虾HMGR氨基酸序列相比一致性达65%, 具有Ⅰ型HMGR保守催化区域、两个HMG-CoA结合基序和两个NADP(H)结合基序。采用实时荧光定量PCR(qRT-PCR)技术, 分析三疣梭子蟹HMGR基因的组织差异表达及在蜕皮周期中的表达水平变化, 结果表明HMGR基因在三疣梭子蟹大颚器(MO)中的表达量最高, 在其它组织中表达量均极低; 在三疣梭子蟹蜕皮周期中, 大颚器中HMGR基因的表达量自A期至D0亚期升至最高, 然后下降, 至D4亚期最低。验证了大颚器是三疣梭子蟹合成甲基法尼酯的唯一器官, 表明HMGR在三疣梭子蟹蜕皮调控中起着重要作用。 |
| 关键词: 三疣梭子蟹 3-羟基-3-甲基戊二酰辅酶A还原酶 基因克隆 蜕皮周期 表达水平 |
| DOI：10.11693/hyhz20130900129 |
| 分类号: |
| 基金项目:国家自然科学基金项目, 41376152 号, 40976098 号; 浙江省自然科学基金项目, LY13C190006 号; 宁波市自然科学基金项目,2012A610137 号 |
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| CLONING AND EXPRESSION OF HMGR GENE IN PORTUNUS TRITUBERCULATUS DURING MOLTING |
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qiu xier,zhu dongfa,cui xiaoyu,tang jie and xie xi
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School of Marine Sciences, Ningbo University,School of Marine Sciences, Ningbo University,School of Marine Sciences, Ningbo University,School of Marine Sciences, Ningbo University,School of Marine Sciences, Ningbo University
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| Abstract: |
| 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is an early rate-limiting enzyme in synthesis of methyl farnesoate, a crustacean homolog of insect JHs. We studied the molecular cloning of a cDNA encoding HMGR from mandibular organ of Portunus trituberculatus (Pt-HMGR) by reversed transcript PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length of Pt-HMGR is 2575bp, including a 5'-untranslated region(UTR) of 53bp, a 3'-UTR of 686bp and an opening reading frame (ORF) of 1836bp (GenBank accession number: KF280756). The ORF of Pt-HMGR encodes 611 amino acid residues and the deduced amino acid sequence showed 65% identity with HMGRs of Homarus americanus. Pt-HMGR contains conserved functional domain of HMGR Class I, as well as the binding motifs of HMG-CoA and NADP (H). Tissue distribution by quantitative real-time PCR (qRT-PCR) revealed that the Pt-HMGR transcript was most highly expressed in the mandibular organ (MO), which is consistent with the observation that MO is the sole site of methyl farnesoate synthesis. During molting, Pt-HMGR transcript in MOs increased gradually from stage A, to the peak in stage D0, then fell to minimum in stage D4, as shown in qRT-PCR. Therefore, HMGR plays an important role in molting regulation of P. Trituberculatus. |
| Key words: Portunus trituberculatus 3-hydroxy-3-methyl glutaryl coenzyme A reductase(HMGR) gene cloning molting cycle expression |
