三疣梭子蟹PtDNMT1基因的克隆及其 在低盐适应中的表达分析

The Cloning of the PtDNMT1 Gene of Portunus trituberculatus and Its Expression Analysis in Low Salinity Adaptation

中文关键词: 三疣梭子蟹  PtDNMT1  盐度胁迫  基因克隆  基因表达

英文关键词: Portunus trituberculatus  Methyltransferase 1  Salinity stress  Gene cloning  Gene expression

中文摘要:

      甲基转移酶是维持基因组甲基化状态的重要基因，本研究利用SMART-RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)甲基转移酶基因(PtDNMT1)。PtDNMT1基因cDNA序列全长5919 bp，包括4832 bp的开放阅读框，编码1610个氨基酸，预测分子量为148.15 kDa，理论等电点为4.68。结构预测发现，PtDNMT1有2个特殊的结构域，分别是锌指结构域(zf-CXXC)和甲基转移酶家族特有的Dcm结构域。进化树分析显示，PtDNMT1基因与昆虫类的DNMT基因聚为一支。组织表达分析发现，PtDNMT1基因在肝胰腺、鳃、卵巢、肌肉、胃、心脏、血液中均有表达，其中在肝胰腺中表达最高，卵巢和鳃次之。进一步研究了低盐胁迫后PtDNMT1基因在鳃、肝胰腺和肌肉组织中的表达变化规律为胁迫6 h时鳃组织中PtDNMT1基因的表达即达到峰值(5.3倍)，并一直持续到12 h (4倍)，随后逐步下降，在72 h时仍显著高于对照组(2.3倍)；PtDNMT1基因在肝胰腺中的表达规律类似于鳃，然而其达到峰值的时间稍晚于鳃(24 h)，且上调倍数高于鳃(8倍)；低盐胁迫后PtDNMT1基因在肌肉中的表达最初呈现下调趋势，之后(24 h)上调表达至峰值(2.2倍)，且一直上调表达至72 h。本研究首次克隆了PtDNMT1基因，根据其在各组织中的表达分布特征以及盐度胁迫后的表达变化情况，推测DNA甲基化在三疣梭子蟹低盐适应中发挥了重要作用。

英文摘要:

      Methyltransferase (DNMT1) is an important gene that maintains the methylation state of the genome. This study uses the SMART-RACE technique to clone the DNMT1 gene of Portunus trituberculatus (PtDNMT1). The cDNA sequence of PtDNMT1 gene is 5919 bp, including the open reading frame of 4832 bp, and 1610 amino acids are encoded. The predicted molecular weight is 148.15 kDa, and the theoretical isoelectric point is 4.68. The structure prediction found that there are two special PtDNMT1 domain structures: the zf-CXXC zinc finger domain and the methyltransferase familial Dcm structure domain. The analysis of the evolutionary tree showed that PtDNMT1 gene and insects DNMT gather into one. PtDNMT1 was expressed in the hepatopancreas, gill, ovary, muscle, stomach, heart, and blood. The highest expression was found in the hepatopancreas, followed by the ovaries and gills. Then we get a further research about the PtDNMT1 gene expression during low salinity stress in gill, l hepatopancreas and muscle tissue. In gill tissue, we found that PtDNMT1 gene expression peaked (5 folds) after 6 h, and continued to 12 h (6 folds), then declined gradually, but was still significantly higher than the control group (3 folds). The expression of PtDNMT1 in the hepatopancreas was similar to the gills, however the peak time was later than in gill tissue (24 h), and the peak was higher than in the gills (8 folds). After low salinity stress, the expression of the PtDNMT1 gene in muscle was firstly reduced, then (24 h) increased to a peak (2.2 times), and the expression was raised to 72 h. This study is the first to clone the PtDNMT1 gene. According to its distribution characteristics and occurrence in various organizations, expression changes under salinity stress. We speculate that low salinity adaptations in DNA methylation in P. trituberculatus played an important role.