罗氏沼虾谷氨酸脱氢酶基因克隆及其在MrTV感染下的组织表达分析

摘要

本研究利用cDNA末端快速扩增技术克隆获得罗氏沼虾(Macrobrachium rosenbergii)谷氨酸脱氢酶(glutamate dehydrogenase,GDH)基因(MrGDH)的cDNA全长序列,对其进行生物信息学分析,并检测该基因在罗氏沼虾不同组织中的表达差异;同时,利用罗氏沼虾太湖病毒(Macrobrachium rosenbergii Taihu virus,MrTV)感染沼虾,研究感染前后沼虾鳃和肝胰腺MrGDH的转录特征.结果显示,MrGDH基因cDNA序列全长2257 bp,拥有一个1662 bp长的开放阅读框,合计编码氨基酸553个,合成的蛋白质分子质量约为61.37 kDa.MrGDH氨基酸序列与中国明对虾(Fenneropenaeus chinensis)、凡纳滨对虾(Litopenaeus vannamei)的同源性较高,达到92%,其二级结构和三维结构与黑腹果蝇(Drosophila melanogaster)以及家蚕(Bombyx mori)高度相似,说明MrGDH氨基酸序列比较保守.实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)结果显示,MrGDH基因在与机体运动和代谢有关的组织中表达量较高,其中在肌肉中表达量最高,而在血淋巴中最低.罗氏沼虾受MrTV病毒感染48 h,肝胰腺和鳃中MrGDH基因的表达在统计学上均极显著高于对照组(P<0.01);感染72 h,鳃中MrGDH基因表达显著高于对照组(P<0.05),肝胰腺中MrGDH基因表达极显著高于对照组(P<0.01):说明MrTV感染胁迫可以刺激罗氏沼虾MrGDH的表达上调.%Macrobrachium rosenbergii is one of the major aquaculture shrimp, whose larva is the susceptible host of Macrobrachium rosenbergii Taihu virus (MrTV) which is one kind of novel RNA virus, and a member of Dicistroviridae owning a single positive strand with two open reading frames. Glutamate dehydrogenase (GDH) is a key enzyme in glutamate biosynthesis and plays an important role in organism's metabolism. But there are still few reports about glutamate dehydrogenase in crustaceans, and almost no reports of M. rosenbergii glutamate dehydrogenase (MrGDH), as well as the interaction between MrGDH and MrTV. In this study, the complete sequence of MrGDH gene was cloned by rapid amplification of cDNA ends (RACE) technology, and the expression of MrGDH gene in different tissues was studied by quantitative real-time polymerase chain reaction (qRT-PCR). Then the transcriptional characteristics of MrGDH gene in the gill and hepatopancreas of M. rosenbergii were analyzed under MrTV infection (positive group), and the negative group was injected with phosphate buffered solution. The results showed that the complete cDNA sequence of MrGDH gene was 2257 bp in length with a 1662 bp open reading frame (ORF) encoding 553 amino acids, which shared 92% homology with Fenneropenaeus chinensis GDH and Litopenaeus vannamei GDH. The protein molecular mass of MrGDH was approximately 61.37 kDa. Its secondary and three-dimensional structures were extremely similar to Drosophila melanogaster GDH and Bombyx mori GDH. The MrGDH gene was expressed in all detected tissues of M. rosenbergii. After 48 h and 72 h of injection, the expression of MrGDH gene in hepatopancreas and gill of positive group was significantly higher than the negative group. In summary, the MrGDH gene is conservative, and its expression distribution indicates that it's highly expressed in the organisms extremely related to movement and metabolism, with the highest in muscle and the lowest in haemolymph. This difference may be caused by the diversity in energy demand of each organization. Overall, it is indicated that MrTV infection could stimulate the expression of MrGDH gene.