罗氏沼虾谷氨酰胺合成酶基因的克隆与表达

Cloning and expression of glutamine synthetase from giant freshwater prawn (Macrobrachium rosenbergii)

谷氨酰胺合成酶(glutamine synthetase, GS)是一种广泛分布在动植物体内的酶,并参与细胞多种代谢调控。在甲壳动物中,GS在能量代谢和渗透压调节过程中起着重要作用。实验克隆了罗氏沼虾GS基因。GS基因cDNA全长1 965 bp,开放读码框(ORF)为1 086 bp,编码361个氨基酸(aa),分子量大小为40.75 ku,等电点为5.81。进化树分析发现,罗氏沼虾GS基因与凡纳滨对虾、斑节对虾和中国明对虾GS聚为一支,氨基酸相似性达到95%。氨基酸多序列比对分析结果显示,罗氏沼虾GS属于无脊椎动物分支的GSⅡ群,有5个保守区域。实验对罗氏沼虾GS蛋白进行表达并制备了多克隆抗体。采用荧光定量PCR技术(qRT-PCR)和免疫印迹(Western blot)对罗氏沼虾蜕壳前后的不同组织GS表达进行检测。qRT-PCR结果显示,GS基因在所检测的8个组织中均有表达;蜕壳前,其相对表达量顺序为:肝胰脏>肌肉>胃>肠>鳃>心脏>脑>血淋巴。蜕壳后和蜕壳前GS基因在组织中的差异表达比较结果显示,除了在肝胰脏表达下调外,其他7个组织都表达升高,其相对表达量的差异顺序为:脑>鳃>胃>肠>肌肉>心脏>血淋巴。Westernblot结果显示,蜕壳后GS蛋白在鳃和肌肉组织表达量上调,与其mRNA表达一致。此外,罗氏沼虾蜕壳后肝胰脏GS酶的活性和谷氨酰胺的含量下调,而其在鳃、肌肉和血淋巴中上调,结果与其基因表达一致。研究表明,GS基因在不同组织中表达的差异可能和罗氏沼虾的能量代谢、渗透压调节有关。

Glutamine synthetase(GS)is a widely distributed enzyme in animals and plants,which is involved in various cellular activities.In crustaceans,GS plays a major role in the regulation of energy metabolism and osmotic pressure.In the present report,a full cDNA of the GS gene with 1 965 bp in length was obtained from giant freshwater prawn(Macrobrachium rosenbergii).The open reading frame(ORF)encoded a 361 amino acid(aa)polypeptide with the predicted molecular weight of 40.75 ku.Analysis of amino acid sequences showed that the GS of M.rosenbergii contained 5 conserved regions that belong to the GS group of invertebrates.Phylogenetic analysis of GS of M.rosenbergii shared the highest identity within the clade of GS from Litopenaeus vannamei,Penaeus monodon,and Fenneropenaeus chinensis with 95%similarities at aa level.Polyclonal antibody against GS of M.rosenbergii was generated.Furthermore,we detected the expression profiles of GS mRNA and proteins in various tissues of M.rosenbergii by quantitative reverse transcription PCR(qRT-PCR)and Western blotting.GS mRNA was differentially expressed in 8 detected tissues of M.rosenbergii before molting in the order of hepatopancreas>muscle>stomach>intestine>gill>heart>brain>haemolymph.The expressions of GS in different tissues were compared before and after molting.The results showed that,except in hepatopancreas,the mRNA expression of GS was up-regulated in the other 7 tissues in the order of brain>gill>stomach>intestine>muscle>heart>haemolymph.By Western blot,the expressions of GS protein in the gill and muscles were up-regulated after molting,which was identical with the expression profiles at mRNA level.In addition,the activity of GS enzyme and the concentration of glutamine were decreased in hepatopancreas after molting,and by contrast,they were increased in the gill,muscle and haemolymph.These results were also identical with the GS mRNA expression.The differentiated expressions of GS gene in different tissues before and after molting suggest that it might be involved in the regulation of energy metabolism and osmotic pressure during the molting.