中华绒螯蟹翻译起始因子4G基因在幼体发育阶段的mRNA表达模式与适应性进化分析

The Identification,Adaptive Evolutionary Analyses and mRNA Expression Pattern of eIF4G-Like Gene in Larval Development Stages of Eriocheir sinensis

真核生物翻译起始因子4G(eIF4G)充当衔接eIF4F复合体的骨架蛋白,在帽依赖性翻译起始中起关键作用.为探究中华绒螯蟹(Eriocheir sinensis)eIF4G基因在幼体发育阶段的作用,本研究利用RACE技术克隆了中华绒螯蟹eIF4G基因(命名为EseIF4G)的全长cDNA序列,利用实时荧光定量PCR技术检测该基因在幼蟹不同发育阶段的mRNA表达模式,并探讨其在节肢动物进化过程中是否发生了适应性进化.生物信息学分析显示,EseIF4G基因的cDNA全长为3769 bp,包含一个2379 bp的开放阅读框,编码792个氨基酸;该编码蛋白具有保守的MIF4G、MA3和W2结构域,无跨膜区、疏水性区域和信号肽,是不稳定且非分泌型蛋白.荧光定量PCR检测显示,EseIF4G基因在中华绒螯蟹受精卵、溞状幼体Ⅰ~Ⅴ期、大眼幼体期和仔蟹Ⅰ~Ⅲ期共10个幼体发育阶段广泛表达,EseIF4G基因mRNA表达量在卵至溞状幼体Ⅳ期相对较高,而在溞状幼体Ⅴ期至仔蟹Ⅱ期相对较低,在溞状幼体Ⅴ期降至最低,与在仔蟹Ⅱ期的表达量没有显著差异(P>0.05).选择压力分析显示,PAML的位点模型和分支位点模型没有鉴定出正选择位点,分支模型在中华绒螯蟹末端分支没有检测到正选择信号;利用Datamonkey在EseIF4G的氨基酸水平鉴定出两个正选择位点(448G和655N).现有数据表明,EseIF4G基因可能参与中华绒螯蟹幼蟹发育过程,特别是短尾化过程,调控mRNA翻译和蛋白质相互作用;同时,其在节肢动物进化过程中进化相对保守.

Eukaryotic translation initiation factor 4 gamma(eIF4 G)plays a critical role in the initiation of cap-dependent translation by acting as a scaffolding protein for the assembly of eIF4 F complex.To investigate the role of eIF4G gene from the Chinese mitten crab during larval development stages,the full-length cDNA of eIF4G-like gene(denoted as EseIF4G)was cloned and identified by RACE(rapid amplification of cDNA ends)PCR approch,and the expression patterns were detected in larval developmental stages.Furthermore,the present study explored whether adaptive evolution of eIF4G gene occurred during arthropods evolution by site model,branch model and branch-site model implemented in CODEML program of the PAML package,and SLAC(fixes effects likelihood),FEL(fixes effects likelihood)and FUBAR(fast unconstrainted bayesian approximation)methods implemented in Datamonkey website.Bioinformatics analyses revealed that the full-length cDNA of EseIF4G is composed of 3769 nucleotides with an open-reading frame of 2379 base pairs,which encoding 792 amino acids and containing MIF4 G,MA3 and W2/eIF5 C domain.EseIF4 G is an unstable,no-secretory and omini-αprotein,and has no transmembrane,hydrophobic and signal region.Real-time PCR analysis indicated that the EseIF4G gene was widely expressed in the embryo(O)and nine different developmental stages of larvae(Z1:the first zoeal stage,Z2:the second zoeal stage,Z3:the third zoeal stage,Z4:the fourth zoeal stage,Z5:the fifth zoeal stage,M:megalopa stage,J1:the first juvenile crab stage,J2:the second juvenile crab stage,J3:the third juvenile crab stage).The expression levels of EseIF4G mRNA were relatively higher from the embryo stage to stage Z4,and were relatively lower at stage Z5,M,J1 and J2.The expression levels of EseIF4G mRNA sharply declined to the lowest at stage Z5,which had no significant difference with stage J2(P>0.05),and then increased significantly at stage M and J1(P<0.05).Segregating sites analysis indicated that site 97 and site 457 were the segregating sites of EseIF4 G protein,which were located in MIF4 G domain and MA3 domain,respectively.Selective pressure analysis showed that overall,there was evidence for strong purifying selection under one-ratio model,with aωvalue of 0.0355(significantly less than 1).No positively selected sites were found by site model in PAML,but two shared amino acid sites(448 G and 655 N)were found by FEL and FUBAR method in Datamonkey.When the terminal branch of E.sinensis was set as foreground,we did not detected any positively selected sites in PAML.Present data suggested that EseIF4G gene involving in the early developmental process of E.sinensis,was possible to serve as a regulator of mRNA translation process and proteins interaction,and was relatively conserved in the evolution of arthropod.These results provide new insights into the mechanisms of brachyurization metamorphosis in decapod crustaceans.