[1]吴海霞,时昕晔,周开亚,等.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[J].南京师大学报(自然科学版),2022,45(03):96-107.[doi:10.3969/j.issn.1001-4616.2022.03.013]
　Wu Haixia,Shi Xinye,Zhou Kaiya,et al.The Identification,Adaptive Evolutionary Analyses and mRNA Expression Pattern of eIF4G-Like Gene in Larval Development Stages of Eriocheir sinensis[J].Journal of Nanjing Normal University(Natural Science Edition),2022,45(03):96-107.[doi:10.3969/j.issn.1001-4616.2022.03.013]
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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()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第45卷
期数:
2022年03期
页码:
96-107
栏目:
生态学
出版日期:
2022-09-15

文章信息/Info

Title:
The Identification,Adaptive Evolutionary Analyses and mRNA Expression Pattern of eIF4G-Like Gene in Larval Development Stages of Eriocheir sinensis
文章编号:
1001-4616(2022)03-0096-12
作者:
吴海霞1; 时昕晔1; 2; 周开亚1; 严 洁1; 李 鹏1
(1.南京师范大学生命科学学院,江苏 南京 210023)(2.南京师范大学海洋科学与工程学院,江苏 南京 210023)
Author(s):
Wu Haixia1; Shi Xinye1; 2; Zhou Kaiya1; Yan Jie1; Li Peng1
(1.School of Life Sciences,Nanjing Normal University,Nanjing 210023,China)(2.School of Ocean Science and Engineering,Nanjing Normal University,Nanjing 210023,China)
关键词:
中华绒螯蟹; 翻译起始因子基因; cDNA克隆; mRNA表达; 适应性进化
Keywords:
Eriocheir sinensis; eukaryotic translation initiation factors; cDNA cloning; mRNA expression; adaptive evolution
分类号:
Q951+.3
DOI:
10.3969/j.issn.1001-4616.2022.03.013
文献标志码:
A
摘要:
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3 769 bp,包含一个2 379 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.
Abstract:
Eukaryotic translation initiation factor 4 gamma(eIF4G)plays a critical role in the initiation of cap-dependent translation by acting as a scaffolding protein for the assembly of eIF4F 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 3 769 nucleotides with an open-reading frame of 2 379 base pairs,which encoding 792 amino acids and containing MIF4G,MA3 and W2/eIF5C domain. EseIF4G is an unstable,no-secretory and omini-α protein,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 EseIF4G protein,which were located in MIF4G domain and MA3 domain,respectively. Selective pressure analysis showed that overall,there was evidence for strong purifying selection under one-ratio model,with a ω value of 0.0355(significantly less than 1). No positively selected sites were found by site model in PAML,but two shared amino acid sites(448G and 655N)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.

参考文献/References:

[1]CHEN Q,YANG B,NASS N,et al. Impact of eukaryotic translation initiation factors on breast cancer:still much to investigate[J]. Cancers,2020,12(7):1-19.
[2]VALÁSEK L S.‘Ribozoomin'-translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors(eIFs)[J]. Current protein & peptide science,2012,13(4):305-330.
[3]SMOLLE M A,CZAPIEWSKI P,LAPIAN'G1SKA-SZUMCZYK S,et al. The prognostic significance of eukaryotic translation initiation factors(eIFs)in endometrial cancer[J]. International journal of molecular sciences,2019,20(24):6169.
[4]吴继华,张建中. 真核翻译起始因子家族与恶性肿瘤相关性的研究进展[J]. 现代肿瘤医学,2015,23(23):3525-3528.
[5]PRÉVT D,DARLIX J L,OHLMANN T. Conducting the initiation of protein synthesis:the role of eIF4G[J]. Biology of the cell,2003,95(3-4):141-156.
[6]VILLA N,DO A,HERSHEY J W,et al. Human eukaryotic initiation factor 4G(eIF4G)protein binds to eIF3c,-d,and -e to promote mRNA recruitment to the ribosome[J]. Journal of biological chemistry,2013,288(46):32932-32940.
[7]HOWARD A,ROGERS A N. Role of translation initiation factor 4G in lifespan regulation and age-related health[J]. Ageing research reviews,2014,13(1):115-124.
[8]SONENBERG N,HINNEBUSCH A G. Regulation of translation initiation in eukaryotes:mechanisms and biological targets[J]. Cell,2009,136(4):731-745.
[9]JACKSON R J,HELLEN C U,PESTOVA T V. The mechanism of eukaryotic translation initiation and principles of its regulation[J]. Nature reviews molecular cell biology,2010,11(2):113-127.
[10]GRADI A,IMATAKA H,SVITKIN Y V,et al. A novel functional human eukaryotic translation initiation factor 4G[J]. Molelular cell biology,1998,18(1):334-342.
[11]COLDWELL M,SACK U,COWAN J,et al. Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters,splice sites and a non-canonical initiation codon[J]. Biochemical journal,2012,448(1):1-11.
[12]KEIPER B D,GAN W,RHOADS R E. Protein synthesis initiation factor 4G[J]. International journal of biochemistry & cell biology,1999,31(1):37-41.
[13]DAS S,DAS B. eIF4G3-an integrator of mRNA metabolism?[J]. Fems yeast research,2016,16(7):1-9.
[14]ORELLANA R A,WILSON F A,GAZZANEO M C,et al. Sepsis and development impede muscle protein synthesis in neonatal pigs by different ribosomal mechanisms[J]. Pediatric research,2011,69(6):473-478.
[15]ALVAREZ-DOMINGUEZ J R,ZHANG X,HU W. Widespread and dynamic translational control of red blood cell development[J]. Blood,2017,129(5):619-629.
[16]GHOSH S,LASKO P. Loss-of-function analysis reveals distinct requirements of the translation initiation factors eIF4E,eIF4E-3,eIF4G and eIF4G2 in Drosophila spermatogenesis[J]. PLoS one,2015,10(4):e0122519.
[17]TANG B,ZHOU K,SONG D,et al. Molecular systematics of the Asian mitten crabs,genus Eriocheir(Crustacea:Brachyura)[J]. Molecular phylogenetics and evolution,2003,29(2):309-316.
[18]GUINOT D,BOUCHARD J M. Evolution of the abdominal holding systems of brachyuran crabs(Crustacea,Decapoda,Brachyura)[J]. Zoosystema,1998,20(4):613-694.
[19]LI P,ZHA J,HUANG H,et al. Identification,mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis[J]. Comparative biochemistry and physiology part b:biochemistry & molecular biology,2009,153(4):332-339.
[20]LI P,ZHA J,KONG Y,et al. Identification,mRNA expression and characterization of proliferating cell nuclear antigen gene from Chinese mitten crab Eriocheir japonica sinensis[J]. Comparative biochemistry and physiology part A:molecular & integrative physiology,2010,157(2):170-176.
[21]江贤峰. 中华绒螯蟹EsScr和EsAntp基因的鉴定及其在幼体发育过程中的表达分析[D]. 南京:南京师范大学,2015.
[22]CHEN M,YUE Y,HE J,et al. Screening and identification of microRNAs during larval metamorphic development of Chinese mitten crab Eriocheir sinensis[J]. Aquaculture research,2020(51):2322-2355.
[23]SONG C,CUI Z,HUI M,et al. Comparative transcriptomic analysis provides insights into the molecular basis of brachyurization and adaptation to benthic lifestyle in Eriocheir sinensis[J]. Gene,2015,558(1):88-98.
[24]GASTEIGER E,HOOGLAND C,GATTIKER A,et al. The proteomics protocols handbook:protein identification and analysis tools on the ExPASy server[M]. Totowa NJ:humana press,2005.
[25]ALMAGRO-ARMENTEROS J J,TSIRIGOS K D,SNDERBY C K,et al. SignalP 5.0 improves signal peptide predictions using deep neural networks[J]. Nature biotechnology,2019,37(4):420-423.
[26]IVICA L,PEER B. 20 years of the SMART protein domain annotation resource[J]. Nucleic acids research,2018,46(D1):D493-D496.
[27]KROGH A,LARSSON B,HEIJNE G V,et al. Predicting transmembrane protein topology with a hidden Markov model:application to complete genomes[J]. Journal of molecular biology,2001,305(3):567-580.
[28]KUMAR S,STECHER G,LI M,et al. MEGA X:molecular evolutionary genetics analysis across computing platforms[J]. Molecular biology and evolution,2018,35(6):1547-1549.
[29]JIN X,LI W,CHENG L,et al. Two novel short C-type lectin from Chinese mitten crab,Eriocheir sinensis,are induced in response to LPS challenged[J]. Fish shellfish immunology,2012,33(5):1149-1158.
[30]LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[31]LÖYTYNOJA A. Phylogeny-aware alignment with PRANK[C]//Russell D. Methods in molecular biology. Totowa NJ:humana press,2014.
[32]GERARD T,JOSE C. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[J]. Systematic biology,2007,56(4):564-577.
[33]LAM-TUNG N,SCHMIDT H A,ARNDT V H,et al. IQ-TREE:a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[J]. Molecular biology and evolution,2015(1):268-274.
[34]KUMAR S,STECHER G,SULESKI M,et al. TimeTree:a resource for timelines,timetrees,and divergence times[J]. Molecular biology and evolution,2017,34(7):1812-1819.
[35]SUN Y. FasParser:a package for manipulating sequence data[J]. Zoological research,2017,38(2):110-112.
[36]YANG Z. PAML 4:phylogenetic analysis by maximum likelihood[J]. Molecular biology and evolution,2007,24(8):1586-1591.
[37]BIELAWSKI J P,YANG Z. Maximum likelihood methods for detecting adaptive evolution after gene duplication[J]. Journal of structural and functional genomics,2003,3(1-4):201-212.
[38]YANG Z,WONG W S W,NIELSEN R. Bayes empirical bayes inference of amino acid sites under positive selection[J]. Molecular biology and evolution,2005,22(4):1107-1118.
[39]WEAVER S,SHANK S D,SPIELMAN S J,et al. Datamonkey 2.0:a modern web application for characterizing selective and other evolutionary processes[J]. Molecular biology and evolution,2018,35(3):773-777.
[40]WARNER G F. The biology of crabs[M]. New York:Van Nostrand Reinhold Company,1977.
[41]PONTING C P. Novel eIF4G domain homologues linking mRNA translation with nonsense-mediated mRNA decay. [J]. Trends in biochemical sciences,2000,25(9):423-426.
[42]SUZUKI C,GARCES R G,EDMONDS K A,et al. PDCD4 inhibits translation initiation by binding to eIF4A using both its MA3 domains[J]. Proceedings of the national academy of sciences of the United States of America,2008,105(9):3274-3279.
[43]ARAVIND L,KOONIN E V. Eukaryote-specific domains in translation initiation factors:implications for translation regulation and evolution of the translation system[J]. Genome research,2000,10:1172-1184.

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备注/Memo

备注/Memo:
收稿日期:2022-01-06.
基金项目:国家自然科学基金项目(31000954)、江苏省高等学校自然科学研究重大项目(19KJA330001)、江苏省研究生科研与实践创新计划项目(KYCX20_1241).
通讯作者:李鹏,博士,副教授,研究方向:动物生态与进化生物学. E-mail:lipeng@njnu.edu.cn
更新日期/Last Update: 2022-09-15