银杏WRKY家族基因密码子使用偏向性分析
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摘要
本研究以银杏基因组数据为基础,利用HMMER软件对银杏WRKY家族进行预测,用保守域分析软件进行确认,再以密码子分析的序列筛选原则对序列进行筛选,共计筛选出34条基因编码序列作为研究对象,对其密码子使用偏向性进行分析。结果显示,银杏WRKY家族基因密码子GC含量范围在40.51%～52.07%,平均含量为45.60%,GC3平均含量为41.75%,说明该家族偏向使用以A或U结尾的密码子;ENC值在49.58～60.45之间,说明其密码子偏向性较弱;相对同义密码子使用度分析显示RSCU值大于1的密码子有23个,其中以A和U为结尾20个;中性绘图分析和ENC-plot曲线分析显示银杏WRKY家族基因GC3与GC12相关性并不显著,并且大部分基因ENC值偏离了理论值。综合分析发现突变和选择压力等诸多因素共同决定着银杏WRKY家族基因的密码子偏向性。银杏WRKY家族基因的最优密码子的确定是结合了高表达优越密码子分析法和高频密码子分析法,并最终确定出UUA、UUG、CCU等7个密码子为银杏WRKY家族基因的最优密码子。研究结果为银杏WRKY家族基因的密码子优化及其转基因研究中载体的选择提供理论依据。
Based on ginkgo genom e data, HMMER software was used to predict ginkgo WRKY family, and conservative domain analysis software were used to confirm these predicted genes. Later, the sequences were screened based on the screen principle of codon analysis. 34 genes coding sequences were filtered as research object for codon usage bias analyzing. The results showed that the GC content in gene codon of ginkgo WRKY family ranged from 40.51% to 52.07%, and the average content was 45.60%, as well as the average GC3 content was 41.75%, suggesting that genes of this family favored the codons end with A or U. The effective number of codons(ENC) value ranged from 49.58 to 60.45, indicating that these genes had low codon bias. The relative synonymous codon usage(RSCU) analysis showed that the number of codons with RSCU value above 1 was 23,and 20 of them ended with ' A' and ' U'. Neutral plot and ENC-plot curve analysis found that there was no significant correlation between GC3 and GC12 of ginkgo WRKY family genes, and most ENC value had deviated from the theoretical value. Comprehensive analysis suggested that many factors such as mutation and selective pressure jointly determined the codon bias of ginkgo WRKY family genes. The determination of the optimal codon of ginkgo WRKY family genes was on the basis of the combination of high expression superior codon analysis method and high-frequency codon analysis method, and seven codons including UUA, UUG, CUU and other codons were determined as the optimal codon of ginkgo WRKY family genes. These results might provide theoretical references for codon optimization of ginkgo WRKY family genes and the selection of vector in transgenic research.
Based on ginkgo genom e data, HMMER software was used to predict ginkgo WRKY family, and conservative domain analysis software were used to confirm these predicted genes. Later, the sequences were screened based on the screen principle of codon analysis. 34 genes coding sequences were filtered as research object for codon usage bias analyzing. The results showed that the GC content in gene codon of ginkgo WRKY family ranged from 40.51% to 52.07%, and the average content was 45.60%, as well as the average GC3 content was 41.75%, suggesting that genes of this family favored the codons end with A or U. The effective number of codons(ENC) value ranged from 49.58 to 60.45, indicating that these genes had low codon bias. The relative synonymous codon usage(RSCU) analysis showed that the number of codons with RSCU value above 1 was 23,and 20 of them ended with ' A' and ' U'. Neutral plot and ENC-plot curve analysis found that there was no significant correlation between GC3 and GC12 of ginkgo WRKY family genes, and most ENC value had deviated from the theoretical value. Comprehensive analysis suggested that many factors such as mutation and selective pressure jointly determined the codon bias of ginkgo WRKY family genes. The determination of the optimal codon of ginkgo WRKY family genes was on the basis of the combination of high expression superior codon analysis method and high-frequency codon analysis method, and seven codons including UUA, UUG, CUU and other codons were determined as the optimal codon of ginkgo WRKY family genes. These results might provide theoretical references for codon optimization of ginkgo WRKY family genes and the selection of vector in transgenic research.
引文
Alberti S.,1997,The origin of the genetic code and protein synthesis,J.Mol.Evol.,45(4):352-358
Baeza M.,Alcaino J.,Barahona S.,Sepulveda D.,and Cifuentes V.,2015,Codon usage and codon context bias in Xanthophyllomyces dendrorhous,BMC Genomics,16(1):293
Comeron J.M.,and AguadéM.,1998,An evaluation of measures of synonymous codon usage bias,J.Mol.Evol.,47(3):268-274
Dong J.,Chen C.,and Chen Z.,2003,Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response,Plant Mol.Biol.,51(1):21-37
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Eulgem T.,Rushton P.J.,Schmelzer E.,Hahlbrock K.,and Somssich I.E.,1999,Early nuclear events in plant defence signalling:rapid gene activation by WRKY transcription factors,EMBO J.,18(17):4689-4699
Finn R.D.,Clements J.,Arndt W.,Miller B.L.,Wheeler T.J.,Schreiber F.,Bateman A.,and Eddy S.R.,2015,HMMERweb server:2015 update,Nucleic Acids Res.,43(1):30-38
Finn R.D.,Coggill P.,Eberhardt R.Y.,Eddy S.R.,Mistry J.,Mitchell A.L.,Potter S.C.,Punta M.,Qureshi M.,Sangrador-Vegas A.,Salazar G.A.,Tate J.,and Bateman A.,2016,The pfam protein families database:towards a more sustainable future,Nucleic Acids Res.,44(D1):279-285
Fu J.M.,Suo Y.J.,Liu H.M.,and Tan X.F.,2017,Analysis on codon usage in the chloroplast protein-coding genes of Diospyros spp.,Jingjilin Yanjiu(Nonwood Forest Research),35(2):38-44(傅建敏,索玉静,刘慧敏,谭晓风,2017,柿属植物叶绿体蛋白质编码基因密码子用法,经济林研究,35(2):38-44)
Fuglsang A.,2004,The effective number of codons'revisited,Biochem.Biophys.Res.Commun.,347(3):957-964
Grantham R.,Gautier C.,and Gouy M.,1980,Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type,Nucleic Acids Res.,8(9):1893-1912
Grantham R.,Gautier C.,Gouy M.,Jacobzone M.,and Mercier R.,1981,Codon catalog usage is a genome strategy modulated for gene expressivity,Nucleic Acids Res.,9(1):43-74
Gustafsson C.,Govindarajan S.,and Minshull J.,2004,Codon bias and heterologous prote in expression,Trends Biotechnol.,22(7):346-353
Kayum M.A.,Jung H.J.,Park J.I.,Ahmed N.U.,Saha G.,Yang T.J.,and Nou I.S.,2015,Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa,Mol.Genet.Genomics,290(1):79-95
Laurent D.,and Dominique M.,1999,Expression pattern and,surprisingly,gene length shape codon usage in Caenorhabditis,Drosophila,and Arabidopsis,Proc.Natl.Acad.Sci.USA,96(8):4482-448
Letunic I.,Doerks T.,and Bork P.,2015,SMART:recent updates,new developments and status in 2015,Nucleic Acids Res.,43(D1):257-260
Li G.Y.,Wang Z.,Zhang Z.Y.,Fang H.D.,and Tan X.L.,2013,The base composition and codon use of the WRKY gene family of the Brassica napus,Shengwuxue Zazhi(Journal of Biology),30(4):42-45,85(李冠英,王政,张志燕,方和娣,谭小力,2013,甘蓝型油菜WRKY基因家族碱基组成及密码子使用特性分析,生物学杂志,30(4):42-45,85)
Li Z.,Li Y.G.,Bo L.,Wang D.H.,and Li W.B.,2014,Analysis of synonymous codon bias of NFYB gene family in Soybean and Arabidopsis,Zuowu Zazhi(Crops),(3):14-21(李真,李永光,薄蕾,王鼎慧,李文滨,2014,大豆和拟南芥NFYB基因家族同义密码子使用偏好性的比较,作物杂志,(3):14-21)
Lin T.,Ni Z.H.,Shen M.S.,and Chen L.,2002,High-frequency codon analysis and its application in codon analysis of tobacco,Xiamen Daxue Xuebao(Journal of Xiamen University(Natural Science)),(5):551-554(林涛,倪志华,沈明山,陈亮,2002,高频密码子分析法及其在烟草密码子分析中的应用,厦门大学学报(自然科学版),(5):551-554)
Luo H.,Hu S.S.,Wu Q.,and Yao H.P.,2015,Analysis of buckwheat chloroplast gene codon bias,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(11):2457-2464(罗洪,胡莎莎,吴琦,姚慧鹏,2015,甜荞叶绿体基因密码子偏爱性分析,基因组学与应用生物学,35(11):2457-2464)
Marchler-Bauer A.,Bo Y.,Han L.,He J.,Lanczycki C.J.,Lu S.,Chitsaz F.,Derbyshire M.K.,Geer R.C.,Gonzales N.R.,Gwadz M.,Hurwitz D.I.,Lu F.,Marchler G.H.,Song J.S.,Thanki N.,Wang Z.,Yamashita R.A.,Zhang D.,Zheng C.,Geer L.Y.,and Bryant S.H.,2017,CDD/SPARCLE:functional classification of proteins via subfamily domain architectures,Nucleic Acids Res.,(D1):200-203
Massimo D.G.,2005,The origin of the genetic code:theories and their relationships,a review,Biosystems,80(2):175-184
Nie J.T.,Bai Y.F.,He F.Y.,Yan J.J.,and Zhang W.F.,2014,Codon bias of pyruvate orthophosphate dikinase gene in A-maranthus hypochondriacus,Zhiwu Xuebao(Chinese Bulletin of Botany),49(6):672-681(聂江婷,白云凤,贺飞燕,闫建俊,张维锋,2014,籽粒苋丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性,植物学报,49(6):672-681)
Prabha R.,Singh D.P.,Sinha S.,Ahmad K.,and Rai A.,2017,Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes,Marine Genomics,32:31-39
Rushton P.J.,Torres J.T.,Parniske M.,Wernert P.,Hahlbrock K.,and Somssich I.,1996,Interaction of elicitor-induced D-NA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes,EMBO J.,15(20):5690-5700
Sharp P.M.,and Li W.H.,1987,The codon adaptation index-a measure of directional synonymous codon usage bias,and its potential applications,Nucleic Acids Res.,15(3):1281-1295
Song H.,Wang P.F.,Ma D.C.,Xia H.,Zhao C.Z.,Zhang Y.,and Zhao S.Z.,2015,Analysis of codon usage bias of WRKYtranscription factors in Medicago truncatula,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),23(2):203-212(宋辉,王鹏飞,马登超,夏晗,赵传志,张烨,赵术珍,2015,蒺藜苜蓿WRKY转录因子密码子使用偏好性分析,农业生物技术学报,23(2):203-212)
Tian Y.,Lu X.Y.,Peng L.S.,and Fang J.,2006,The structure and function of plant WRKY transcription factors,Yichuan(Hereditas),28(12):1607-1612(田云,卢向阳,彭丽莎,方俊,2006,植物WRKY转录因子结构特点及其生物学功能,遗传,28(12):1607-1612)
Ulker B.,and Somssich I.E.,2004,WRKY transcription factors:from DNA binding towards biological function,Curr.Opin.Plant Biol.,7(5):491-498
Wright F.,1990,The'effective number of codons''used in a gene,Gene,87(1):23-29
Wu X.M.,Wu S.F.,Ren D.M.,Zhu Y.P.,and He F.C.,2007,The analysis method and progress in the study of codon bias,Yichuan(Hereditas),29(4):420-426(吴宪明,吴松锋,任大明,朱云平,贺福初,2007,密码子偏性的分析方法及相关研究进展,遗传,29(4):420-426)
Xu C.,Cai X.,Chen Q.,Zhou H.,Cai Y.,and Ben A.,2011,Factors affecting synonymous codon usage bias in chloroplast genome of Oncidium Gower Ramsey,Evol.Bioinform.Online,(7):271-278
Yang G.F.,Su K.L.,Zhao Y.R.,Song Z.B.,and Sun J.,2015,Analysis of codon usage in the chloroplast genome of Medicago truncatula,Caoye Xuebao(Acta Prataculturae Sinica),24(12):171-179(杨国锋,苏昆龙,赵怡然,宋智斌,孙娟,2015,蒺藜苜蓿叶绿体密码子偏好性分析,草业学报,24(12):171-179)
Zhao W.S.,Zhai Q.L.,An Z.W.,Fang J.L.,and Huang H.S.,2015,Cloning and characteristics of HbWRKY9 transcription factor in the rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):599-606(赵武帅,翟琪麟,安泽伟,方家林,黄华孙,2015,橡胶树转录因子HbWRKY9的克隆与特性分析,基因组学与应用生物学,34(3):599-606)
Zhu Y.X.,Li Y.,and Zheng X.F.,eds.,2007,Modern molecular biology,High Education Press,Beijing,China,pp.108(朱玉贤,李毅,郑晓峰,编著,2007,现代分子生物学,高等教育出版社,中国,北京,pp.108)
Baeza M.,Alcaino J.,Barahona S.,Sepulveda D.,and Cifuentes V.,2015,Codon usage and codon context bias in Xanthophyllomyces dendrorhous,BMC Genomics,16(1):293
Comeron J.M.,and AguadéM.,1998,An evaluation of measures of synonymous codon usage bias,J.Mol.Evol.,47(3):268-274
Dong J.,Chen C.,and Chen Z.,2003,Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response,Plant Mol.Biol.,51(1):21-37
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Eulgem T.,Rushton P.J.,Schmelzer E.,Hahlbrock K.,and Somssich I.E.,1999,Early nuclear events in plant defence signalling:rapid gene activation by WRKY transcription factors,EMBO J.,18(17):4689-4699
Finn R.D.,Clements J.,Arndt W.,Miller B.L.,Wheeler T.J.,Schreiber F.,Bateman A.,and Eddy S.R.,2015,HMMERweb server:2015 update,Nucleic Acids Res.,43(1):30-38
Finn R.D.,Coggill P.,Eberhardt R.Y.,Eddy S.R.,Mistry J.,Mitchell A.L.,Potter S.C.,Punta M.,Qureshi M.,Sangrador-Vegas A.,Salazar G.A.,Tate J.,and Bateman A.,2016,The pfam protein families database:towards a more sustainable future,Nucleic Acids Res.,44(D1):279-285
Fu J.M.,Suo Y.J.,Liu H.M.,and Tan X.F.,2017,Analysis on codon usage in the chloroplast protein-coding genes of Diospyros spp.,Jingjilin Yanjiu(Nonwood Forest Research),35(2):38-44(傅建敏,索玉静,刘慧敏,谭晓风,2017,柿属植物叶绿体蛋白质编码基因密码子用法,经济林研究,35(2):38-44)
Fuglsang A.,2004,The effective number of codons'revisited,Biochem.Biophys.Res.Commun.,347(3):957-964
Grantham R.,Gautier C.,and Gouy M.,1980,Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type,Nucleic Acids Res.,8(9):1893-1912
Grantham R.,Gautier C.,Gouy M.,Jacobzone M.,and Mercier R.,1981,Codon catalog usage is a genome strategy modulated for gene expressivity,Nucleic Acids Res.,9(1):43-74
Gustafsson C.,Govindarajan S.,and Minshull J.,2004,Codon bias and heterologous prote in expression,Trends Biotechnol.,22(7):346-353
Kayum M.A.,Jung H.J.,Park J.I.,Ahmed N.U.,Saha G.,Yang T.J.,and Nou I.S.,2015,Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa,Mol.Genet.Genomics,290(1):79-95
Laurent D.,and Dominique M.,1999,Expression pattern and,surprisingly,gene length shape codon usage in Caenorhabditis,Drosophila,and Arabidopsis,Proc.Natl.Acad.Sci.USA,96(8):4482-448
Letunic I.,Doerks T.,and Bork P.,2015,SMART:recent updates,new developments and status in 2015,Nucleic Acids Res.,43(D1):257-260
Li G.Y.,Wang Z.,Zhang Z.Y.,Fang H.D.,and Tan X.L.,2013,The base composition and codon use of the WRKY gene family of the Brassica napus,Shengwuxue Zazhi(Journal of Biology),30(4):42-45,85(李冠英,王政,张志燕,方和娣,谭小力,2013,甘蓝型油菜WRKY基因家族碱基组成及密码子使用特性分析,生物学杂志,30(4):42-45,85)
Li Z.,Li Y.G.,Bo L.,Wang D.H.,and Li W.B.,2014,Analysis of synonymous codon bias of NFYB gene family in Soybean and Arabidopsis,Zuowu Zazhi(Crops),(3):14-21(李真,李永光,薄蕾,王鼎慧,李文滨,2014,大豆和拟南芥NFYB基因家族同义密码子使用偏好性的比较,作物杂志,(3):14-21)
Lin T.,Ni Z.H.,Shen M.S.,and Chen L.,2002,High-frequency codon analysis and its application in codon analysis of tobacco,Xiamen Daxue Xuebao(Journal of Xiamen University(Natural Science)),(5):551-554(林涛,倪志华,沈明山,陈亮,2002,高频密码子分析法及其在烟草密码子分析中的应用,厦门大学学报(自然科学版),(5):551-554)
Luo H.,Hu S.S.,Wu Q.,and Yao H.P.,2015,Analysis of buckwheat chloroplast gene codon bias,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(11):2457-2464(罗洪,胡莎莎,吴琦,姚慧鹏,2015,甜荞叶绿体基因密码子偏爱性分析,基因组学与应用生物学,35(11):2457-2464)
Marchler-Bauer A.,Bo Y.,Han L.,He J.,Lanczycki C.J.,Lu S.,Chitsaz F.,Derbyshire M.K.,Geer R.C.,Gonzales N.R.,Gwadz M.,Hurwitz D.I.,Lu F.,Marchler G.H.,Song J.S.,Thanki N.,Wang Z.,Yamashita R.A.,Zhang D.,Zheng C.,Geer L.Y.,and Bryant S.H.,2017,CDD/SPARCLE:functional classification of proteins via subfamily domain architectures,Nucleic Acids Res.,(D1):200-203
Massimo D.G.,2005,The origin of the genetic code:theories and their relationships,a review,Biosystems,80(2):175-184
Nie J.T.,Bai Y.F.,He F.Y.,Yan J.J.,and Zhang W.F.,2014,Codon bias of pyruvate orthophosphate dikinase gene in A-maranthus hypochondriacus,Zhiwu Xuebao(Chinese Bulletin of Botany),49(6):672-681(聂江婷,白云凤,贺飞燕,闫建俊,张维锋,2014,籽粒苋丙酮酸磷酸二激酶(PPDK)基因的密码子偏好性,植物学报,49(6):672-681)
Prabha R.,Singh D.P.,Sinha S.,Ahmad K.,and Rai A.,2017,Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes,Marine Genomics,32:31-39
Rushton P.J.,Torres J.T.,Parniske M.,Wernert P.,Hahlbrock K.,and Somssich I.,1996,Interaction of elicitor-induced D-NA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes,EMBO J.,15(20):5690-5700
Sharp P.M.,and Li W.H.,1987,The codon adaptation index-a measure of directional synonymous codon usage bias,and its potential applications,Nucleic Acids Res.,15(3):1281-1295
Song H.,Wang P.F.,Ma D.C.,Xia H.,Zhao C.Z.,Zhang Y.,and Zhao S.Z.,2015,Analysis of codon usage bias of WRKYtranscription factors in Medicago truncatula,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),23(2):203-212(宋辉,王鹏飞,马登超,夏晗,赵传志,张烨,赵术珍,2015,蒺藜苜蓿WRKY转录因子密码子使用偏好性分析,农业生物技术学报,23(2):203-212)
Tian Y.,Lu X.Y.,Peng L.S.,and Fang J.,2006,The structure and function of plant WRKY transcription factors,Yichuan(Hereditas),28(12):1607-1612(田云,卢向阳,彭丽莎,方俊,2006,植物WRKY转录因子结构特点及其生物学功能,遗传,28(12):1607-1612)
Ulker B.,and Somssich I.E.,2004,WRKY transcription factors:from DNA binding towards biological function,Curr.Opin.Plant Biol.,7(5):491-498
Wright F.,1990,The'effective number of codons''used in a gene,Gene,87(1):23-29
Wu X.M.,Wu S.F.,Ren D.M.,Zhu Y.P.,and He F.C.,2007,The analysis method and progress in the study of codon bias,Yichuan(Hereditas),29(4):420-426(吴宪明,吴松锋,任大明,朱云平,贺福初,2007,密码子偏性的分析方法及相关研究进展,遗传,29(4):420-426)
Xu C.,Cai X.,Chen Q.,Zhou H.,Cai Y.,and Ben A.,2011,Factors affecting synonymous codon usage bias in chloroplast genome of Oncidium Gower Ramsey,Evol.Bioinform.Online,(7):271-278
Yang G.F.,Su K.L.,Zhao Y.R.,Song Z.B.,and Sun J.,2015,Analysis of codon usage in the chloroplast genome of Medicago truncatula,Caoye Xuebao(Acta Prataculturae Sinica),24(12):171-179(杨国锋,苏昆龙,赵怡然,宋智斌,孙娟,2015,蒺藜苜蓿叶绿体密码子偏好性分析,草业学报,24(12):171-179)
Zhao W.S.,Zhai Q.L.,An Z.W.,Fang J.L.,and Huang H.S.,2015,Cloning and characteristics of HbWRKY9 transcription factor in the rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):599-606(赵武帅,翟琪麟,安泽伟,方家林,黄华孙,2015,橡胶树转录因子HbWRKY9的克隆与特性分析,基因组学与应用生物学,34(3):599-606)
Zhu Y.X.,Li Y.,and Zheng X.F.,eds.,2007,Modern molecular biology,High Education Press,Beijing,China,pp.108(朱玉贤,李毅,郑晓峰,编著,2007,现代分子生物学,高等教育出版社,中国,北京,pp.108)