野生夏葡萄PR3和PR5基因的克隆及生物信息学分析
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摘要
根据NCBI数据库相关信息,我们设计了特异引物,从野生夏葡萄叶片克隆了PR3和PR5基因。利用公共数据库数据,采用生物信息学的理论和方法,对获得的野生夏葡萄病程相关蛋白基因进行了较全面的性质分析。为后续的功能研究提供理论基础,也为利用生物信息学方法深入研究功能基因提供新的思路。本文主要研究结果如下:
1.根据数据库信息,成功克隆出野生夏葡萄PR3和PR5基因。
2.采用生物信息学方法进行信号肽分析,结果表明,PR3很可能是具有信号肽的分泌型蛋白,而PR5具有信号肽的可能性很小。
3.运用生物信息学的方法和工具对野生夏葡萄PR3和PR5基因的核苷酸和氨基酸序列进行分析;并对它们的疏水性/亲水性、跨膜结构域、蛋白质二级结构、三级结构和功能结构域进行预测和分析。这些预测和分析结果表明:野生夏葡萄PR3的功能域是chitinase,PR5的功能域是thaumatin。
4.NCBI上的电子表达谱分析表明野生夏葡萄PR3和PR5可能在葡萄多种器官中表达,但不同的PR基因在不同的组织中表达强弱不同。
5.用PlantCARE分析野生夏葡萄PR3和PR5基因的启动子,发现它们具有启动子的基本元件TATA-box、CAAT-box,并含有真菌诱导元件Box-W1及与防御和胁迫相关的作用元件TC-rich repeats,还包含与水杨酸相关的作用元件TGA-element,与脱落酸相关的作用元件ABRE,与茉莉酸甲酯相关的作用元件CGTCA-motif和TGACG-motif等很多顺式作用元件。这些顺式作用元件的存在,表明PR3和PR5基因的表达可能受到不良环境和植物激素的诱导和调节。
According to the data in NCBI,we designed primers and cloned PR3 and PR5 genes from Vitis aestivalis.Based on the public databases,we conducted comprehensive analysis of PR3 and PR5 by using the methods of bioinformatics.It will provide some useful information for its following function research and new methods for the researches of other gene families.The results of this study are as follows:
1.According to the information from NCBI,we successfully cloned PR3 and PR5 genes from V.aestivalis.
2.The results of signal peptide analysis indicated that PR3 has signal peptide sequence while PR5 may have no signal peptide sequence.
3.By the bioinformatics methods and tools,the composition and sequences of amino acids and nucleotides,hydrophobicity and hydrophilicity, transmembrane domains,the secondary and tertiary structures of PR3 and PR5 were analyzed and predicted. The results showed that PR3 contains chitinase functional domain and that PR5 contains thaumatin functional domain.
4.The predicted expression profiles showed that these two genes might be expressed in most grape organs.However, the different PR was expressed in different organs at quite different levels.
5.The functional cis-acting elements in the promoters of PR3 and PR5 gene were analyzed by using PlantCARE software.Many cis-acting elements were found such as: TATA-box;CAAT-box;Box-W1,a fungal elicitor responsive element;TC-rich repeats,a defense and stress responsive element;TGA element,an auxin-responsive element;ABRE,an abscisic acid responsive element;CGTCA-motif and TGACG-motif, MeJA-responsive elements and so on.
1.根据数据库信息,成功克隆出野生夏葡萄PR3和PR5基因。
2.采用生物信息学方法进行信号肽分析,结果表明,PR3很可能是具有信号肽的分泌型蛋白,而PR5具有信号肽的可能性很小。
3.运用生物信息学的方法和工具对野生夏葡萄PR3和PR5基因的核苷酸和氨基酸序列进行分析;并对它们的疏水性/亲水性、跨膜结构域、蛋白质二级结构、三级结构和功能结构域进行预测和分析。这些预测和分析结果表明:野生夏葡萄PR3的功能域是chitinase,PR5的功能域是thaumatin。
4.NCBI上的电子表达谱分析表明野生夏葡萄PR3和PR5可能在葡萄多种器官中表达,但不同的PR基因在不同的组织中表达强弱不同。
5.用PlantCARE分析野生夏葡萄PR3和PR5基因的启动子,发现它们具有启动子的基本元件TATA-box、CAAT-box,并含有真菌诱导元件Box-W1及与防御和胁迫相关的作用元件TC-rich repeats,还包含与水杨酸相关的作用元件TGA-element,与脱落酸相关的作用元件ABRE,与茉莉酸甲酯相关的作用元件CGTCA-motif和TGACG-motif等很多顺式作用元件。这些顺式作用元件的存在,表明PR3和PR5基因的表达可能受到不良环境和植物激素的诱导和调节。
According to the data in NCBI,we designed primers and cloned PR3 and PR5 genes from Vitis aestivalis.Based on the public databases,we conducted comprehensive analysis of PR3 and PR5 by using the methods of bioinformatics.It will provide some useful information for its following function research and new methods for the researches of other gene families.The results of this study are as follows:
1.According to the information from NCBI,we successfully cloned PR3 and PR5 genes from V.aestivalis.
2.The results of signal peptide analysis indicated that PR3 has signal peptide sequence while PR5 may have no signal peptide sequence.
3.By the bioinformatics methods and tools,the composition and sequences of amino acids and nucleotides,hydrophobicity and hydrophilicity, transmembrane domains,the secondary and tertiary structures of PR3 and PR5 were analyzed and predicted. The results showed that PR3 contains chitinase functional domain and that PR5 contains thaumatin functional domain.
4.The predicted expression profiles showed that these two genes might be expressed in most grape organs.However, the different PR was expressed in different organs at quite different levels.
5.The functional cis-acting elements in the promoters of PR3 and PR5 gene were analyzed by using PlantCARE software.Many cis-acting elements were found such as: TATA-box;CAAT-box;Box-W1,a fungal elicitor responsive element;TC-rich repeats,a defense and stress responsive element;TGA element,an auxin-responsive element;ABRE,an abscisic acid responsive element;CGTCA-motif and TGACG-motif, MeJA-responsive elements and so on.
引文
[1]王建文,王有年.欧洲葡萄栽培和酿酒在中国的传播.北京农学院学报.2006(1),21(1):242-251.
[2]王金生.植物抗病性分子机制.植物病理学报,1995,25(4):289-295.
[3]Key J,Kosuge T.Cellular and molecular biology of plant stress.New York:Alan R Liss Inc,1985.303-327.
[4]Van Loon L C.The nom enclosure of pathogenesis-related proteins.Plant Mol Biol,1990,37:229.
[5]Bowles D J.Defense-related proteins in higher plants.Annual Review of Biochemistry,1990,59:873-907
[6]Guo H,Ecker J R.The ethylene signaling pathway:new insights.Cur opinion plant boil,2004,7:40-49.
[7]Van Loon L C,Van Strien E A.The families of pathogenesis-related proteins,their activities,and comparative analysis of PR-1types proteins.Physiology Mol Plant Pathol.1999,55:85-97
[8]Webber J P,Jostens M hajj,V an Kan Jal,et al.Sub cellar localization of plant chitinases and 1,3—β—gluconase C lad osporium fulvum infected tomato leaves.Physiology Mol Plant Patrol.1992,41:23.
[9]Sikorski M M,Biesiadka J,Kasperska A E,et al.Expression of genes encoding PR10 class pathogenesis—related proteins is inhibited in yellow lupine root nodules.Plant Science Limerick,1999,149(2):125-137.
[10]Lo S C,Hips kind J D,Nicholson R L.cDNA cloning of a sorghum pathogenesis—related protein(PR—10) and differential expression of defense—related genes following inoculation with Cochliobolus heterotrophy bus or Colletotrichum sublineolum.Lo SCC Molecular Plant Microbe Interactions,1999,12(6):479 -489.
[11]Uknes S,D incher S,F ride rich L,et al.Regulation of pathogenesis related protein lagene expression in tobacco.Plant Cell,1993,5:159-169.
[12]Pritsch C,Muehlbauer G,Bushnell W R,Somers D A,Vance C P.Fungal development and induction of defense response gene during early infection of wheat spikes by Fusarium graminearum.Mol Microbe Interact,2000,13:159-169
[13]Steven W Hutcheson.Current concepts of active defense in plants.A nun Rev Phytopathol,1998,36:59-90.
[14]Kitajima S,Sato F.Plant pathogenesis-related proteins:molecular mechanisms of gene expression and protein function.Journal of Biochemistry Tokyo,1999,125(1):1-8.
[15]王关林,方宏筠.植物基因工程(第二版).北京:科学出版社,2002.
[16]梁国栋主编,最新分子生物学实验技术.科学出版社,2001,129-149.
[17]李明才,何韶衡.一种高效、快速的大肠杆菌感受态细胞制备及质粒转化方法.汕头大学医学院学报,2005,18(4):228-241.
[18]梁建庆,叶志华,江秀梅等.转化条件对质粒DNA转化大肠杆菌的影响.微生物学志,2004,7(24):15-17.
[19]温建新,李俊,周顺等.大肠杆菌质粒DNA提取方法的优选.西南农业学报,2007,20(4):825-828.
[20]朱帆,刘忠纯,李健.大肠杆菌质粒的快速提取.微生物学杂志,2004,5(24):59-60.
[21]陈润生.生物信息学.生物物理学报1999,15(1):6-12.
[22]张春霆.生物信息学-重大科学意义与经济效益兼备的新学科.中国科学基金,1999(2),:65-68.
[23]孙啸,陆祖宏,谢建明.生物信息学基础.北京:清华大学出版社,2005.
[24]郝柏林,张淑誉.生物信息学手册.2000.
[25]赵国屏.生物信息学.北京,2000.
[26]何红波,谭晓超,李斌,李义兵.生物信息学对计算机科学发展的机遇与挑战.生物信息学,2005.
[27]朱佳鸣,郭勇,唐权等.国内外生物信息学WWW服务器及数据库.微生物学通报,2002,29(1):99-103.
[28]张春霆.生物信息学的现状与展望.世界科技研究与发展,2000.
[29]施晓秋,孔繁胜.计算机科学在生物信息学中的应用.浙江工业大学学报,2001.
[30]黄原.生物信息学-信息时代生命科学研究的公共平台.中学生物教学,2004.
[31]司徒琳莉,于敦亮.跨学科时代的基因工程研究.牡丹江师范学院学报,自然科学版,1997.
[32]Huynen M A and Bork.Measuring genome evolution,Proc.Natl.Acad.Sci.USA,1998,95:5849-5856.
[33]侯国清.欧盟加强生物信息学研究.全球科技经济展望,2001,10:47.
[34]Kyte J,Doolittle R F.A Simple Method for Displaying the Hydropathical Character of a Protein.Journal of Molecular Biology,1982,157(6):105-142
[35]Clare A,King R D.Machine learning functional class from phenotype data.Bioinformatics,2002,18(1):160-166
[36]Rost B,Sander C.Prediction of protein secondary structure at better than 70%accuracy.J Mol Biol,1993,232(2):584-599.
[37]Levin J,Robson B,Gamier J.An algorithm for secondary structure determination in protein based on sequence similarity.FEBS Lett,1986,205:303-308.
[38]Gibran J F,Gamier J,Robson B.Further developments of protein secondary structure prediction using information theory.New parameters and consideration of residue pairs,Mol Biol,1987,198(3):425-443.
[39]Chou P Y,Fasman G B.Conformational parameters for amino acids inhelical,betasheet,andrandomcoilregionscalculatedproteins.B iochemistry,1974,13:211-222.
[40]Gamier J,Osguthorpe D J,Robson B.Analysis of the accuracy and implications of simple methods for prediction the secondary structure of globular proteins Mol Biol,1978,120:97-120.
[41]Lim V I.Algorithm for prediction of a-helical and β-structural regions in Globular proteins Mol Biol,1974,88:873-894.
[42]Batalia M A,Monzingo A F,Ernst S,Roberts W,Roberts J D.The crystal structure of the antifungal protein zeamatin,a member of the thaumatin-like,PR5 protein family.Nat Struct Biol.1996,3(1):19-23.
[43]Koiwa H,Kato H,Nakatsu T,Oda J,Yamada Y,Sato F.Crystal structure of tobacco PR5d protein at 1.8A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins Mol Bio 1999,286:1137-1145.
[44]Ogata C M,Gordon P F,de Voss AM Kim S M.Crystal structure of a sweet tasting protein thaumatin I,at 1.65 A resolution.J Mol Bio 1992,228:893-908.
[45]Leonea P,Menu-Bouaouicheb L,Van Dammec E.And Rougeb P,Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7.,2006,88:45-52.
[46]Pad dam M.Chemically regulated gene expression in plants.Curt Opin.Plant Mol.Biol,(2003),6:169-177.
[47]胡廷章,罗凯,甘丽萍,石汝杰.植物基因启动子的类型及其应用.湖北农业科学.2007(1),46(1):99-103.
[48]Lescot M,Dehais P,Thijs G,et al.Plant CARE,a database of plant cis-acting regulatory elements and portal to tools for in silicon analysis of promoter sequences.Nucl Acids Res,(2002),30(1):325-327.
[2]王金生.植物抗病性分子机制.植物病理学报,1995,25(4):289-295.
[3]Key J,Kosuge T.Cellular and molecular biology of plant stress.New York:Alan R Liss Inc,1985.303-327.
[4]Van Loon L C.The nom enclosure of pathogenesis-related proteins.Plant Mol Biol,1990,37:229.
[5]Bowles D J.Defense-related proteins in higher plants.Annual Review of Biochemistry,1990,59:873-907
[6]Guo H,Ecker J R.The ethylene signaling pathway:new insights.Cur opinion plant boil,2004,7:40-49.
[7]Van Loon L C,Van Strien E A.The families of pathogenesis-related proteins,their activities,and comparative analysis of PR-1types proteins.Physiology Mol Plant Pathol.1999,55:85-97
[8]Webber J P,Jostens M hajj,V an Kan Jal,et al.Sub cellar localization of plant chitinases and 1,3—β—gluconase C lad osporium fulvum infected tomato leaves.Physiology Mol Plant Patrol.1992,41:23.
[9]Sikorski M M,Biesiadka J,Kasperska A E,et al.Expression of genes encoding PR10 class pathogenesis—related proteins is inhibited in yellow lupine root nodules.Plant Science Limerick,1999,149(2):125-137.
[10]Lo S C,Hips kind J D,Nicholson R L.cDNA cloning of a sorghum pathogenesis—related protein(PR—10) and differential expression of defense—related genes following inoculation with Cochliobolus heterotrophy bus or Colletotrichum sublineolum.Lo SCC Molecular Plant Microbe Interactions,1999,12(6):479 -489.
[11]Uknes S,D incher S,F ride rich L,et al.Regulation of pathogenesis related protein lagene expression in tobacco.Plant Cell,1993,5:159-169.
[12]Pritsch C,Muehlbauer G,Bushnell W R,Somers D A,Vance C P.Fungal development and induction of defense response gene during early infection of wheat spikes by Fusarium graminearum.Mol Microbe Interact,2000,13:159-169
[13]Steven W Hutcheson.Current concepts of active defense in plants.A nun Rev Phytopathol,1998,36:59-90.
[14]Kitajima S,Sato F.Plant pathogenesis-related proteins:molecular mechanisms of gene expression and protein function.Journal of Biochemistry Tokyo,1999,125(1):1-8.
[15]王关林,方宏筠.植物基因工程(第二版).北京:科学出版社,2002.
[16]梁国栋主编,最新分子生物学实验技术.科学出版社,2001,129-149.
[17]李明才,何韶衡.一种高效、快速的大肠杆菌感受态细胞制备及质粒转化方法.汕头大学医学院学报,2005,18(4):228-241.
[18]梁建庆,叶志华,江秀梅等.转化条件对质粒DNA转化大肠杆菌的影响.微生物学志,2004,7(24):15-17.
[19]温建新,李俊,周顺等.大肠杆菌质粒DNA提取方法的优选.西南农业学报,2007,20(4):825-828.
[20]朱帆,刘忠纯,李健.大肠杆菌质粒的快速提取.微生物学杂志,2004,5(24):59-60.
[21]陈润生.生物信息学.生物物理学报1999,15(1):6-12.
[22]张春霆.生物信息学-重大科学意义与经济效益兼备的新学科.中国科学基金,1999(2),:65-68.
[23]孙啸,陆祖宏,谢建明.生物信息学基础.北京:清华大学出版社,2005.
[24]郝柏林,张淑誉.生物信息学手册.2000.
[25]赵国屏.生物信息学.北京,2000.
[26]何红波,谭晓超,李斌,李义兵.生物信息学对计算机科学发展的机遇与挑战.生物信息学,2005.
[27]朱佳鸣,郭勇,唐权等.国内外生物信息学WWW服务器及数据库.微生物学通报,2002,29(1):99-103.
[28]张春霆.生物信息学的现状与展望.世界科技研究与发展,2000.
[29]施晓秋,孔繁胜.计算机科学在生物信息学中的应用.浙江工业大学学报,2001.
[30]黄原.生物信息学-信息时代生命科学研究的公共平台.中学生物教学,2004.
[31]司徒琳莉,于敦亮.跨学科时代的基因工程研究.牡丹江师范学院学报,自然科学版,1997.
[32]Huynen M A and Bork.Measuring genome evolution,Proc.Natl.Acad.Sci.USA,1998,95:5849-5856.
[33]侯国清.欧盟加强生物信息学研究.全球科技经济展望,2001,10:47.
[34]Kyte J,Doolittle R F.A Simple Method for Displaying the Hydropathical Character of a Protein.Journal of Molecular Biology,1982,157(6):105-142
[35]Clare A,King R D.Machine learning functional class from phenotype data.Bioinformatics,2002,18(1):160-166
[36]Rost B,Sander C.Prediction of protein secondary structure at better than 70%accuracy.J Mol Biol,1993,232(2):584-599.
[37]Levin J,Robson B,Gamier J.An algorithm for secondary structure determination in protein based on sequence similarity.FEBS Lett,1986,205:303-308.
[38]Gibran J F,Gamier J,Robson B.Further developments of protein secondary structure prediction using information theory.New parameters and consideration of residue pairs,Mol Biol,1987,198(3):425-443.
[39]Chou P Y,Fasman G B.Conformational parameters for amino acids inhelical,betasheet,andrandomcoilregionscalculatedproteins.B iochemistry,1974,13:211-222.
[40]Gamier J,Osguthorpe D J,Robson B.Analysis of the accuracy and implications of simple methods for prediction the secondary structure of globular proteins Mol Biol,1978,120:97-120.
[41]Lim V I.Algorithm for prediction of a-helical and β-structural regions in Globular proteins Mol Biol,1974,88:873-894.
[42]Batalia M A,Monzingo A F,Ernst S,Roberts W,Roberts J D.The crystal structure of the antifungal protein zeamatin,a member of the thaumatin-like,PR5 protein family.Nat Struct Biol.1996,3(1):19-23.
[43]Koiwa H,Kato H,Nakatsu T,Oda J,Yamada Y,Sato F.Crystal structure of tobacco PR5d protein at 1.8A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins Mol Bio 1999,286:1137-1145.
[44]Ogata C M,Gordon P F,de Voss AM Kim S M.Crystal structure of a sweet tasting protein thaumatin I,at 1.65 A resolution.J Mol Bio 1992,228:893-908.
[45]Leonea P,Menu-Bouaouicheb L,Van Dammec E.And Rougeb P,Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7.,2006,88:45-52.
[46]Pad dam M.Chemically regulated gene expression in plants.Curt Opin.Plant Mol.Biol,(2003),6:169-177.
[47]胡廷章,罗凯,甘丽萍,石汝杰.植物基因启动子的类型及其应用.湖北农业科学.2007(1),46(1):99-103.
[48]Lescot M,Dehais P,Thijs G,et al.Plant CARE,a database of plant cis-acting regulatory elements and portal to tools for in silicon analysis of promoter sequences.Nucl Acids Res,(2002),30(1):325-327.