辣椒疫霉NPP效应子基因家族生物信息学分析

2014-09-02 00:51冯宝珍李培谦成娟丽等
江苏农业科学 2014年7期
关键词:生物信息学

冯宝珍+李培谦+成娟丽等

摘要:NPP(necrosis-inducing Phytophthora protein)是一类效应子,在卵菌疫霉属病原菌中普遍存在,该类蛋白能够激发植物的防御反应,如引起植物细胞死亡、促使寄主防御基因表达及产生乙烯。利用生物信息学方法对辣椒疫霉(Phytophthora capsici)基因组内的NPP基因家族存在状况进行分析,并对其氨基酸序列组成、基本理化性质、蛋白质三级结构等进行预测分析,旨在了解其结构特征,进而发掘辣椒疫霉的致病相关基因。结果表明:NPP基因家族有25个成员,其中大部分成员存在多个拷贝。对25个NPP氨基酸序列进行分析发现,其分子量在12.45~88.9 ku之间;对其中15个NPP蛋白进行三级结构预测发现,它们的三维结构类似,其主要的结构元件为α-螺旋和β-片层;构建了25个NPP蛋白的进化树,进行了分子进化分析。

关键词:辣椒疫霉;NPP基因家族;效应子;生物信息学

中图分类号: S436.418.1+9 文献标志码: A 文章编号:1002-1302(2014)07-0028-04

收稿日期:2013-10-02

基金项目:山西省高校科技项目(编号:20121025);山西省青年科技研究基金(编号:2013021024-6)

作者简介:冯宝珍(1981—),女,山东临沂人,博士,讲师,主要从事分子植物病理学研究。E-mail:fengbaozhen@126.com。效应子(effector)是病原物产生的各种能够压制或平衡寄主防御系统的分子。在侵染过程中,病原物以效应子为关键武器突破寄主的防御系统,一方面能减弱寄主抵抗力以成功侵入,另一方面还能利用寄主的养分以利于自身增殖[1-2]。许多病原物都能分泌效应子,在细菌、真菌、卵菌及线虫中都发现了效应子[3]。病原物中存在的效应子有许多种,包括NPP[4]、无毒基因(Avr)[5]、CRN[6]、PcF[7]、ScR[8]、糖类水解酶、果胶酶、几丁质酶以及酯酶[7]等,其中NPP(necrosis-inducing Phytophthora protein)也被称为NLP(NEP1-like protein),此类基因在卵菌、真菌、细菌中普遍存在[8],但在细菌、真菌基因组内的数量较少,一般为1~4个,而在卵菌基因组中的数量较多,且多数以基因家族形式存在,如在大豆疫霉(Phytophthora sojea)中约有29个,在橡胶疫霉(Phytohthora ramorum)中约有40个[9]。多数植物被NPP蛋白处理后表现为产生乙烯、MAP激酶活化、植保素合成、PR基因诱导表达、胞质Ca2+释放以及多种双子叶植物的叶片坏死反应等现象。最早报道的NPP蛋白NEP1来自镰刀菌(Fusarisum oxysporium),能够引起双子叶植物叶片坏死[10]。后来在腐霉(Pythium aphanidermatum)中分离了该类基因PaNie,发现其原核表达产物能使胡萝卜、拟南芥及烟草等细胞死亡[11]。Fellbrich等研究了Phytohthora parasitica的NPP1基因,发现其原核表达产物能够诱导欧芹及拟南芥体内产生pathogenesis-related(PR)蛋白、活性氧(ROS)和乙烯,并引起过敏性坏死反应[12]。Keates等用NeP1处理斑点矢车菊、蒲公英、拟南芥,电镜结果显示,这些植物细胞壁结构发生了明显变化,可引起角质层变薄、叶绿体降解等现象[13]。Bailey等分别用NeP1和Phytohthora megakarya游动孢子处理不同生长时期的可可叶片,发现不同生长时期可可叶片的各类抗逆基因表达水平不同,2种处理叶片中的抗逆基因表达模式相似,因此推测NeP1可能是一种感病因子[14]。棉花黄萎病菌(Verticillium dahliae)VdNEP基因的表达产物能使棉花、烟草、拟南芥叶片萎蔫,因此认为VdNEP是棉花与病原物互作过程中的重要诱导因子[15]。敲除细菌Erwinia carotovora ssp. carotovora和E. carotovora ssp. atroseptica的NPP基因后再分别接种马铃薯块茎,发现其毒性明显下降[16]。研究发现,大豆疫霉(P. sojae)的PsojNIP基因表达是在活体营养向死体营养转换的过渡时期,并且只能引起双子叶植物细胞死亡,因此认为该基因在大豆疫霉半活体营养生活过程中起到辅助病原在寄主植物中定殖的作用[17]。目前有人研究了这类蛋白对植物的过敏反应,但是这类蛋白的功能及在植物中的作用模式还是未知的,更没有足够的证据证实NPP蛋白对疫霉毒性起重要作用。

辣椒疫霉(Phytohthora capsici)属于卵菌纲,是一类区别于真菌的真核生物,除了能够引起辣椒疫病,还能够引起多种茄科及葫芦科植物疫病。辣椒疫病是一种土传植物病害,广泛分布于世界各地,且发病快、流行广,易造成作物严重损失。大量使用传统的化学药剂不仅使病原物产生了药物抗性,而且造成的农药残留更会严重威胁人类的生存环境。随着基因工程及蛋白质组学的迅速发展,从基因和蛋白水平解释植物病原的致病机理、挖掘致病相关基因、开展抗病育种及研究抗病基因过程势在必行。随着辣椒疫霉全基因组测序的完成[9],有关致病重要基因家族及靶基因预测分析等逐渐成为研究热点。分析植物病原真菌关键致病基因编码的蛋白质结构及其关键功能结构基团的修饰特性,对于探索植物病原真菌致病性变异的机理具有重要的意义。本试验在前人研究的基础上,开展与辣椒疫霉致病性变异相关的基因簇大小、组成、亚组划分及其关键基因的研究。

1材料与方法

1.1数据库

辣椒疫霉基因组序列信息从DOE Joint Genome Institute(http://genome.jgi.doe.gov/)下载。

虽然有些蛋白质的基本元件个数相同,但它们的α-螺旋、β-片层以及无规则卷曲的长度都存在一定差异,这些相似或者差异可能导致它们家族成员之间功能的多样性。

3结论与讨论

NPP效应子的研究是当前卵菌功能基因组学领域的热点。NPP效应子基因家族在微生物中广泛存在,目前已知的微生物有卵菌腐霉属(P. aphanidermatum)[4]、疫霉属(Phytophthora spp.)、真菌镰刀菌(F. oxysporum)、链孢霉属(Neurospora crassa)[21],以及革兰氏阳性菌芽孢杆菌(Bacillus halodurans)、链霉菌属(Streptomyces coelicolor),革兰氏阴性菌欧文氏菌属(Erwinia)[4]和弧菌属(Vibrio pommerensis sp.)[22],这些微生物中部分是植物病原菌,部分是动物病原物,其侵染方式各异,表明NPP蛋白功能的多样性。目前的研究集中于对部分植物病原NPP基因功能的分析,大部分NPP蛋白的功能都是未知的。据报道,NPP蛋白可参与寄主防御反应,引起寄主的过敏性反应。也有研究表明,有些病原物中的NPP蛋白与病原物致病性有关。本研究对辣椒疫霉NPP基因家族生物信息学进行了分析,为深入开展NPP基因在辣椒疫霉中的克隆、表达分析及生物学功能鉴定等方面的研究提供了重要基础。

本研究以大豆疫霉NPP基因编码蛋白为探针,对辣椒疫霉基因组数据库进行检索得到了25个NPP蛋白序列,利用相关数据库和软件对这些序列进行基因鉴定和蛋白质三级结构预测分析,并构建系统进化树对该家族不同成员之间的相互关系和演化历程进行了探讨。对发掘辣椒疫霉致病基因、解析其关键基因对于探索植物病原真菌致病性变异的机理及开展抗病基因工程研究具有重要的意义。

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[14]Bailey B A,Bae H,Strem M D,et al. Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya[J]. Plant Physiology and Biochemistry,2005,43(6):611-622.

[15]Wang J Y,Cai Y,Gou J Y,et al. VdNEP,an elicitor from Verticillium dahliae,induces cotton plant wilting[J]. Applied and Environmental Microbiology,2004,70(8):4989-4995.

[16]Mattinen L,Tshuikina M,Me A,et al. Identification and characterization of Nip,necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora[J]. Molecular Plant-Microbe Interactions,2004,17(12):1366-1375.

[17]Qutob D,Kamoun S,Gijzen M. Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy[J]. Plant Journal,2002,32(3):361-373.

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[21]Galagan J E,Calvo S E,Borkovich K A,et al. The genome sequence of the filamentous Neurospora crassa[J]. Nature,2003,422(6934):859-868.

[22]Jores J L A,Appel B,Lewin A.Cloning and molecular Characterization of a Unique Hemolysin Gene of Vibrio pommerensis sp. nov. development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp. from the Baltic Sea[J]. FEMS Micro Lett,2003,229(2):223-229.

[11]Veit S,Wrle J M,Nürnberger T,et al. A novel protein elicitor(PaNie)from Pythium aphanidermatum induces multiple defense responses in carrot,Arabidopsis,and tobacco[J]. Plant Physiology,2001,127(3):832-841.

[12]Fellbrich G,Romanski A,Varet A,et al. NPP1,a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis[J]. Plant Journal,2002,32(3):375-390.

[13]Keates S E,Kostman T A,Anderson J D,et al. Altered gene expression in three plant species in response to treatment with Nep1,a fungal protein that causes necrosis[J]. Plant Physiology,2003,132(3):1610-1622.

[14]Bailey B A,Bae H,Strem M D,et al. Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya[J]. Plant Physiology and Biochemistry,2005,43(6):611-622.

[15]Wang J Y,Cai Y,Gou J Y,et al. VdNEP,an elicitor from Verticillium dahliae,induces cotton plant wilting[J]. Applied and Environmental Microbiology,2004,70(8):4989-4995.

[16]Mattinen L,Tshuikina M,Me A,et al. Identification and characterization of Nip,necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora[J]. Molecular Plant-Microbe Interactions,2004,17(12):1366-1375.

[17]Qutob D,Kamoun S,Gijzen M. Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy[J]. Plant Journal,2002,32(3):361-373.

[18]Bendtsen J D,Nielsen H,von Heijne G,et al. Improved prediction of signal peptides:SignalP 3.0[J]. Journal of Molecular Biology,2004,340(4):783-795.

[19]Marchler-Bauer A,Anderson J B,Chitsaz F,et al. CDD:specific functional annotation with the Conserved Domain Database[J]. Nucleic Acids Research,2009,37:D205-D210.

[20]Letunic I,Doerks T,Bork P. SMART 7:recent updates to the protein domain annotation resource[J]. Nucleic Acids Research,2012,40:D302-D305.

[21]Galagan J E,Calvo S E,Borkovich K A,et al. The genome sequence of the filamentous Neurospora crassa[J]. Nature,2003,422(6934):859-868.

[22]Jores J L A,Appel B,Lewin A.Cloning and molecular Characterization of a Unique Hemolysin Gene of Vibrio pommerensis sp. nov. development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp. from the Baltic Sea[J]. FEMS Micro Lett,2003,229(2):223-229.

[11]Veit S,Wrle J M,Nürnberger T,et al. A novel protein elicitor(PaNie)from Pythium aphanidermatum induces multiple defense responses in carrot,Arabidopsis,and tobacco[J]. Plant Physiology,2001,127(3):832-841.

[12]Fellbrich G,Romanski A,Varet A,et al. NPP1,a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis[J]. Plant Journal,2002,32(3):375-390.

[13]Keates S E,Kostman T A,Anderson J D,et al. Altered gene expression in three plant species in response to treatment with Nep1,a fungal protein that causes necrosis[J]. Plant Physiology,2003,132(3):1610-1622.

[14]Bailey B A,Bae H,Strem M D,et al. Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya[J]. Plant Physiology and Biochemistry,2005,43(6):611-622.

[15]Wang J Y,Cai Y,Gou J Y,et al. VdNEP,an elicitor from Verticillium dahliae,induces cotton plant wilting[J]. Applied and Environmental Microbiology,2004,70(8):4989-4995.

[16]Mattinen L,Tshuikina M,Me A,et al. Identification and characterization of Nip,necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora[J]. Molecular Plant-Microbe Interactions,2004,17(12):1366-1375.

[17]Qutob D,Kamoun S,Gijzen M. Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy[J]. Plant Journal,2002,32(3):361-373.

[18]Bendtsen J D,Nielsen H,von Heijne G,et al. Improved prediction of signal peptides:SignalP 3.0[J]. Journal of Molecular Biology,2004,340(4):783-795.

[19]Marchler-Bauer A,Anderson J B,Chitsaz F,et al. CDD:specific functional annotation with the Conserved Domain Database[J]. Nucleic Acids Research,2009,37:D205-D210.

[20]Letunic I,Doerks T,Bork P. SMART 7:recent updates to the protein domain annotation resource[J]. Nucleic Acids Research,2012,40:D302-D305.

[21]Galagan J E,Calvo S E,Borkovich K A,et al. The genome sequence of the filamentous Neurospora crassa[J]. Nature,2003,422(6934):859-868.

[22]Jores J L A,Appel B,Lewin A.Cloning and molecular Characterization of a Unique Hemolysin Gene of Vibrio pommerensis sp. nov. development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp. from the Baltic Sea[J]. FEMS Micro Lett,2003,229(2):223-229.

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