馮寶珍+李培謙+成娟麗等
摘要:NPP(necrosis-inducing Phytophthora protein)是一類效應(yīng)子,在卵菌疫霉屬病原菌中普遍存在,該類蛋白能夠激發(fā)植物的防御反應(yīng),如引起植物細(xì)胞死亡、促使寄主防御基因表達(dá)及產(chǎn)生乙烯。利用生物信息學(xué)方法對辣椒疫霉(Phytophthora capsici)基因組內(nèi)的NPP基因家族存在狀況進(jìn)行分析,并對其氨基酸序列組成、基本理化性質(zhì)、蛋白質(zhì)三級結(jié)構(gòu)等進(jìn)行預(yù)測分析,旨在了解其結(jié)構(gòu)特征,進(jìn)而發(fā)掘辣椒疫霉的致病相關(guān)基因。結(jié)果表明:NPP基因家族有25個成員,其中大部分成員存在多個拷貝。對25個NPP氨基酸序列進(jìn)行分析發(fā)現(xiàn),其分子量在12.45~88.9 ku之間;對其中15個NPP蛋白進(jìn)行三級結(jié)構(gòu)預(yù)測發(fā)現(xiàn),它們的三維結(jié)構(gòu)類似,其主要的結(jié)構(gòu)元件為α-螺旋和β-片層;構(gòu)建了25個NPP蛋白的進(jìn)化樹,進(jìn)行了分子進(jìn)化分析。
關(guān)鍵詞:辣椒疫霉;NPP基因家族;效應(yīng)子;生物信息學(xué)
中圖分類號: S436.418.1+9 文獻(xiàn)標(biāo)志碼: A 文章編號:1002-1302(2014)07-0028-04
收稿日期:2013-10-02
基金項(xiàng)目:山西省高??萍柬?xiàng)目(編號:20121025);山西省青年科技研究基金(編號:2013021024-6)
作者簡介:馮寶珍(1981—),女,山東臨沂人,博士,講師,主要從事分子植物病理學(xué)研究。E-mail:fengbaozhen@126.com。效應(yīng)子(effector)是病原物產(chǎn)生的各種能夠壓制或平衡寄主防御系統(tǒng)的分子。在侵染過程中,病原物以效應(yīng)子為關(guān)鍵武器突破寄主的防御系統(tǒng),一方面能減弱寄主抵抗力以成功侵入,另一方面還能利用寄主的養(yǎng)分以利于自身增殖[1-2]。許多病原物都能分泌效應(yīng)子,在細(xì)菌、真菌、卵菌及線蟲中都發(fā)現(xiàn)了效應(yīng)子[3]。病原物中存在的效應(yīng)子有許多種,包括NPP[4]、無毒基因(Avr)[5]、CRN[6]、PcF[7]、ScR[8]、糖類水解酶、果膠酶、幾丁質(zhì)酶以及酯酶[7]等,其中NPP(necrosis-inducing Phytophthora protein)也被稱為NLP(NEP1-like protein),此類基因在卵菌、真菌、細(xì)菌中普遍存在[8],但在細(xì)菌、真菌基因組內(nèi)的數(shù)量較少,一般為1~4個,而在卵菌基因組中的數(shù)量較多,且多數(shù)以基因家族形式存在,如在大豆疫霉(Phytophthora sojea)中約有29個,在橡膠疫霉(Phytohthora ramorum)中約有40個[9]。多數(shù)植物被NPP蛋白處理后表現(xiàn)為產(chǎn)生乙烯、MAP激酶活化、植保素合成、PR基因誘導(dǎo)表達(dá)、胞質(zhì)Ca2+釋放以及多種雙子葉植物的葉片壞死反應(yīng)等現(xiàn)象。最早報(bào)道的NPP蛋白NEP1來自鐮刀菌(Fusarisum oxysporium),能夠引起雙子葉植物葉片壞死[10]。后來在腐霉(Pythium aphanidermatum)中分離了該類基因PaNie,發(fā)現(xiàn)其原核表達(dá)產(chǎn)物能使胡蘿卜、擬南芥及煙草等細(xì)胞死亡[11]。Fellbrich等研究了Phytohthora parasitica的NPP1基因,發(fā)現(xiàn)其原核表達(dá)產(chǎn)物能夠誘導(dǎo)歐芹及擬南芥體內(nèi)產(chǎn)生pathogenesis-related(PR)蛋白、活性氧(ROS)和乙烯,并引起過敏性壞死反應(yīng)[12]。Keates等用NeP1處理斑點(diǎn)矢車菊、蒲公英、擬南芥,電鏡結(jié)果顯示,這些植物細(xì)胞壁結(jié)構(gòu)發(fā)生了明顯變化,可引起角質(zhì)層變薄、葉綠體降解等現(xiàn)象[13]。Bailey等分別用NeP1和Phytohthora megakarya游動孢子處理不同生長時期的可可葉片,發(fā)現(xiàn)不同生長時期可可葉片的各類抗逆基因表達(dá)水平不同,2種處理葉片中的抗逆基因表達(dá)模式相似,因此推測NeP1可能是一種感病因子[14]。棉花黃萎病菌(Verticillium dahliae)VdNEP基因的表達(dá)產(chǎn)物能使棉花、煙草、擬南芥葉片萎蔫,因此認(rèn)為VdNEP是棉花與病原物互作過程中的重要誘導(dǎo)因子[15]。敲除細(xì)菌Erwinia carotovora ssp. carotovora和E. carotovora ssp. atroseptica的NPP基因后再分別接種馬鈴薯塊莖,發(fā)現(xiàn)其毒性明顯下降[16]。研究發(fā)現(xiàn),大豆疫霉(P. sojae)的PsojNIP基因表達(dá)是在活體營養(yǎng)向死體營養(yǎng)轉(zhuǎn)換的過渡時期,并且只能引起雙子葉植物細(xì)胞死亡,因此認(rèn)為該基因在大豆疫霉半活體營養(yǎng)生活過程中起到輔助病原在寄主植物中定殖的作用[17]。目前有人研究了這類蛋白對植物的過敏反應(yīng),但是這類蛋白的功能及在植物中的作用模式還是未知的,更沒有足夠的證據(jù)證實(shí)NPP蛋白對疫霉毒性起重要作用。
辣椒疫霉(Phytohthora capsici)屬于卵菌綱,是一類區(qū)別于真菌的真核生物,除了能夠引起辣椒疫病,還能夠引起多種茄科及葫蘆科植物疫病。辣椒疫病是一種土傳植物病害,廣泛分布于世界各地,且發(fā)病快、流行廣,易造成作物嚴(yán)重?fù)p失。大量使用傳統(tǒng)的化學(xué)藥劑不僅使病原物產(chǎn)生了藥物抗性,而且造成的農(nóng)藥殘留更會嚴(yán)重威脅人類的生存環(huán)境。隨著基因工程及蛋白質(zhì)組學(xué)的迅速發(fā)展,從基因和蛋白水平解釋植物病原的致病機(jī)理、挖掘致病相關(guān)基因、開展抗病育種及研究抗病基因過程勢在必行。隨著辣椒疫霉全基因組測序的完成[9],有關(guān)致病重要基因家族及靶基因預(yù)測分析等逐漸成為研究熱點(diǎn)。分析植物病原真菌關(guān)鍵致病基因編碼的蛋白質(zhì)結(jié)構(gòu)及其關(guān)鍵功能結(jié)構(gòu)基團(tuán)的修飾特性,對于探索植物病原真菌致病性變異的機(jī)理具有重要的意義。本試驗(yàn)在前人研究的基礎(chǔ)上,開展與辣椒疫霉致病性變異相關(guān)的基因簇大小、組成、亞組劃分及其關(guān)鍵基因的研究。
1材料與方法
1.1數(shù)據(jù)庫
辣椒疫霉基因組序列信息從DOE Joint Genome Institute(http://genome.jgi.doe.gov/)下載。
雖然有些蛋白質(zhì)的基本元件個數(shù)相同,但它們的α-螺旋、β-片層以及無規(guī)則卷曲的長度都存在一定差異,這些相似或者差異可能導(dǎo)致它們家族成員之間功能的多樣性。
3結(jié)論與討論
NPP效應(yīng)子的研究是當(dāng)前卵菌功能基因組學(xué)領(lǐng)域的熱點(diǎn)。NPP效應(yīng)子基因家族在微生物中廣泛存在,目前已知的微生物有卵菌腐霉屬(P. aphanidermatum)[4]、疫霉屬(Phytophthora spp.)、真菌鐮刀菌(F. oxysporum)、鏈孢霉屬(Neurospora crassa)[21],以及革蘭氏陽性菌芽孢桿菌(Bacillus halodurans)、鏈霉菌屬(Streptomyces coelicolor),革蘭氏陰性菌歐文氏菌屬(Erwinia)[4]和弧菌屬(Vibrio pommerensis sp.)[22],這些微生物中部分是植物病原菌,部分是動物病原物,其侵染方式各異,表明NPP蛋白功能的多樣性。目前的研究集中于對部分植物病原NPP基因功能的分析,大部分NPP蛋白的功能都是未知的。據(jù)報(bào)道,NPP蛋白可參與寄主防御反應(yīng),引起寄主的過敏性反應(yīng)。也有研究表明,有些病原物中的NPP蛋白與病原物致病性有關(guān)。本研究對辣椒疫霉NPP基因家族生物信息學(xué)進(jìn)行了分析,為深入開展NPP基因在辣椒疫霉中的克隆、表達(dá)分析及生物學(xué)功能鑒定等方面的研究提供了重要基礎(chǔ)。
本研究以大豆疫霉NPP基因編碼蛋白為探針,對辣椒疫霉基因組數(shù)據(jù)庫進(jìn)行檢索得到了25個NPP蛋白序列,利用相關(guān)數(shù)據(jù)庫和軟件對這些序列進(jìn)行基因鑒定和蛋白質(zhì)三級結(jié)構(gòu)預(yù)測分析,并構(gòu)建系統(tǒng)進(jìn)化樹對該家族不同成員之間的相互關(guān)系和演化歷程進(jìn)行了探討。對發(fā)掘辣椒疫霉致病基因、解析其關(guān)鍵基因?qū)τ谔剿髦参锊≡婢虏⌒宰儺惖臋C(jī)理及開展抗病基因工程研究具有重要的意義。
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[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.
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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.