花魔芋NBS-LRR类抗病基因同源序列的克隆和初步分析
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摘要
通过同源克隆法从花魔芋抗病植株中得到抗软腐病基因的同源片段,为筛选魔芋抗软腐病基因提供了科学依据。根据已知植物NBS-LRR类抗病基因同源序列(RGA)的保守区域设计简并引物,从抗软腐病花魔芋植株的基因组DNA与cDNA上得到抗病基因同源片段(RGAs),并对其结构进行分析。从抗软腐病花魔芋植株基因组中分离得到了一条500 bp的NBS-LRR类抗病基因同源序列,共获得6条特异序列,分别命名为RGAR1、RGAR2、RGAR3、RGAR4、RGAR5、RGAR6,6条魔芋RGAs在氨基酸水平上的同源性表现出多态性,序列相似性分析结果表明,这些魔芋抗病基因同源序列均包含P-loop、Kinase-2、Kinase-3及疏水结构域HD等保守结构域,与已知的抗病基因在氨基酸水平上的同源性为33%～63%。通过构建系统进化树分析,将这6个序列分为3个亚组,均属于TIR-NBS-LRR类抗病基因。在花魔芋上成功获得了抗病基因同源序列,将为魔芋中抗病基因的克隆、功能分析及定位等提供帮助。
The homologous fr agments of soft rot resistance genes were obtained from Amorphophallus konjac resistant plants by homologous cloning, which laid a foundation for screening the Amorphophallus konjac plant with resistance to soft rot disease. A degenerate primer was designed according to the conserved domains of(RGA) homologous sequence of plant NBS-LRR type resistance gene. The homologous fragment of resistance gene(RGAs), was obtained from the genomic DNA and cDNA of the plant resistant to soft rot and its structure was analyzed. A 500 bp NBS-LRR type resistance gene homologous sequence was isolated from the genome of Amorphophallus konjac plant with resistance to soft rot disease, and six specific sequences were obtained,respectively named RGAR1, RGAR2, RGAR3, RGAR4, RGAR5, RGAR6. The homology of 6 RGAs was polymorphic at amino acid level. The results of sequence similarity analysis showed that 6 RGAs contained conserved motifs such as P-loop, Kinase-2, Kinase-3 and hydrophobic domain(HD). The homology of these genes with known resistant genes was 33% ～63% at the amino acid level. By constructing phylogenetic tree, the six sequences were divided into three subgroups, all of which belonged to TIR-NBS-LRR resistance genes. The homologous sequences of resistance genes were successfully obtained from Amorphophallus konjac, which will be helpful for cloning, functional analysis and location of disease resistance genes in in A. konjac.
The homologous fr agments of soft rot resistance genes were obtained from Amorphophallus konjac resistant plants by homologous cloning, which laid a foundation for screening the Amorphophallus konjac plant with resistance to soft rot disease. A degenerate primer was designed according to the conserved domains of(RGA) homologous sequence of plant NBS-LRR type resistance gene. The homologous fragment of resistance gene(RGAs), was obtained from the genomic DNA and cDNA of the plant resistant to soft rot and its structure was analyzed. A 500 bp NBS-LRR type resistance gene homologous sequence was isolated from the genome of Amorphophallus konjac plant with resistance to soft rot disease, and six specific sequences were obtained,respectively named RGAR1, RGAR2, RGAR3, RGAR4, RGAR5, RGAR6. The homology of 6 RGAs was polymorphic at amino acid level. The results of sequence similarity analysis showed that 6 RGAs contained conserved motifs such as P-loop, Kinase-2, Kinase-3 and hydrophobic domain(HD). The homology of these genes with known resistant genes was 33% ～63% at the amino acid level. By constructing phylogenetic tree, the six sequences were divided into three subgroups, all of which belonged to TIR-NBS-LRR resistance genes. The homologous sequences of resistance genes were successfully obtained from Amorphophallus konjac, which will be helpful for cloning, functional analysis and location of disease resistance genes in in A. konjac.
引文
An H.S.,and Yang K.Q.,2014,Sequence analysis of NBS-Type RGAs and their relationship with anthracnose resistance in walnut,Zhongguo Nongye Kexue(Scientia Agriculture Sinica),47(2):344-356(安海山,杨克强,2014,核桃NBS类抗病基因类似物的序列特征及其与炭疽病的抗性,中国农业科学,47(2):344-356)
Ben dahmane A.,Kanyuka K.,and Baulcombe D.C.,1999,The Rx gene from potato controls separate virus resistance and cell death responses,Plant Cell,11(5):781-792
Brugmans B.,Wouters D.,van Os H.,Hutten R.,van der Linden G.,Visser R.G.,van Eck H.J.,and van der Vossen E.A.,2008,Genetic mapping and transcription analysis of resistance gene loci in potato using NBS profiling,Theor.Appl.Genet.,117(8):1379-1388
Chai X.L.,Zhou Y.,Lin Y.J.,Zhou Y.,Ruan L.F.,Zou Y.L.,Yu Z.N.,and Sun M.,2007,Regeneration of transgenic Amorphophallus konjac expressing synthesized acyl-homoderine lactonase(aiiA)gene from Bacillus thuringiensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),5(5):613-618(柴鑫莉,周盈,林拥军,周燚,阮丽芳,邹玉兰,喻子牛,孙明,2007,苏云金芽胞杆菌抗软腐病aiiA基因转花魔芋研究,分子植物育种,5(5):613-618)
Collins N.C.,Webb C.A.,Seah S.,Ellis J.G.,Helbert S.H.,and Pryor T.,1998,The isolation and mapping of disease resistance gene analogs in maize,Mol.Plant Microbe Interact.,11(10):968-978
Deng H.,and Tu Z.,2006,Study on the preparation of green packaging materials konjac glucomannan degradable film,Shipin Gongye Keji(Science and Technology of Food Industry),27(11):148-151(邓红,涂追,2006,绿色包装材料魔芋葡甘聚糖可降解膜的制备研究,食品工业科技,27(11):148-151)
Eitas T.K.,and Dangl J.L.,2010,NB-LRR proteins:pairs,pieces,perception,partners,and pathways,Plant Biol.,13(4):472-477
Feuillet C.,Schachermayr G.,and Keller B.,1997,Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat,Plant J.,11(1):45-52
Gong J.X.,Liu P.Y.,and Liu Q.J.,2004,Study on the stabilizing mechanism of konjac glucomannan in tea drink,Chaye Kexue(Journal of Tea Science),24(2):141-146(龚加顺,刘佩瑛,刘勤晋,2004,魔芋多糖在茶饮料中的稳定作用机理研究,茶叶科学,24(2):141-146)
Jacobs M.M.,Vosman B.,Vleeshouwers V.G.,Visser R.G.,Henken B.,and van der Berg R.G.,2010,A novel approach to locate phytophthora infestans resistance genes on the potato genetic map,Theor.Appl.Genet.,120(4):785-796
Jupe F.,Pritchard L.,Etherington G.J.,Mackenzie K.,Cock P.J.,Wright F.,Sharma S.K.,Bolser D.,Bryan G.J.,Jones J.D.,and Hein I.,2012,Identification and localization of the NB-LRR gene family within the potato genome,BMC Genomics,13(75):1-14
Kanazin V.,Marek L.F.,and Shoemaker R.C.,1996,Resistance gene analogs are conserved and clustered in soybean,Proc.Natl.Acad.Sci.USA,93(21):11746-11750
Lei Z.Z.,Ye J.L.,Cheng H.L.,Chen Y.,Wang H.X.,Xu K.J.,and Yue C.Y.,2013,Characterization of Soft-rot-resistant Amorphophallus konjac and preliminary analysis of the resistance mechanism,Zhiwu Xuebao(Chinese Bulletin of Botany),48(3):295-302(雷珍珍,叶晶龙,程海丽,陈云,望彗星,许克静,乐超银,2013,花魔芋抗软腐病植株的鉴定及其抗性机理的初步研究,植物学报,48(3):295-302)
Li Z.X.,and Zhang X.G.,2004,Genetic transformation of Amorphophallus mediated by gene-gun,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),23(6):659-662(李贞霞,张兴国,2004,基因枪法介导的魔芋遗传转化研究,华中农业大学学报,23(6):659-662)
Liu Y.,Yao Q.S.,Su J.B.,Hong Y.N.,and Lei X.T.,2013,Isolation and characterization of NBS type resistance gene analogs from mango(Mangifera indica Linn.),Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(3):571-576(刘洋,姚全胜,苏俊波,洪亚楠,雷新涛,2013,芒果NBS类抗病基因同源序列克隆与分析,植物遗传资源学报,14(3):571-576)
Liu Y.F.,Wan H.J.,Wei Y.P.,Li Z.M.,Ye Q.J.,Wang R.Q.,Ru an M.Y.,Yao Z.P.,Zhou G.Z.,and Yang Y.J.,2014,Genomewide analysis of NBS-LRR resistance genes in tomato,Henongxue Bao(Journal of Nuclear Agricultural Sciences),28(5):790-799(刘云飞,万红建,韦艳萍,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,杨悦俭,2014,番茄NBS-LRR抗病基因家族全基因组分析,核农学报,28(5):790-799)
Martin G.B.,Brommonschenkel S.H.,Chunwongse J.,Frary A.,Ganal M.W.,Spivey R.,Wu T.,Earle E.D.,and Tanksley S.D.,1993,Map-based cloning of a protein kinase gene conferring disease resistance in tomato,Science,262(5138):1432-1436
Plocik A.,Layden J.,and Kesseli R.,2004,Comparative analysis of NBS domain sequences of NBS-LRR disease resistance genes from sunflower,lettuce and chicory,Mol.Phylogenet Evol.,31(1):153-163
Song W.Y.,Wang G.L.,Chen L.L.,Kim H.S.,Pi L.Y.,Holsten T.,Gardner J.,Wang B.,Zhai W.X.,Fauquet C.,and Ronald P.,1995,A receptor kinase-like protein encoded by the rice disease resistance gene,Xa21,Science,270(5243):1804-1806
Swiderski M.R.,and Innes R.W.,2001,The Arabidopsis PBS1resistance gene encodes a member of a novel protein kinase subfamily,Plant J.,26(1):101-112
Wang C.T.,Cao G.Q.,Liang C.M.,Xie B.X.,and Yan F.X.,2014,Bioinformatics analysis of the NBS resistance gene analogs in cucumber,Beifang Yuanyi(Northern Horticulture),(14):101-104(王昶童,曹刚强,梁从敏,谢冰心,闫飞翔,2014,黄瓜NBS类抗病基因类似物的生物信息学分析,北方园艺,(14):101-104)
Zhang X.J.,Liu M.X.,Zhang Y.X.,Wang J.Z.,and Zhao H.,2017,Cloning of disease resistance gene analogs and research progress of its application in crops,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(8):3138-3144(张晓娟,刘梦昕,张云霄,王佳州,赵辉,2017,抗病基因同源序列的克隆及其在作物中的应用研究进展,基因组学与应用生物学,36(8):3138-3144)
Zhang Y.,Li F.,Liu C.H.,Fan X.C.,Sun H.S.,Jiang J.F.,and Zhang G.H.,2013,Isolation and identification of NBS-LRRresistance gene analogs sequences from Vitis davidii,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),46(4):780-789(张颖,李峰,刘崇怀,樊秀彩,孙海生,姜建福,张国海,2013,中国野生刺葡萄抗白腐病NBS-LRR类抗病基因同源序列的分离与鉴定,中国农业科学,46(4):780-789)
Zhou Y.,Sun M.,and Yu Z.N.,2004,Activity of Baccillus thuringiensis Aii A protein against soft rot disease for Amorphophallus konjac caused by Ervinia.carotovora pv.carovotova,Wuhan Daxue Xuebao(Wuhan University Journal(Natural Science Edition)),50(6):761-764(周燚,孙明,喻子牛,2004,苏云金芽胞杆菌Aii A蛋白对魔芋软腐病菌的抗病活性,武汉大学学报(理学版),50(6):761-764)
Ben dahmane A.,Kanyuka K.,and Baulcombe D.C.,1999,The Rx gene from potato controls separate virus resistance and cell death responses,Plant Cell,11(5):781-792
Brugmans B.,Wouters D.,van Os H.,Hutten R.,van der Linden G.,Visser R.G.,van Eck H.J.,and van der Vossen E.A.,2008,Genetic mapping and transcription analysis of resistance gene loci in potato using NBS profiling,Theor.Appl.Genet.,117(8):1379-1388
Chai X.L.,Zhou Y.,Lin Y.J.,Zhou Y.,Ruan L.F.,Zou Y.L.,Yu Z.N.,and Sun M.,2007,Regeneration of transgenic Amorphophallus konjac expressing synthesized acyl-homoderine lactonase(aiiA)gene from Bacillus thuringiensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),5(5):613-618(柴鑫莉,周盈,林拥军,周燚,阮丽芳,邹玉兰,喻子牛,孙明,2007,苏云金芽胞杆菌抗软腐病aiiA基因转花魔芋研究,分子植物育种,5(5):613-618)
Collins N.C.,Webb C.A.,Seah S.,Ellis J.G.,Helbert S.H.,and Pryor T.,1998,The isolation and mapping of disease resistance gene analogs in maize,Mol.Plant Microbe Interact.,11(10):968-978
Deng H.,and Tu Z.,2006,Study on the preparation of green packaging materials konjac glucomannan degradable film,Shipin Gongye Keji(Science and Technology of Food Industry),27(11):148-151(邓红,涂追,2006,绿色包装材料魔芋葡甘聚糖可降解膜的制备研究,食品工业科技,27(11):148-151)
Eitas T.K.,and Dangl J.L.,2010,NB-LRR proteins:pairs,pieces,perception,partners,and pathways,Plant Biol.,13(4):472-477
Feuillet C.,Schachermayr G.,and Keller B.,1997,Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat,Plant J.,11(1):45-52
Gong J.X.,Liu P.Y.,and Liu Q.J.,2004,Study on the stabilizing mechanism of konjac glucomannan in tea drink,Chaye Kexue(Journal of Tea Science),24(2):141-146(龚加顺,刘佩瑛,刘勤晋,2004,魔芋多糖在茶饮料中的稳定作用机理研究,茶叶科学,24(2):141-146)
Jacobs M.M.,Vosman B.,Vleeshouwers V.G.,Visser R.G.,Henken B.,and van der Berg R.G.,2010,A novel approach to locate phytophthora infestans resistance genes on the potato genetic map,Theor.Appl.Genet.,120(4):785-796
Jupe F.,Pritchard L.,Etherington G.J.,Mackenzie K.,Cock P.J.,Wright F.,Sharma S.K.,Bolser D.,Bryan G.J.,Jones J.D.,and Hein I.,2012,Identification and localization of the NB-LRR gene family within the potato genome,BMC Genomics,13(75):1-14
Kanazin V.,Marek L.F.,and Shoemaker R.C.,1996,Resistance gene analogs are conserved and clustered in soybean,Proc.Natl.Acad.Sci.USA,93(21):11746-11750
Lei Z.Z.,Ye J.L.,Cheng H.L.,Chen Y.,Wang H.X.,Xu K.J.,and Yue C.Y.,2013,Characterization of Soft-rot-resistant Amorphophallus konjac and preliminary analysis of the resistance mechanism,Zhiwu Xuebao(Chinese Bulletin of Botany),48(3):295-302(雷珍珍,叶晶龙,程海丽,陈云,望彗星,许克静,乐超银,2013,花魔芋抗软腐病植株的鉴定及其抗性机理的初步研究,植物学报,48(3):295-302)
Li Z.X.,and Zhang X.G.,2004,Genetic transformation of Amorphophallus mediated by gene-gun,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),23(6):659-662(李贞霞,张兴国,2004,基因枪法介导的魔芋遗传转化研究,华中农业大学学报,23(6):659-662)
Liu Y.,Yao Q.S.,Su J.B.,Hong Y.N.,and Lei X.T.,2013,Isolation and characterization of NBS type resistance gene analogs from mango(Mangifera indica Linn.),Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(3):571-576(刘洋,姚全胜,苏俊波,洪亚楠,雷新涛,2013,芒果NBS类抗病基因同源序列克隆与分析,植物遗传资源学报,14(3):571-576)
Liu Y.F.,Wan H.J.,Wei Y.P.,Li Z.M.,Ye Q.J.,Wang R.Q.,Ru an M.Y.,Yao Z.P.,Zhou G.Z.,and Yang Y.J.,2014,Genomewide analysis of NBS-LRR resistance genes in tomato,Henongxue Bao(Journal of Nuclear Agricultural Sciences),28(5):790-799(刘云飞,万红建,韦艳萍,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,杨悦俭,2014,番茄NBS-LRR抗病基因家族全基因组分析,核农学报,28(5):790-799)
Martin G.B.,Brommonschenkel S.H.,Chunwongse J.,Frary A.,Ganal M.W.,Spivey R.,Wu T.,Earle E.D.,and Tanksley S.D.,1993,Map-based cloning of a protein kinase gene conferring disease resistance in tomato,Science,262(5138):1432-1436
Plocik A.,Layden J.,and Kesseli R.,2004,Comparative analysis of NBS domain sequences of NBS-LRR disease resistance genes from sunflower,lettuce and chicory,Mol.Phylogenet Evol.,31(1):153-163
Song W.Y.,Wang G.L.,Chen L.L.,Kim H.S.,Pi L.Y.,Holsten T.,Gardner J.,Wang B.,Zhai W.X.,Fauquet C.,and Ronald P.,1995,A receptor kinase-like protein encoded by the rice disease resistance gene,Xa21,Science,270(5243):1804-1806
Swiderski M.R.,and Innes R.W.,2001,The Arabidopsis PBS1resistance gene encodes a member of a novel protein kinase subfamily,Plant J.,26(1):101-112
Wang C.T.,Cao G.Q.,Liang C.M.,Xie B.X.,and Yan F.X.,2014,Bioinformatics analysis of the NBS resistance gene analogs in cucumber,Beifang Yuanyi(Northern Horticulture),(14):101-104(王昶童,曹刚强,梁从敏,谢冰心,闫飞翔,2014,黄瓜NBS类抗病基因类似物的生物信息学分析,北方园艺,(14):101-104)
Zhang X.J.,Liu M.X.,Zhang Y.X.,Wang J.Z.,and Zhao H.,2017,Cloning of disease resistance gene analogs and research progress of its application in crops,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(8):3138-3144(张晓娟,刘梦昕,张云霄,王佳州,赵辉,2017,抗病基因同源序列的克隆及其在作物中的应用研究进展,基因组学与应用生物学,36(8):3138-3144)
Zhang Y.,Li F.,Liu C.H.,Fan X.C.,Sun H.S.,Jiang J.F.,and Zhang G.H.,2013,Isolation and identification of NBS-LRRresistance gene analogs sequences from Vitis davidii,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),46(4):780-789(张颖,李峰,刘崇怀,樊秀彩,孙海生,姜建福,张国海,2013,中国野生刺葡萄抗白腐病NBS-LRR类抗病基因同源序列的分离与鉴定,中国农业科学,46(4):780-789)
Zhou Y.,Sun M.,and Yu Z.N.,2004,Activity of Baccillus thuringiensis Aii A protein against soft rot disease for Amorphophallus konjac caused by Ervinia.carotovora pv.carovotova,Wuhan Daxue Xuebao(Wuhan University Journal(Natural Science Edition)),50(6):761-764(周燚,孙明,喻子牛,2004,苏云金芽胞杆菌Aii A蛋白对魔芋软腐病菌的抗病活性,武汉大学学报(理学版),50(6):761-764)