番茄NBS-LRR抗根结线虫基因同源序列的克隆与分析
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摘要
根据已知抗病基因的NBS-LRR保守结构域设计简并引物,从抗南方根结线虫病的番茄材料cDNA中克隆抗病基因同源序列(resistance gene analogs,RGAs),并通过qRT-PCR技术检测RGAs与南方根结线虫侵染的相关性。序列分析发现,2条序列具有NBS-LRR保守结构域,分别命名为F5-8和F5-20;保守区域氨基酸比对结果显示,F5-8(GenBank登录号为MG860907)和F5-20(GenBank登录号为MG860908)与已知抗病基因保守区域具有很高的相似性,其中F5-8与Mi-1相应区域氨基酸相似性为94%,F5-20与Hero基因相应区域氨基酸相似性为96%。qRT-PCR结果发现,F5-8和F5-20在接种南方根结线虫2龄幼虫24h后其表达量均发生了变化。初步推测F5-8和F5-20为南方根线线虫侵染相关基因同源序列。
Degenerated primers were designed according to the conserved regions of NBS-LRR domain from known plant resistance genes.Then these primers were used for amplification of the NBSLRR analogs from Lycopersicon esculentum.Sequence analysis showed that 5 fragments contained the resistance gene analogs,two of which have the ORF region and conserved NBS-LRR domain.F5-8(MG860907)shared 94%identity with the resistance genes of Mi-1,and F5-20(MG860908)shared96%identity with the resistance genes of Hero.The quantitative real-time PCR results showed that expression of F5-8 and F5-20 was changed after Meloidogyne incognita infection,suggesting that F5-8 and F5-20 might be related with nematode infection.
Degenerated primers were designed according to the conserved regions of NBS-LRR domain from known plant resistance genes.Then these primers were used for amplification of the NBSLRR analogs from Lycopersicon esculentum.Sequence analysis showed that 5 fragments contained the resistance gene analogs,two of which have the ORF region and conserved NBS-LRR domain.F5-8(MG860907)shared 94%identity with the resistance genes of Mi-1,and F5-20(MG860908)shared96%identity with the resistance genes of Hero.The quantitative real-time PCR results showed that expression of F5-8 and F5-20 was changed after Meloidogyne incognita infection,suggesting that F5-8 and F5-20 might be related with nematode infection.
引文
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[23]PAN Q,LIU Y S,BUDAI H,et al.Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons:tomato and Arabidopsis[J].Genetics,2000,155:309-322.
[24]ANDOLFO G,SANSEVERINO W,AVERSANO R,et al.Genome-wide identification and analysis of candidate genes for disease resistance in tomato[J].Mol Breed,2013,33:227-233.
[25]LIU J J,EKRAMODDOULLAH A K.Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine(Pinus monticola Dougl.ex.D.Don)[J].Mol Genet Genom,2003,270:432-441.
[26]MARONE D,RUSSO M A,LAIDO G,et al.Plant nucleotide binding site-leucine-rich repeat(NBS-LRR)genes:active guardians in host defense responses[J].Mol Sci,2013,14:7302-7326.
[27]NORDBERG H,CANTOR M,DUSHEYKO S,et al.The genome portal of the department of energy joint genome institute:2014updates[J].Nucleic Acids Res,2014,42:D26-D31.
[28]GOODSTEIN D M,SHU S,HOWSON R,et al.Phytozome:a comparative platform for green plant genomics[J].Nucleic Acids Res,2012,40:D1178-D1186.
[29]MONACO M K,STEIN J,NAITHANI S,et al.Gramene2013:comparative plant genomics resources[J].Nucleic Acids Res,2014,42:D1193-D1199.
[2]刘维志.植物病原线虫学[M].北京:中国农业出版社,2000:157-160.
[3]赵统敏,王银磊,杨玛丽,等.番茄根结线虫病抗性基因的研究进展[J].江苏农业学报,2012,28(6):1492-1497.
[4]MUHAMMAD A,FARRUKH A,AMJAD A,et al.Transgenic strategies for enhancement of nematode resistance in plants[J].Frontiers in plant science,2017,5:e00750.
[5]WILLIAMSON V M.Root-knot nematode resistance genes in tomato and their potential for future use[J].Phytopathol,1998,36:277-293.
[6]SOBCZAK M,AVROVA A,JUPOWICZ J,et al.Characterization of susceptibility and resistance responses to potatocyst nematode(Globodera spp.)infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene[J].Plant Microbe Interact,2005,18:158-168.
[7]CAI D G,KLEINE M,KIFLE S,et al.Positional cloning of a gene for nematode resistance in sugar beet[J].Science,1997,275:832-834.
[8]VANDER V,VANDER V,KANYUKA K,et al.Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens:a virus and a nematode[J].Plant J,2000,23:567-576.
[9]HAMMOND K K E,JONES J D.Plant disease resistance genes[J].Plant Mol Biol,1997,48:575-607.
[10]LEAH M,XIAOPING T,PATRICE K,et al.Plant NBS-LRRproteins:adaptable guards[J].Genome biology,2006,7(4):212-222.
[11]MANDEEP S,AMIT A,BUNYAMIN T.Genetic analysis of NBS-LRR gene family in chickpea and their expression profiles in response to ascochyta blight infection[J].Front Plant Sci,2017,8:838.
[12]ROBERTO L,OLGA P,MANUEL R,et al.Genome-wide identification and mapping of NBS encoding resistance genes in Solanum tuberosum group Phureja[J].PLoS One,2012,7(4):e34775.
[13]GIUSEPPE A,FLORIAN J,KAMIL W,et al.Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq[J].BMC Plant Biol,2014,14:120.
[14]MANOJ K,SEKHWALI,PINGCHUAN L,et al.Disease resistance gene analogs(RGAs)in plants[J].Int J Mol Sci,2015,16:19248-19290.
[15]赵洪海,武侠,迟晓红.不同番茄品种对根结线虫的感染性测定简报[J].莱阳农学院学报,2004,21(2):180-181.
[16]王新荣,郑静君,汪国平,等.华南地区主要番茄品种对南方根结线虫的抗性评价[J].植物保护,2009,35(1):124-126.
[17]WANG S S,WANG F,TAN S J,et al.Transcript profiles of maize embryo sacs and preliminary identification of genes involved in the embryo sac-pollen tube interaction[J].Plant Sci,2014,5:702-716.
[18]ZHOU T,WANG Y,CHEN J Q,et al.Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes[J].Mol Genet Genom,2004,271:402-415.
[19]FRITZ-LAYLIN L K,KRISHNAMURTHY N,TOR M,et al.Phylogenomic analysis of the receptor-like proteins of rice and Arabidopsis[J].Plant Physiol,2005,138:611-623.
[20]YU J,TEHRIM S,ZHANG F,et al.Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana[J].BMC Genom,2014,15:3-21.
[21]MEYERS B C.Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis[J].Plant cell,2003,15:809-834.
[22]KOHLER A,RINALDI C,DUPLESSIS S,et al.Genome-wide identification of NBS resistance genes in Populus trichocarpa[J].Plant Mol Biol,2008,66:619-636.
[23]PAN Q,LIU Y S,BUDAI H,et al.Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons:tomato and Arabidopsis[J].Genetics,2000,155:309-322.
[24]ANDOLFO G,SANSEVERINO W,AVERSANO R,et al.Genome-wide identification and analysis of candidate genes for disease resistance in tomato[J].Mol Breed,2013,33:227-233.
[25]LIU J J,EKRAMODDOULLAH A K.Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine(Pinus monticola Dougl.ex.D.Don)[J].Mol Genet Genom,2003,270:432-441.
[26]MARONE D,RUSSO M A,LAIDO G,et al.Plant nucleotide binding site-leucine-rich repeat(NBS-LRR)genes:active guardians in host defense responses[J].Mol Sci,2013,14:7302-7326.
[27]NORDBERG H,CANTOR M,DUSHEYKO S,et al.The genome portal of the department of energy joint genome institute:2014updates[J].Nucleic Acids Res,2014,42:D26-D31.
[28]GOODSTEIN D M,SHU S,HOWSON R,et al.Phytozome:a comparative platform for green plant genomics[J].Nucleic Acids Res,2012,40:D1178-D1186.
[29]MONACO M K,STEIN J,NAITHANI S,et al.Gramene2013:comparative plant genomics resources[J].Nucleic Acids Res,2014,42:D1193-D1199.