基于转录产物的野生稻NBS-LRR类和STK类序列的克隆与分析
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摘要
野生稻含有许多栽培稻中已消失的有利基因,特别是对各种生物逆境和非生物逆境的抗性基因。但由于野生稻的遗传背景复杂,利用传统的克隆方法存在着很多问题和不足。候选抗性基因克隆策略是最近十年发展出来的克隆抗病基因的新方法,利用抗病基因的保守序列从而获得抗病基因同源序列并最终克隆获得新基因。本研究首先从野生稻中提取总RNA,反转录以获得cDNA;根据NBS-LRR类和STK激酶类抗病基因的保守序列设计引物,PCR扩增野生稻cDNA得到了预计大小的目的条带,再经克隆和测序。共分别获得了11条NBS-LRR类序列和26条STK激酶类序列。经过分析将NBS-LRR类抗病基因同源序列分为3类,而STK激酶类抗病基因同源序列可分为13类。利用BLAST比对分析发现三类NBS-LRR类序列均与水稻抗病基因RPR1有很高的同源性,经电子定位分析将其定位到水稻11号染色体RPR1基因座位上,推测为RPR1基因家族在进化过程中复制与分化重组的结果。STK激酶类抗病基因同源序列除一类序列在水稻基因组上无法找到相似性极高的序列外,其余均可以找到90%以上的同源序列且有10类序列可电子定位于RAP-DB预测基因内。这些序列可为进一步的基因克隆提供探针以及发展成为分子标记,或者作为电子克隆的初始序列,为克隆野生稻的新抗性基因提供支持。
Wild rice contains a large number of favorable gene which have disappeared in the cultivated rice,especially the resistance genes to biotic stesses and abiotic stresses. Because the wild rice genetic background is complex, there are many problems and shortcomings in the use of traditional methods to clone them. The strategy of cloning by the candidates of Resistance gene is a new method to clone the resistance gene that developed in the last decade years, which using the conserved resistance gene sequence to gain resistance gene analogs and ultimately clone the new genes. We extracted total RNA from Wild rice at first, reverse transcript to obtain cDNA; then designed PCR primers by the NBS-LRR and STK kinase gene conserved domains and amplified cDNA of the wild rice.the expected size of the zone is obtained, and clone into the vector,then sequenced it,finally we obtained 11 NBS-LRR resistance gene sequence homology and 26 STK kinase resistance gene homologous. the NBS-LRR resistance gene homology sequences can be divided into three categories, and STK kinase resistance gene homologous sequences can be divided into 13 categories. using BLAST alignment to analyse the NBS-LRR sequences,we found all three types of NBS-LRR sequences have high homologous to the resistance gene RPR1 of rice, and mapped them preliminary into the region near the RPR1 in the chromosome 11 of the rice genome by the method of electronic mapping. we infered it is the result of replication and differentiation of the RPR1 gene family in the process of Evolutionary. in addition to a category of sequence could not find the high sequence similarity in the rice genome , STK kinase resistance gene analogs could be found more than 90% homologous to the rice genome, and 10 categories can be mapped to hypothetical gene. These sequences may provide as probes in the further gene cloning、develop to be molecular markers, or as an initial sequence of the silico cloning,which can provide support to the cloning of new resistance gene from wild rice.
Wild rice contains a large number of favorable gene which have disappeared in the cultivated rice,especially the resistance genes to biotic stesses and abiotic stresses. Because the wild rice genetic background is complex, there are many problems and shortcomings in the use of traditional methods to clone them. The strategy of cloning by the candidates of Resistance gene is a new method to clone the resistance gene that developed in the last decade years, which using the conserved resistance gene sequence to gain resistance gene analogs and ultimately clone the new genes. We extracted total RNA from Wild rice at first, reverse transcript to obtain cDNA; then designed PCR primers by the NBS-LRR and STK kinase gene conserved domains and amplified cDNA of the wild rice.the expected size of the zone is obtained, and clone into the vector,then sequenced it,finally we obtained 11 NBS-LRR resistance gene sequence homology and 26 STK kinase resistance gene homologous. the NBS-LRR resistance gene homology sequences can be divided into three categories, and STK kinase resistance gene homologous sequences can be divided into 13 categories. using BLAST alignment to analyse the NBS-LRR sequences,we found all three types of NBS-LRR sequences have high homologous to the resistance gene RPR1 of rice, and mapped them preliminary into the region near the RPR1 in the chromosome 11 of the rice genome by the method of electronic mapping. we infered it is the result of replication and differentiation of the RPR1 gene family in the process of Evolutionary. in addition to a category of sequence could not find the high sequence similarity in the rice genome , STK kinase resistance gene analogs could be found more than 90% homologous to the rice genome, and 10 categories can be mapped to hypothetical gene. These sequences may provide as probes in the further gene cloning、develop to be molecular markers, or as an initial sequence of the silico cloning,which can provide support to the cloning of new resistance gene from wild rice.
引文
[1]Katsuo K,Mizushima U.Studies on the cytoplasmic difference among rice varieties,Oryza sativa L.I.On the fertility of hybrids obtained reciprocally between cultivated and wild varieties.Japan Breed,1958,8:1-5
[2]Xiao Jinhua,Li Jiming,Silvana Grandillo et al.Identification of trait-improving quantitative trait loci alleles from a wild rice relative Oryza rufipogon.Genetics,1998,150:899-909.
[3]Martinez CP,Tohme J,Lopez J et al.Current status of rice improvement through use of wild rice species at CIAT.Agronomia Mesoamerlcana,1998,9(1):10-17
[4]Yong G.Yu,Glennr.Buss,M.A.Saghai Maroof.Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site.Proc Natl Acad Sci USA,1996,98:11751-11756
[5]谭明谱.水稻白叶枯病抗性基因Xa22(t)的克隆:[博士学位论文].武汉:华中农业大学,2004
[6]易图永,谢丙炎等.植物抗病基因同源序列及其在抗病基因克隆与定位中的应用.生物技术通报,2002,2:16-20
[7]丁国华,池春玉等.植物抗病基因同源序列研究进展.农业生物技术科学,2006.22(1):38-41
[8]Deng Z,Gmitter F G.Cloning and characterization of receptor kinase class disease resistance gene candidates in Citrus.Theor Appl genet,2003,108:53-61
[9]Vladimir Kanazin,Laura Fredrick Marek,Randy C.Shoemaker.Resistance gene analogs are conserved and clustered in soybean Proc.Natl Acad Sci,1996,93:11746-11750
[10]Catherine Feuillet,Gabriele Schachermayr,Beat Keller.Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat.The Plant Journal,1997,11(1):45-52
[11]李春来,张怀渝.植物抗病基因同源序列(RGA)研究进展.分子植物育种,2004,2(6):853-860
[12]SY Lee et al.Comparative analysis of superfamilies of NBS-encoding disease resistance gene analogs in cultivated and wild apple species.Mol Gen Genomics,2003,269:101-108
[13]Lili Maleki et al.Physical and Genetic Mapping of Wheat Kinase Analogs and NBS-LRR Resistance Gene.Analogs Crop Sci,2003,43:660-670
[14]Z.Deng.Cloning and characterization of NBS-LRR class resistance gene.Theor Appl Genet,2000,101:814-822
[15]易图永,张宝玺等.利用差异显示技术克隆辣椒抗疫病相关DNA序列的研究.中国蔬菜,2005,(12):11-13
[16]程志强等.水稻受白叶枯病菌诱导抗性相关基因片段的克隆.农业生物技术学报,2000,8(1):45-48
[17]YU Ling et al.Cloning,Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat.Acta Botanica Sinica,2002,44(12):1438-1444
[18]李子银,陈受宜.水稻抗病基因同源序列的克隆、定位及其表达.科学通报,1999,44(7):727-733
[19]Dario heister et al.Rapid reorganization of resistance gene homologues in cereal genomes.Proc Natl Acad Sci USA,1998,95:370-375
[20]ChaoYing He et al.Isolation and characterization of a full-length resistance gene homolog from soybean.Theor Appl Genet,2003,106:786-793
[21]WANG Bang Jun et al.cloning and analysis of a disease resistance gene homolog from soybean.Acta botanica sinica,2003,45(7):864-870
[22]汪旭升,吴为人,金谷雷等.水稻全基因组R基因鉴定及候选RGA标记开发.科学通报,2005,50(11):1085-1089
[23]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能.生物工程进展,1997,17(6):43-47
[24]左示敏等.植物数量抗病基因克隆及其抗性机理的研究进展.分子植物育种,2006,4(5):603-613
[25]Shaohong Qu et al.The Broad-Spectrum Blast Resistance Gene Pi9Encodes a Nucleotide-Binding Site-Leucine-Rich Repeat Protein and Is a Member of a Multigene Family in Rice.Genetics,2006,172:1901-1914
[26]David Mackey et al.RIN4 Interacts with Pseudomonas syringae Type ⅢEffector Molecules and Is Required for RPM1-Mediated Resistance in Arabidopsis.Cell,2002,108:743-754
[27]Yulin Jia et al.Direct interaction of resistance gene and avirulence gene products confers rice blast resistance,the EMBO Journal,2000,19(15):4004-4014
[28]Joanne E Luck et al.Regions outside of the Leucine-Rich Repeats of Flax Rust Resistance Proteins Play a Role in Specificity Determination.The Plant Cell,2000,12:1367-1377
[29]Marc J Orbach et al.A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta.The Plant Cell,2000,12:2019-2032
[30]ZK Li et al.Complex ganetic networks underlying the defensive system of rice(Oryza sativa L)to Xanthomonas oryzae pv.Oryzae.PNAS,2006,103(21):7994-7999
[31]韩德俊,曹莉,陈耀锋等.植物抗病基因与病原菌无毒基因互作的分子基础.遗传学报,2005,32(12):1319-1326
[32]王友红,张鹏飞,陈建群.植物抗病基因及其作用机理.植物学通报,2005,22(1):92-99
[33]李文凤,牛永春,吴立人.植物抗病基因克隆与功能研究进展.生命科学,2001,13(4):151-153
[34]庄军,刘志昕.植物抗病基因的进化.遗传,2004,26(6):962-968
[35]赵茂俊,马永毅,吴明君等.植物抗病左右机制与信号传导研究进展.草业与畜牧,2006,(9):1-7
[36]赵淑清,郭剑波.植物系统性获得抗性及其信号转导途径.中国农业科学,2003,36(7):781-78
[37]许丽,李明莹,林凤.植物抗病的分子基础与研究进展.杂粮作物,2006,26(6):428-432
[38]宋东辉,宋凤鸣,郑重.MAP激酶在植物信号传递网络中的功能.浙江大学学报,2004,30(2):119-126.
[39]郭泽建,李德葆.活性氧与植物抗病性.植物学报,2000,42(9):881-891
[40]尉万聪,李润植.一氧化氮在植物抗病反应中的信号作用.生物工程进展,2001,21(1):25-28
[41]陈英,黄敏仁,诸葛强等.植物抗病信号传导途径及其相互作用.南京林业大学学报,2002,26(3):85-90
[42]彭金英,黄勇平.植物防御反应的两种信号转导途径及其相互作用.植物生理与分子生物学学报,2005,3l(4):347-353
[43]王燕华,何水林.植物诱导抗病性的分子生物学研究进展.应用与环境生物学 报,2004,10(6):811-815
[44]夏启中,吴家和,张献龙.与植物超敏反应(HR)相关的细胞编程性死亡.华中农业大学学报,2005,24(1):97-103
[45]王文娟,张飞云.植物抗病分子机制研究进展.生物技术通报,2007,1:19-23
[46]Guo Liana Wang,David J,Mackill J et al.RFLP Mapping of Genes Conferring Complete and Partial Resistance to Blast in a Durably Resistant Rice Cultivar.Genetics,1994,136:1421-1434
[47]石海燕.植物抗病基因的克隆技术.生命的化学,2005,25(4):330-333
[48]王冬冬,朱延明,李勇等.电子克隆技术及其在植物基因工程中的应用.东北农业大学学报,2006,37(3):403-408
[49]李鑫,章涛.新基因的克隆策略和方法.海峡药学,2004,16(3):16-20
[50]钟平安,黄英金,陈大洲.东乡野生稻遗传资源的保护及其在育种上的利用.江西农业大学学报,2003,25(1):12-16
[51]梁昭全.电子定位的原理与方法.分子植物育种,2004,2(4):581-592
[52]Yulin Jia,Gregory T Bryan,Leonard Farrall et al.Natural Variation at the Pi-ta Rice Blast Resistance Locus.Phytopathology,2003,93(11):1452-1459
[53]Koji Sakamoto,Yuichi Tada,Yumi Yokozeki et al.Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats.Plant Molecular Biology,1999,40(5):847-855
[54]The Rice Chromosomes 11 and 12 Sequencing Consortia.The sequence of rice chromosomes 11 and 12,rich in disease resistance genes and recent gene duplications.BMC Biology,2005,3:20
[55]R.Todd Leister,Frederick M Ausubel,Fumiaki Katagiri.Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsisgenes RPS2 and RPM1.Proc.Natl.Acad.Sci.USA,1996,93:15497-15502
[56]Michael A Ayliffe,Evans S Lagudah.Molecular Genetics of Disease Resistance in Cereals.Annals of Botany,2004,94:765-773
[57]王志坤,秦智伟等.植物抗病基因同源序列及其研究进展.生物技术.2004,14(4):80-82
[58]Meyers B C et al.Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.Plant Cell,1998b,11:1833-1846
[59]钟平安,黄英金等.东乡野生稻遗传资源的保护及其在育种上的利用.江西农业大学学报,2003(1):12-16
[2]Xiao Jinhua,Li Jiming,Silvana Grandillo et al.Identification of trait-improving quantitative trait loci alleles from a wild rice relative Oryza rufipogon.Genetics,1998,150:899-909.
[3]Martinez CP,Tohme J,Lopez J et al.Current status of rice improvement through use of wild rice species at CIAT.Agronomia Mesoamerlcana,1998,9(1):10-17
[4]Yong G.Yu,Glennr.Buss,M.A.Saghai Maroof.Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site.Proc Natl Acad Sci USA,1996,98:11751-11756
[5]谭明谱.水稻白叶枯病抗性基因Xa22(t)的克隆:[博士学位论文].武汉:华中农业大学,2004
[6]易图永,谢丙炎等.植物抗病基因同源序列及其在抗病基因克隆与定位中的应用.生物技术通报,2002,2:16-20
[7]丁国华,池春玉等.植物抗病基因同源序列研究进展.农业生物技术科学,2006.22(1):38-41
[8]Deng Z,Gmitter F G.Cloning and characterization of receptor kinase class disease resistance gene candidates in Citrus.Theor Appl genet,2003,108:53-61
[9]Vladimir Kanazin,Laura Fredrick Marek,Randy C.Shoemaker.Resistance gene analogs are conserved and clustered in soybean Proc.Natl Acad Sci,1996,93:11746-11750
[10]Catherine Feuillet,Gabriele Schachermayr,Beat Keller.Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat.The Plant Journal,1997,11(1):45-52
[11]李春来,张怀渝.植物抗病基因同源序列(RGA)研究进展.分子植物育种,2004,2(6):853-860
[12]SY Lee et al.Comparative analysis of superfamilies of NBS-encoding disease resistance gene analogs in cultivated and wild apple species.Mol Gen Genomics,2003,269:101-108
[13]Lili Maleki et al.Physical and Genetic Mapping of Wheat Kinase Analogs and NBS-LRR Resistance Gene.Analogs Crop Sci,2003,43:660-670
[14]Z.Deng.Cloning and characterization of NBS-LRR class resistance gene.Theor Appl Genet,2000,101:814-822
[15]易图永,张宝玺等.利用差异显示技术克隆辣椒抗疫病相关DNA序列的研究.中国蔬菜,2005,(12):11-13
[16]程志强等.水稻受白叶枯病菌诱导抗性相关基因片段的克隆.农业生物技术学报,2000,8(1):45-48
[17]YU Ling et al.Cloning,Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat.Acta Botanica Sinica,2002,44(12):1438-1444
[18]李子银,陈受宜.水稻抗病基因同源序列的克隆、定位及其表达.科学通报,1999,44(7):727-733
[19]Dario heister et al.Rapid reorganization of resistance gene homologues in cereal genomes.Proc Natl Acad Sci USA,1998,95:370-375
[20]ChaoYing He et al.Isolation and characterization of a full-length resistance gene homolog from soybean.Theor Appl Genet,2003,106:786-793
[21]WANG Bang Jun et al.cloning and analysis of a disease resistance gene homolog from soybean.Acta botanica sinica,2003,45(7):864-870
[22]汪旭升,吴为人,金谷雷等.水稻全基因组R基因鉴定及候选RGA标记开发.科学通报,2005,50(11):1085-1089
[23]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能.生物工程进展,1997,17(6):43-47
[24]左示敏等.植物数量抗病基因克隆及其抗性机理的研究进展.分子植物育种,2006,4(5):603-613
[25]Shaohong Qu et al.The Broad-Spectrum Blast Resistance Gene Pi9Encodes a Nucleotide-Binding Site-Leucine-Rich Repeat Protein and Is a Member of a Multigene Family in Rice.Genetics,2006,172:1901-1914
[26]David Mackey et al.RIN4 Interacts with Pseudomonas syringae Type ⅢEffector Molecules and Is Required for RPM1-Mediated Resistance in Arabidopsis.Cell,2002,108:743-754
[27]Yulin Jia et al.Direct interaction of resistance gene and avirulence gene products confers rice blast resistance,the EMBO Journal,2000,19(15):4004-4014
[28]Joanne E Luck et al.Regions outside of the Leucine-Rich Repeats of Flax Rust Resistance Proteins Play a Role in Specificity Determination.The Plant Cell,2000,12:1367-1377
[29]Marc J Orbach et al.A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta.The Plant Cell,2000,12:2019-2032
[30]ZK Li et al.Complex ganetic networks underlying the defensive system of rice(Oryza sativa L)to Xanthomonas oryzae pv.Oryzae.PNAS,2006,103(21):7994-7999
[31]韩德俊,曹莉,陈耀锋等.植物抗病基因与病原菌无毒基因互作的分子基础.遗传学报,2005,32(12):1319-1326
[32]王友红,张鹏飞,陈建群.植物抗病基因及其作用机理.植物学通报,2005,22(1):92-99
[33]李文凤,牛永春,吴立人.植物抗病基因克隆与功能研究进展.生命科学,2001,13(4):151-153
[34]庄军,刘志昕.植物抗病基因的进化.遗传,2004,26(6):962-968
[35]赵茂俊,马永毅,吴明君等.植物抗病左右机制与信号传导研究进展.草业与畜牧,2006,(9):1-7
[36]赵淑清,郭剑波.植物系统性获得抗性及其信号转导途径.中国农业科学,2003,36(7):781-78
[37]许丽,李明莹,林凤.植物抗病的分子基础与研究进展.杂粮作物,2006,26(6):428-432
[38]宋东辉,宋凤鸣,郑重.MAP激酶在植物信号传递网络中的功能.浙江大学学报,2004,30(2):119-126.
[39]郭泽建,李德葆.活性氧与植物抗病性.植物学报,2000,42(9):881-891
[40]尉万聪,李润植.一氧化氮在植物抗病反应中的信号作用.生物工程进展,2001,21(1):25-28
[41]陈英,黄敏仁,诸葛强等.植物抗病信号传导途径及其相互作用.南京林业大学学报,2002,26(3):85-90
[42]彭金英,黄勇平.植物防御反应的两种信号转导途径及其相互作用.植物生理与分子生物学学报,2005,3l(4):347-353
[43]王燕华,何水林.植物诱导抗病性的分子生物学研究进展.应用与环境生物学 报,2004,10(6):811-815
[44]夏启中,吴家和,张献龙.与植物超敏反应(HR)相关的细胞编程性死亡.华中农业大学学报,2005,24(1):97-103
[45]王文娟,张飞云.植物抗病分子机制研究进展.生物技术通报,2007,1:19-23
[46]Guo Liana Wang,David J,Mackill J et al.RFLP Mapping of Genes Conferring Complete and Partial Resistance to Blast in a Durably Resistant Rice Cultivar.Genetics,1994,136:1421-1434
[47]石海燕.植物抗病基因的克隆技术.生命的化学,2005,25(4):330-333
[48]王冬冬,朱延明,李勇等.电子克隆技术及其在植物基因工程中的应用.东北农业大学学报,2006,37(3):403-408
[49]李鑫,章涛.新基因的克隆策略和方法.海峡药学,2004,16(3):16-20
[50]钟平安,黄英金,陈大洲.东乡野生稻遗传资源的保护及其在育种上的利用.江西农业大学学报,2003,25(1):12-16
[51]梁昭全.电子定位的原理与方法.分子植物育种,2004,2(4):581-592
[52]Yulin Jia,Gregory T Bryan,Leonard Farrall et al.Natural Variation at the Pi-ta Rice Blast Resistance Locus.Phytopathology,2003,93(11):1452-1459
[53]Koji Sakamoto,Yuichi Tada,Yumi Yokozeki et al.Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats.Plant Molecular Biology,1999,40(5):847-855
[54]The Rice Chromosomes 11 and 12 Sequencing Consortia.The sequence of rice chromosomes 11 and 12,rich in disease resistance genes and recent gene duplications.BMC Biology,2005,3:20
[55]R.Todd Leister,Frederick M Ausubel,Fumiaki Katagiri.Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsisgenes RPS2 and RPM1.Proc.Natl.Acad.Sci.USA,1996,93:15497-15502
[56]Michael A Ayliffe,Evans S Lagudah.Molecular Genetics of Disease Resistance in Cereals.Annals of Botany,2004,94:765-773
[57]王志坤,秦智伟等.植物抗病基因同源序列及其研究进展.生物技术.2004,14(4):80-82
[58]Meyers B C et al.Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.Plant Cell,1998b,11:1833-1846
[59]钟平安,黄英金等.东乡野生稻遗传资源的保护及其在育种上的利用.江西农业大学学报,2003(1):12-16