稻瘟病新抗性基因Pi39候选基因Pi39E长片段载体的构建
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摘要
稻瘟病新抗性基因Pi39来源于粳稻品种Q15,定位于水稻第12号染色体,前期研究发现候选基因区域预测有12个抗病相关候选基因,其中候选基因Pi39E最大,克隆的难度较大。为了构建Pi39E长片段载体,本实验通过加大反应底物的浓度,目的片段的质量浓度为124 ng/μL,质粒载体的质量浓度为158 ng/μL,目的片断与质粒DNA按1.15∶1的摩尔浓度比进行连接反应,在9μL的总体系中加入1μL的PEG4000,同时采用电击大肠杆菌DH5α转化方法优化实验方案,成功构建该载体。本结果对新抗性基因Pi39精细定位及其功能的研究具有重要意义。
The new rice blast resistance gene Pi39 originates from indica rice cultivar Q15 and is located on chromosome 12 of rice. Previous studies have found that there are 12 candidate genes related to disease resistance in candidate gene regions, among which Pi39 E is the largest candidate gene, and the cloning is more difficult. To construct the Pi39 E long fragment vector, the concentration of the reaction substrate was increased in this experiment. The target fragment mass concentration was 124 ng/μL, the plasmid vector mass concentration was 158 ng/μL,and the ligation reaction between the target fragment and plasmid DNA was carried out at the molar concentration ratio of 1.15∶1. A total of 9 μL of the total system was added with 1 μL of PEG4000. At the same time, E. coli DH5α transformation method was used to optimize the experimental protocol and the vector was successfully constructed. This study was of great significance to the fine mapping and function of the new resistance gene Pi39.
The new rice blast resistance gene Pi39 originates from indica rice cultivar Q15 and is located on chromosome 12 of rice. Previous studies have found that there are 12 candidate genes related to disease resistance in candidate gene regions, among which Pi39 E is the largest candidate gene, and the cloning is more difficult. To construct the Pi39 E long fragment vector, the concentration of the reaction substrate was increased in this experiment. The target fragment mass concentration was 124 ng/μL, the plasmid vector mass concentration was 158 ng/μL,and the ligation reaction between the target fragment and plasmid DNA was carried out at the molar concentration ratio of 1.15∶1. A total of 9 μL of the total system was added with 1 μL of PEG4000. At the same time, E. coli DH5α transformation method was used to optimize the experimental protocol and the vector was successfully constructed. This study was of great significance to the fine mapping and function of the new resistance gene Pi39.
引文
Dang H.J.,Liu Q.,Xu W.R.,Zhang C.L.,Wang S.,Tu L.D.,and Niu X.L.,2017,Construction of a fast and high efficient cloning system based on the CcdB lethal gene and SmaⅠrestriction enzyme cutting site,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(7):2874-2879(党会杰,刘秦,许文茸,张传玲,王森,涂鲁迪,牛晓磊,2017,一种快速高效的基于CcdB致死基因和SmaⅠ酶切位点的克隆体系构建,基因组学与应用生物学,36(7):2874-2879)
Deng X.X.,Liu Z.L.,Wang C.T.,and Liu X.Q.,2017,Isolation and cloning of OsAAA1 gene promoter in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):2907-2911(邓霄霄,刘早利,王春台,刘新琼,2017,水稻OsAAA1基因启动子的分离与克隆,分子植物育种,15(8):2907-2911)
Jiang W.J.,2016,CATCH enables targeted isolation and cloning of large genomic DNA fragments,Dissertation for Ph.D.,Tsinghua University,Supervisor:Zhu T.,pp.1(姜文君,2016,应用CATCH技术靶向分离及克隆基因组大片段DNA,博士学位论文,清华大学,导师:朱听,pp.1)
Liu X.Q.,Yang Q.Z.,Lin F.,Hua L.X.,Wang C.T.,Wang L.,and Pan Q.H.,2007,Identification and fine mapping of Pi39(t),a major gene conferring the broad-spectrum resistance to Magnaporthe oryzae,Molecular Genetics and Genomics,4(278):403-410
Liu Z.L.,2017,Screening and functional preliminary research of candidate gene of the novel blast resistance gene Pi39 in rice,Thesis for M.S.,South-Central University for Nationalities,Supervisor:Liu X.Q.,pp.9-11(刘早利,2017,水稻稻瘟病新抗性基因Pi39候选基因的筛选及功能初步研究,硕士学位论文,中南民族大学,导师:刘新琼,pp.9-11)
Puchta H.,Dujon B.,and Hohn B.,1996,Two different but related mechanisms are used in plants for the repair of genomic double strand breaks by homologous recombination,Proc.Natl.Acad.Sci.USA,93(10):5055-5060
Skamnioti P.,and Gurr S.J.,2009,Against the grain:safeguarding rice from rice blast disease,Trends Biotechnol.,27(3):141-150
Sun G.C.,Du X.F.,Tao R.X.,Mao X.Q.,and Sun S.R.,2000,Advances in research on prevention and control of rice blast and research ideas in the early 21stcentury,Zhiwu Baohu(Plant Protection),26(1):33-35(孙国昌,杜新法,陶荣祥,毛雪琴,孙漱沅,2000,水稻稻瘟病防治研究进展和21世纪初研究设想,植物保护,26(1):33-35)
Wang B.H.,Ebbole D.J.,and Wang Z.H.,2017,The arms race between Magnaporthe oryzae and rice:diversity and interaction of Avr and R genes,J.Integr.Agric.,16:2746-2760
Zhang Y.,Yang J.Y.,Shan Z.L.,Chen S.,and Qiao W.,2012,Substitution mapping of QTLs for blast resistance with SSSLs in rice(Oryza sativa L.),Euphytica,184(1):141-150
Zhu X.L.,Tao Y.Z.,and Zhou B.,2015,Distribution pattern of multiple alleles of rice blast resistance genes and its application in gene cloning,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),27(10):1789-1796(朱小丽,陶跃之,周波,2015,水稻抗稻瘟病基因的复等位分布特征及其在基因挖掘中的应用,浙江农业学报,27(10):1789-1796)
Deng X.X.,Liu Z.L.,Wang C.T.,and Liu X.Q.,2017,Isolation and cloning of OsAAA1 gene promoter in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):2907-2911(邓霄霄,刘早利,王春台,刘新琼,2017,水稻OsAAA1基因启动子的分离与克隆,分子植物育种,15(8):2907-2911)
Jiang W.J.,2016,CATCH enables targeted isolation and cloning of large genomic DNA fragments,Dissertation for Ph.D.,Tsinghua University,Supervisor:Zhu T.,pp.1(姜文君,2016,应用CATCH技术靶向分离及克隆基因组大片段DNA,博士学位论文,清华大学,导师:朱听,pp.1)
Liu X.Q.,Yang Q.Z.,Lin F.,Hua L.X.,Wang C.T.,Wang L.,and Pan Q.H.,2007,Identification and fine mapping of Pi39(t),a major gene conferring the broad-spectrum resistance to Magnaporthe oryzae,Molecular Genetics and Genomics,4(278):403-410
Liu Z.L.,2017,Screening and functional preliminary research of candidate gene of the novel blast resistance gene Pi39 in rice,Thesis for M.S.,South-Central University for Nationalities,Supervisor:Liu X.Q.,pp.9-11(刘早利,2017,水稻稻瘟病新抗性基因Pi39候选基因的筛选及功能初步研究,硕士学位论文,中南民族大学,导师:刘新琼,pp.9-11)
Puchta H.,Dujon B.,and Hohn B.,1996,Two different but related mechanisms are used in plants for the repair of genomic double strand breaks by homologous recombination,Proc.Natl.Acad.Sci.USA,93(10):5055-5060
Skamnioti P.,and Gurr S.J.,2009,Against the grain:safeguarding rice from rice blast disease,Trends Biotechnol.,27(3):141-150
Sun G.C.,Du X.F.,Tao R.X.,Mao X.Q.,and Sun S.R.,2000,Advances in research on prevention and control of rice blast and research ideas in the early 21stcentury,Zhiwu Baohu(Plant Protection),26(1):33-35(孙国昌,杜新法,陶荣祥,毛雪琴,孙漱沅,2000,水稻稻瘟病防治研究进展和21世纪初研究设想,植物保护,26(1):33-35)
Wang B.H.,Ebbole D.J.,and Wang Z.H.,2017,The arms race between Magnaporthe oryzae and rice:diversity and interaction of Avr and R genes,J.Integr.Agric.,16:2746-2760
Zhang Y.,Yang J.Y.,Shan Z.L.,Chen S.,and Qiao W.,2012,Substitution mapping of QTLs for blast resistance with SSSLs in rice(Oryza sativa L.),Euphytica,184(1):141-150
Zhu X.L.,Tao Y.Z.,and Zhou B.,2015,Distribution pattern of multiple alleles of rice blast resistance genes and its application in gene cloning,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),27(10):1789-1796(朱小丽,陶跃之,周波,2015,水稻抗稻瘟病基因的复等位分布特征及其在基因挖掘中的应用,浙江农业学报,27(10):1789-1796)
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