感染重组型大豆花叶病毒本氏烟的酵母双杂文库构建及评价
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摘要
为了建立以本氏烟为模式作物研究SMV侵染机制的平台,以可系统侵染本氏烟的重组型SMV分离物HBRS为毒源,构建感染HB-RS本氏烟的酵母双杂c DNA文库。利用SMART技术获得本氏烟的双链c DNA文库,通过双酶切手段将cDNA文库构建至pGADT7-SfiI载体上。检测结果显示,文库容量约为3.2×106cfu,插入cDNA片段长度为0.75~2.00 kb,平均长度大于1.00 kb,且多态性丰富,表明文库质量较好。该酵母双杂文库的构建为钓取与SMV互作的寄主因子和系统性研究SMV侵染机制奠定了物质基础。
In order to establish a platform for studying on the mechanism of SMV infection on the model crop of Nicotiana benthamiana. In this study,a recombinant SMV isolate HB-RS,which could systematically infect Nicotiana benthamiana,was used as virus source to construct a yeast two hybrid cDNA library of Nicotiana benthamiana with HB-RS infection. Double stranded cDNA library of the Nicotiana benthamiana were obtained through SMART technology and cDNA library were cloned into p GADT7-SfiI vector by double enzyme digestion method. Testing result showed that the cDNA library contained more than 3. 2 × 106 cfu and the cDNA fragments length inserted were from 0. 75- 2. 00 kb. The fragment was polymorphism and average length was more than 1. 00 kb indicating that the cDNA library was high quality. In all,cDNA library of the yeast two hybrids provided a material basis for screening host factor with SMV proteins and systematic studying of SMV infection mechanism.
In order to establish a platform for studying on the mechanism of SMV infection on the model crop of Nicotiana benthamiana. In this study,a recombinant SMV isolate HB-RS,which could systematically infect Nicotiana benthamiana,was used as virus source to construct a yeast two hybrid cDNA library of Nicotiana benthamiana with HB-RS infection. Double stranded cDNA library of the Nicotiana benthamiana were obtained through SMART technology and cDNA library were cloned into p GADT7-SfiI vector by double enzyme digestion method. Testing result showed that the cDNA library contained more than 3. 2 × 106 cfu and the cDNA fragments length inserted were from 0. 75- 2. 00 kb. The fragment was polymorphism and average length was more than 1. 00 kb indicating that the cDNA library was high quality. In all,cDNA library of the yeast two hybrids provided a material basis for screening host factor with SMV proteins and systematic studying of SMV infection mechanism.
引文
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[3]Jeong S C,Maroof M A S.Detection and genotyping of SNPs tightly linked to two disease resistance loci,Rsv1and Rsv3,of soybean[J].Plant Breeding,2004,123(4):305-310.
[4]Gore M A,Hayes A J,Jeong S C,et al.Mapping tightly linked genes controlling potyvirus infection at the Rsv1and Rpv1 region in soybean[J].Genome,2002,45(3):592-599.
[5]Hajimorad M R,Hill J H.Rsv1-mediated resistance against soybean mosaic virus-N is hypersensitive response-independent at inoculation site,but has the potential to initiate a hypersensitive response-like mechanism[J].Molecular plant-microbe Interactions,2001,14(5):587-598.
[6]Hayes A J,Ma G R,Buss G R,et al.Molecular marker mapping of Rsv4,agene conferring resistance to all known strains of soybean mosaic virus[J].Crop Sci,2000,40(4):1434-1437.
[7]Yu Y G,Saghai Maroof M A,Buss G R,et al.RFLP and micro satellite mapping of a gene for soybean mosaic virus resistance[J].Phytopathology,1994,84(1):60-64.
[8]Chen P,Buss G R,Roane C W,et al.Allelism among genes for resistance to soybean mosaic virus in strain-differential soybean cultivars[J].Crop Sci,1991,31(2):305-309.
[9]Kiihl R A,Hartwig E E.Inheritance of reaction to soybean mosaic virus in soybeans[J].Crop Sci,1979,19(1):372-379.
[10]Gao L,Zhai R,Zhong Y K,et al.Screening Isolates of Soybean mosaic virus for Infectivity in a Model Plant,Nicotiana benthamiana[J].Plant Dis,2015,99(4):442-446.
[11]Fields S,Song O.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245-246.
[12]Cui X Y,Wei T Y,Chowda-Reddy R V,et al.The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments[J].Virology,2010,397(1):56-63.
[13]Lin L,Shi Y,Luo Z,et al.Protein-protein interactions in two potyviruses using the yeast two-hybrid system[J].Virus Research,2009,142(1-2):36-40.
[14]Guo D,Rajamaki M L,Saarma M,et al.Towards a protein interaction map of potyviruses:protein interaction matrixes of two potyviruses based on the yeast two-hybrid system[J].The Journal of general Virology,2001,82(4):935-939.
[15]Arbatova J,Lehto K,Pehu E,et al.Localization of the P1protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells[J].The Journal of general Virology 1998,79(Pt10):2319-2323.
[16]崔晓艳.马铃薯Y病毒属编码的膜相关蛋白的功能研究[D].西安:西北农林科技大学,2010.
[17]杨永庆,侯文焕,边全楽,等.河北地区大豆花叶病毒株系的组成与分布[J].大豆科学,2014,33(1):87-90.
[18]林静,杨永庆,侯文焕,等.重组型大豆花叶病毒河北分离物序列特征及侵染力比较研究[J].作物学报,2015,41(11):1657-1662.