水稻条纹病毒四川省会东县分离物p3基因序列分析及其蛋白抗体制备
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摘要
为探讨水稻条纹病毒(Rice stripe virus,RSV)四川省会东县分离物p3基因的遗传变异并获得其蛋白抗体,采用斑点酶联免疫吸附测定(dot enzyme-linked immunosorbent assay,Dot-ELISA)检测四川省会东县水稻植株;利用反转录PCR(reverse transcription-PCR,RT-PCR)技术克隆了RSV p3基因,并利用MEGA 5.0软件构建其发育进化树;通过原核表达方法获得纯化蛋白,免疫家兔获得其多克隆抗体,并用Western blot方法检测其效价。结果显示,从四川省会东县水稻种植区共采集13份水稻植株病样,经Dot-ELISA检测有3份样品呈阳性;通过RT-PCR技术从阳性样品中扩增获得RSV四川省会东县分离物Huid04(GenBank登录号KX611339)p3基因全长,长度为636 nt;系统进化分析显示该分离物与KunM08(GenBank登录号为JQ927423)和DaL08(GenBank登录号为JQ927420)2个云南分离物的核苷酸相似性最高,分别为96.86%和97.01%;将重组质粒pET-32a/p3转化至大肠杆菌Escherichia coli表达菌株BL21pLysS,经异丙基硫代-β-半乳糖苷诱导后,获得了分子量为40 kD的融合p3蛋白;并将纯化蛋白免疫家兔后获取抗血清,经ELISA及Western blot检测验证其为RSV p3蛋白的多克隆抗体,且效价为1∶2 000。以转RSV p3基因本氏烟为材料,利用Western blot技术证明了该抗体可用于检测RSV p3蛋白。
In order to clarify the genetic variation in p3 gene of Rice stripe virus(RSV) isolate from Huidong County, Sichuan Province and obtain its antibody, the dot-enzyme linked immunosorbent assay(Dot-ELISA) was used to detect virus-infected rice samples from Huidong County; the fragment of p3 gene was obtained with reverse transcription-PCR(RT-PCR), and a phylogenetic tree was constructed by MEGA 5.0. The p3 fusion protein was obtained and purified with prokaryotic expression system,and the antibody was made in rabbits and detected by ELISA and Western blot. The results showed that three of the 13 samples of diseased rice plants collected from Huidong County were positive and infect-ed with RSV. Furthermore, the RSV p3 gene named Huid04(GenBank accession number KX611339)was isolated from these positive samples using RT-PCR, and it contained 636 nucleotides(nt). The analysis of phylogenetic relationships showed that there was a high similarity between the nucleotide sequence of Huid04 isolate with those of KunM08(GenBank accession number JQ927423) and DaL08(GenBank accession number JQ927420) two other isolates from Yunnan Province(96.86% and 97.01%). The recombinant plasmid pET-32 a/p3 was transformed into Escherichia coli BL21 pLysS.Then, the fusion protein with a molecular weight of 40 kD was induced with isopropyl β-D-thiogalactoside(IPTG) and purified, which was used as an antigen to raise the antibody in rabbits. ELISA and Western blot analysis showed that the anti-RSV p3 polyclonal antibody was successfully prepared, and its titer was 1∶2 000. In addition, Western blot detection showed that this antibody could specifically recognize the p3 protein in transgenic plants.
In order to clarify the genetic variation in p3 gene of Rice stripe virus(RSV) isolate from Huidong County, Sichuan Province and obtain its antibody, the dot-enzyme linked immunosorbent assay(Dot-ELISA) was used to detect virus-infected rice samples from Huidong County; the fragment of p3 gene was obtained with reverse transcription-PCR(RT-PCR), and a phylogenetic tree was constructed by MEGA 5.0. The p3 fusion protein was obtained and purified with prokaryotic expression system,and the antibody was made in rabbits and detected by ELISA and Western blot. The results showed that three of the 13 samples of diseased rice plants collected from Huidong County were positive and infect-ed with RSV. Furthermore, the RSV p3 gene named Huid04(GenBank accession number KX611339)was isolated from these positive samples using RT-PCR, and it contained 636 nucleotides(nt). The analysis of phylogenetic relationships showed that there was a high similarity between the nucleotide sequence of Huid04 isolate with those of KunM08(GenBank accession number JQ927423) and DaL08(GenBank accession number JQ927420) two other isolates from Yunnan Province(96.86% and 97.01%). The recombinant plasmid pET-32 a/p3 was transformed into Escherichia coli BL21 pLysS.Then, the fusion protein with a molecular weight of 40 kD was induced with isopropyl β-D-thiogalactoside(IPTG) and purified, which was used as an antigen to raise the antibody in rabbits. ELISA and Western blot analysis showed that the anti-RSV p3 polyclonal antibody was successfully prepared, and its titer was 1∶2 000. In addition, Western blot detection showed that this antibody could specifically recognize the p3 protein in transgenic plants.
引文
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Chen SH,Cheng ZB,Ma XW,Zhou YJ,Fan YJ.2013.The difference analysis on the prevalence of rice stripe disease in Jiangsu and Yunnan provinces.Jiangsu Agricultural Sciences,41(3):88-90(in Chinese)[陈思宏,程兆榜,马学文,周益军,范永坚.2013.江苏和云南地区水稻条纹叶枯病流行差异分析.江苏农业科学,41(3):88-90]
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Cheng ZB,Yang RM,Zhou YJ,Diao CY,Xiong RY.2002.New occurrence pattern of rice stripe disease in Jiangsu Province.Jiangsu Agricultural Sciences,(1):39-41(in Chinese)[程兆榜,杨荣明,周益军,刁春友,熊如意.2002.江苏稻区水稻条纹叶枯病发生新规律.江苏农业科学,(1):39-41]
Falk BW,Tsai JH.1998.Biology and molecular biology of viruses in the genus Tenuivirus.Annual Review of Phytopathology,36:139-163
Guo SY,Tong Y,Huang Y,Luo XF,Qing L.2015.Preliminary identification and analyses of viruses causing pepper virus disease in Chongqing,China.Acta Horticulturae Sinica,42(2):263-270(in Chinese)[郭思瑶,童艳,黄娅,罗信福,青玲.2015.重庆辣椒病毒病病原初步鉴定和分析.园艺学报,42(2):263-270]
Huang LZ,Li ZF,Wu JX,Xu Y,Yang XL,Fan LJ,Fang RX,Zhou XP.2015.Analysis of genetic variation and diversity of Rice stripe virus populations through high-throughput sequencing.Frontiers in Plant Science,6:176
Jiang SS,Xie L,Wu B,Xin XQ,Chen JP,Zhao JH.2017.Identification and genetic diversity analysis on sweet potato viruses in Shandong Province.Journal of Plant Protection,44(1):93-102(in Chinese)[姜珊珊,谢礼,吴斌,辛相启,陈剑平,赵玖华.2017.山东甘薯主要病毒的鉴定及多样性分析.植物保护学报,44(1):93-102]
Jiang T,He ZL,Zhang XX,Xia WW,Li XY.2017.Prokaryotic expression of protein encoded by gene S7-1 and cloning of genome S7of Anhui isolate of Southern rice black-streaked dwarf virus.Journal of Plant Protection,44(3):467-472(in Chinese)[江彤,何志龙,张享享,夏伟伟,李祥宇.2017.南方水稻黑条矮缩病毒安徽分离物S7片段的克隆及其S7-1基因编码蛋白的原核表达.植物保护学报,44(3):467-472]
Lian S,Jonson MG,Cho WK,Choi HS,Je YH,Kim KH.2011.Generation of antibodies against Rice stripe virus proteins based on recombinant proteins and synthetic polypeptides.Plant Pathology Journal,27(1):37-43
Lin L,Zheng HY,Chen J,Chen JP.2004.The prokaryotic expression of the coat protein gene of Garlic virus E and the antiserum preparation.Acta Microbiologica Sinica,44(4):533-535(in Chinese)[林林,郑红英,陈炯,陈剑平.2004.大蒜E病毒外壳蛋白基因的原核表达及抗血清制备.微生物学报,44(4):533-535]
Ma J,Song YZ,Li KD,Zhu CX,Wen FJ.2008.The analysis of molecular variability of Rice stripe virus isolate(RSV-SD-JN2)in Jining,Shandong.Journal of Plant Protection,35(5):415-420(in Chinese)[马进,宋云枝,李开东,朱常香,温孚江.2008.水稻条纹病毒山东济宁分离物RSV-SD-JN2分子变异分析.植物保护学报,35(5):415-420]
Mallory AC,Reinhart BJ,Bartel D,Vance VB,Bowman LH.2002.Aviral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco.Proceedings of the National Academy of Sciences of the United States of America,99(23):15228-15233
Meng Y,Xin X,Yang J,Zhang HM,Wang JM,Chen JP.2013.Preparation and application of antiserum against P12 protein of Rice gall dwarf virus.Journal of Plant Protection,40(5):413-417(in Chinese)[孟媛,辛星,羊健,张恒木,王建明,陈剑平.2013.水稻瘤矮病毒P12蛋白抗血清制备及其应用.植物保护学报,40(5):413-417]
Ren CM,Cheng ZB,Yang L,Miao Q,Zhou YJ.2016.Molecular variality of NS2 and NS3 genes from Rice stripe virus in Laodelphax striatellus.Southwest China Journal of Agricultural Sciences,29(8):1854-1863(in Chinese)[任春梅,程兆榜,杨柳,缪倩,周益军.2016.灰飞虱携带的水稻条纹病毒NS2和NS3基因的分子变异.西南农业学报,29(8):1854-1863]
Ruan YL,Jin DD,Xu RY.1984.The hosts of Rice stripe virus.Plant Protection,(3):22-23(in Chinese)[阮义理,金登迪,许如银.1984.水稻条纹叶枯病毒的寄主植物.植物保护,(3):22-23]
Shen M,Xu Y,Jia R,Zhou XP,Ye KQ.2010.Size-independent and noncooperative recognition of dsRNA by the Rice stripe virus RNA silencing suppressor NS3.Journal of Molecular Biology,404(4):665-679
Song YZ,Jiang YY,Bi LJ,Wen FJ,Zhu CX.2014.Cloning and prokaryotic expression of coat protein gene of Rice black-streaked dwarf virus and preparation of its polyclonal antibody.Journal of Plant Protection,41(1):35-40(in Chinese)[宋云枝,姜雅元,毕吕杰,温孚江,朱常香.2014.水稻黑条矮缩病毒外壳蛋白基因的克隆、原核表达及抗体制备.植物保护学报,41(1):35-40]
Toriyama S,Takahashi M,Sano Y,Shimizu T,Ishihama A.1994.Nucleotide sequence of RNA 1,the largest genomic segment of Rice stripe virus,the prototype of the tenuiviruses.Journal of Genernal Virology,75(Pt12):3569-3579
Wei TY.2003.Genomic structure and molecular population genetics of Rice stripe virus.Ph.D Thesis.Fuzhou:Fujian Agriculture and Forestry University(in Chinese)[魏太云.2003.水稻条纹病毒的基因组结构及其分子群体遗传.博士学位论文.福州:福建农林大学]
Wei TY,Yang JG,Liao FR,Gao FL,Lu LM,Zhang XT,Li F,Wu ZJ,Lin QY,Xie LH,et al.2009.Genetic diversity and population structure of Rice stripe virus in China.Journal of Genernal Virology,90(Pt4):1025-1034
Wu GT,Lu YW,Zheng HY,Lin L,Yan F,Chen JP.2013.Transcription of ORFs on RNA2 and RNA4 of Rice stripe virus terminate at an AUCCGGAU sequence that is conserved in the genus Tenuivirus.Virus Research,175(1):71-77
Wu GT,Wang JY,Yang Y,Dong B,Wang YL,Sun GC,Yan CQ,Yan F,Chen JP.2014.Transgenic rice expressing Rice stripe virus NS3protein,a suppressor of RNA silencing,shows resistance to rice blast disease.Virus Genes,48(3):566-569
Xiao CL,Zheng HY,Wu XH,Li GJ,Yan F,Chen JP.2016.The complete genomic sequence analysis of three isolates of Cucumber green mottle mosaic virus in Zhejiang and application of prepared antiserum.Journal of Plant Protection,43(4):529-536(in Chinese)[肖彩丽,郑红英,吴晓花,李国景,燕飞,陈剑平.2016.黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查.植物保护学报,43(4):529-536]
Xiong RY,Wu JX,Zhou YJ,Zhou XP.2009.Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus.Virology,387(1):29-40
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