南方水稻黑条矮缩病毒安徽分离物S5片段的克隆及S5-2基因的原核表达
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摘要
【目的】探讨南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)安徽分离物(SRBSDVAn Hui-HN2)的遗传特性,并获得原核表达的P5-2蛋白。【方法】RT-PCR扩增SRBSDV S5片段,克隆、测序并进行序列分析。将S5-2基因插入原核表达载体,重组载体转化大肠埃希菌并用IPTG诱导,Ni2+-NTA亲和柱纯化融合蛋白,SDS-PAGE分析P5-2蛋白的表达情况。【结果】SRBSDV-An Hui-HN2 S5片段全长3 167 bp,包含S5-2基因全长612 bp,编码204个氨基酸。序列比对结果显示,SRBSDV-An Hui-HN2 S5片段与其他SRBSDV分离物S5片段的序列相似性极高,达99.0%~99.7%,而与斐济病毒属(Fijivirus)其他成员S5片段的序列相似性较低,仅为38.0%~71.3%;构建的S5片段系统发育树表明SRBSDV和RBSDV聚成1个分支,其中6个SRBSDV分离物聚成1个亚分支。原核表达获得相对分子质量约为47 000的重组蛋白,Western blot分析显示,GST单抗能够与重组融合蛋白发生特异性反应。【结论】SRBSDV各分离物之间亲缘关系非常近,而与Fijivirus其他成员亲缘关系较远,原核表达获得的融合蛋白为靶标蛋白。
【Objective 】To study genetic characteristics of the isolates of Southern rice black-streaked dwarf virus(SRBSDV) from Anhui province,and to obtain P5-2 protein by prokaryotic expression.【Method】The S5 segment of SRBSDV was amplified by RT-PCR,and it was cloned,sequenced and analyzed.Gene S5-2 was inserted into prokaryotic expression vector.The recombinant vector was transformed into Escherichia coli and was induced by IPTG.The fusion protein was purified by Ni2 +-NTA affinity column.The expression of P5-2 protein was analyzed by SDS-PAGE.【Result】The S5 segment from Anhui isolate of SRBSDV(SRBSDV-An Hui-HN2) was 3 167 bp in full length and contained a 612 bp S5-2 gene encoding 204 amino acids.Sequence comparison showed that the S5 segment of SRBSDVAn Hui-HN2 shared high sequence similarity(99.0%-99.7%) with other SRBSDV isolates,while had relatively low sequence similarity(38.0%-71.3%) to other Fijivirus members.The phylogenetic tree based on S5 segment sequences showed that SRBSDV and RBSDV clustered into a branch,and six isolates of SRBSDV clustered into a sub-branch.The recombinant protein with approximately 47 000 relative molecular mass was obtained by prokaryotic expression.Western blot analysis revealed that GSTmonoclonal antibody could specifically bind to the fusion protein.【Conclusion】All isolates of SRBSDV are closely related,and they have relatively far relationship to other Fijivirus members.The fusion protein obtained by prokaryotic expression is the target protein.
【Objective 】To study genetic characteristics of the isolates of Southern rice black-streaked dwarf virus(SRBSDV) from Anhui province,and to obtain P5-2 protein by prokaryotic expression.【Method】The S5 segment of SRBSDV was amplified by RT-PCR,and it was cloned,sequenced and analyzed.Gene S5-2 was inserted into prokaryotic expression vector.The recombinant vector was transformed into Escherichia coli and was induced by IPTG.The fusion protein was purified by Ni2 +-NTA affinity column.The expression of P5-2 protein was analyzed by SDS-PAGE.【Result】The S5 segment from Anhui isolate of SRBSDV(SRBSDV-An Hui-HN2) was 3 167 bp in full length and contained a 612 bp S5-2 gene encoding 204 amino acids.Sequence comparison showed that the S5 segment of SRBSDVAn Hui-HN2 shared high sequence similarity(99.0%-99.7%) with other SRBSDV isolates,while had relatively low sequence similarity(38.0%-71.3%) to other Fijivirus members.The phylogenetic tree based on S5 segment sequences showed that SRBSDV and RBSDV clustered into a branch,and six isolates of SRBSDV clustered into a sub-branch.The recombinant protein with approximately 47 000 relative molecular mass was obtained by prokaryotic expression.Western blot analysis revealed that GSTmonoclonal antibody could specifically bind to the fusion protein.【Conclusion】All isolates of SRBSDV are closely related,and they have relatively far relationship to other Fijivirus members.The fusion protein obtained by prokaryotic expression is the target protein.
引文
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[13]张恒木,雷娟利,陈剑平,等.浙江和河北发生的一种水稻,小麦,玉米矮缩病是水稻黑条矮缩病毒引起的[J].中国病毒学,2001,16(3):246-251.
[14]章松柏,王开放,刘小娟,等.南方水稻黑条矮缩病毒非结构蛋白的亚细胞定位研究[J].热带作物学报2013,34(11):2102-2107.
[15]卢嫣红,张金凤,熊如意,等.南方水稻黑条矮缩病毒S6编码一个沉默抑制子[J].中国农业科学,2011,44(14):2909-2917.
[16]羊健,张恒木,陈剑平,等.水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性[J].植物保护学报,2007,34(3):252-258.
[17]张蔚明,刘燕娟,周倩,等.南方水稻黑条矮缩病毒外壳蛋白P10的原核表达和抗血清制备及应用[J].湖南农业大学学报(自然科学版),2011,37(4):400-402.
[18]LIU X Y,YANG J,XIE L,et al.P5-2 of Rice blackstreaked dwarf virus is a non-structural protein targeted to chloroplasts[J].Arch Virol,2015,160(5):1211-1217.
[19]洪健,李德葆,周雪平,等.植物病毒分类图谱[M].北京:科学出版社,2001:64-67.
[20]NAKASHIMA N,KOIZUMI M,WATANABE H,et al.Complete nucleotide sequence of the Nilaparvata lugens reovirus:A putative member of the genus Fijivirus[J].J Gen Virol,1996,77(1):139-146.
[21]HARDING R M,BURNS P,GEIJSKES R J,et al.Molecular analysis of Fiji disease virus segments 2,4 and7 completes the genome sequence[J].Virus Genes,2006,32(1):43-47.
[22]DISTEFANO A J,CONCI L R,HIDALGO M M,et al.Sequence analysis of genome segments S4 and S8 of Mal de Rio Cuarto virus(MRCV):Evidence that the virus should be a separate Fijivirus species[J].Arch Virol,2002,147(9):1699-1709.
[23]张上林,孙丽英,陈剑平.四种水稻蛋白与水稻黑条矮缩病毒编码非结构蛋白P7-2的互作分析[J].浙江农业学报,2013,25(6):1298-1303.
[24]陈秀,饶雪琴,阮小蕾,等.香蕉线条病毒MP功能域基因的克隆、原核表达及抗血清制备[J].华南农业大学学报,2014,35(2):47-52.
[2]季英华,高瑞珍,张野,等.一种快速同步检测水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的方法[J].中国水稻科学,2011,25(1):91-94.
[3]王强,周国辉,张曙光.南方水稻黑条矮缩病毒一步双重RT-PCR检测技术及其应用[J].植物病理学报,2012,42(1):84-87.
[4]周国辉,张曙光,邹寿发,等.水稻新病害南方水稻黑条矮缩病发生特点及危害趋势分析[J].植物保护,2010,36(2):144-146.
[5]ZHOU G H,XU D L,XU D G,et al.Southern rice black-streaked dwarf virus:A white-backed planthoppertransmitted fijivirus threatening rice production in Asia[J].Front Microbiol,2013,4:270.
[6]刘欢,倪跃群,饶黎霞,等.南方水稻黑条矮缩病毒和水稻条矮缩病毒的单抗制备及其检测应用[J].植物病理学报,2013,43(1):27-34.
[7]杨迎青,兰波,孟凡,等.江西省白背飞虱消长动态及南方水稻黑条矮缩病带毒率的测定[J].华中农业大学学报,2013,32(6):60-64.
[8]郑兆阳,张启勇,沈光斌.安徽省南方水稻黑条矮缩病发生现状和综防对策[J].安徽农学通报,2011,17(7):132-188.
[9]丁铭,尹跃艳,方琦,等.云南水稻上检测到南方水稻黑条矮缩病毒[J].植物病理学报,2011,41(6):640-644.
[10]LIU Y,JIA D S,CHEN H Y,et al.The P7-1 protein of Southern rice black-streaked dwarf virus,a fijivirus,induces the formation of tubular structures in insect cells[J].Arch Virol,2011,156(10):1729-1736.
[11]羊健.水稻黑条矮缩病毒S5基因组功能的研究[D].长沙:湖南农业大学,2007:1-57.
[12]ZHANG P,MAR T T,LIU W W,et al.Simultaneous detection and differentiation of Rice black streaked dwarf virus(RBSDV)and Southern rice black streaked dwarf virus(SRBSDV)by duplex real time RT-PCR[J].Virol J,2013(10):24-35.
[13]张恒木,雷娟利,陈剑平,等.浙江和河北发生的一种水稻,小麦,玉米矮缩病是水稻黑条矮缩病毒引起的[J].中国病毒学,2001,16(3):246-251.
[14]章松柏,王开放,刘小娟,等.南方水稻黑条矮缩病毒非结构蛋白的亚细胞定位研究[J].热带作物学报2013,34(11):2102-2107.
[15]卢嫣红,张金凤,熊如意,等.南方水稻黑条矮缩病毒S6编码一个沉默抑制子[J].中国农业科学,2011,44(14):2909-2917.
[16]羊健,张恒木,陈剑平,等.水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性[J].植物保护学报,2007,34(3):252-258.
[17]张蔚明,刘燕娟,周倩,等.南方水稻黑条矮缩病毒外壳蛋白P10的原核表达和抗血清制备及应用[J].湖南农业大学学报(自然科学版),2011,37(4):400-402.
[18]LIU X Y,YANG J,XIE L,et al.P5-2 of Rice blackstreaked dwarf virus is a non-structural protein targeted to chloroplasts[J].Arch Virol,2015,160(5):1211-1217.
[19]洪健,李德葆,周雪平,等.植物病毒分类图谱[M].北京:科学出版社,2001:64-67.
[20]NAKASHIMA N,KOIZUMI M,WATANABE H,et al.Complete nucleotide sequence of the Nilaparvata lugens reovirus:A putative member of the genus Fijivirus[J].J Gen Virol,1996,77(1):139-146.
[21]HARDING R M,BURNS P,GEIJSKES R J,et al.Molecular analysis of Fiji disease virus segments 2,4 and7 completes the genome sequence[J].Virus Genes,2006,32(1):43-47.
[22]DISTEFANO A J,CONCI L R,HIDALGO M M,et al.Sequence analysis of genome segments S4 and S8 of Mal de Rio Cuarto virus(MRCV):Evidence that the virus should be a separate Fijivirus species[J].Arch Virol,2002,147(9):1699-1709.
[23]张上林,孙丽英,陈剑平.四种水稻蛋白与水稻黑条矮缩病毒编码非结构蛋白P7-2的互作分析[J].浙江农业学报,2013,25(6):1298-1303.
[24]陈秀,饶雪琴,阮小蕾,等.香蕉线条病毒MP功能域基因的克隆、原核表达及抗血清制备[J].华南农业大学学报,2014,35(2):47-52.