干扰水稻齿叶矮缩病毒(RRSV)非结构蛋白Pns6的表达阻碍病毒在褐飞虱体内的增殖
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摘要
水稻齿叶矮缩病毒(Rice ragged stunt virus,RRSV)由介体褐飞虱(Nilaparvata lugens)以持久增殖型方式传播。RRSV编码的非结构蛋白6(nonstructural protein 6,Pns6)是病毒原质的组分,但其在介体昆虫传毒中的功能未知。本研究利用ds RNA诱导的RNA干扰(RNA interference,RNAi)技术研究Pns6蛋白在病毒侵染介体昆虫中的功能。通过膜饲喂法将源于Pns6基因的ds RNA(ds Pns6)以及源于绿色荧光蛋白(green fluorescent protein,GFP)基因的ds RNA(ds GFP)分别导入2龄褐飞虱体内,并以饲喂10%蔗糖的处理为对照,之后于病株上获毒2 d。应用免疫荧光标记技术检测发现,昆虫获毒9 d后,ds GFP和10%蔗糖处理组褐飞虱的带毒率分别为27%和30%,而ds Pns 6处理组褐飞虱的带毒率仅为10%;同时q RT-PCR检测发现,Pns6蛋白的表达量受到抑制后,病毒编码的结构蛋白P8和P9的表达量也显著下降(P<0.05)。此外,ds GFP和10%蔗糖处理组褐飞虱的传毒率分别为20%和22%,干扰Pns6蛋白的表达后褐飞虱的传毒率仅为6%。研究结果表明,Pns6是RRSV在褐飞虱体内增殖的重要蛋白,抑制Pns6的表达显著降低褐飞虱的带毒率并抑制病毒的增殖,同时削弱褐飞虱的传毒能力,明确了Pns6可作为理想的靶标用于阻断RRSV在褐飞虱体内的增殖。
Rice ragged stunt virus(RRSV) is transmitted by Nilaparvata lugens in a persistent propagative manner. The nonstructural protein 6(Pns6) encoded by RRSV is a constituent of viroplasm, but the functional role of Pns6 in viral infection of insect vector is still unkown. In this study, RNA interference(RNAi) induced by synthesized ds RNAs was used to investigate the function of Pns6 in N. lugens. Second-instar nymphs of N.lugens were fed with ds RNAs targeting the Pns6 gene(ds Pns6), green fluorescent protein(GFP) gene(ds GFP)or 10% sucrose diet control through a membrane- feeding method, then fed on diseased rice(Oryza sativa)plants for 2 d. At 9 d post- first access to diseased plants, immunofluorescence assay showed that the viruliferous rates of insects treated by ds GFP and 10% sucrose diet control were 27% and 30%, respectively,while the viruliferous rate of insects treated by ds Pns6 was 10%. The q RT- PCR results displayed that inhibiting the expression of Pns6 could also significantly reduced(P<0.05) the expression levels of viral structural proteins P8 and P9. In addition, the transmission rate of insects treated with ds GFP and diet control were 20% and 22%, respectively. In contrast, the transmission rate of insects treated with ds Pns6 was 6%.These results revealed that Pns6 was an important protein essential for RRSV multiplication in N. lugens.Inhibition of Pns6 expression could significantly reduce(P<0.05) the viruliferous rate and block viral multiplication, finally prevent the transmitting ability of insects. The results also confirmed that Pns6 of RRSV could act as a good target for blocking the multiplication of RRSV in its insect vector.
Rice ragged stunt virus(RRSV) is transmitted by Nilaparvata lugens in a persistent propagative manner. The nonstructural protein 6(Pns6) encoded by RRSV is a constituent of viroplasm, but the functional role of Pns6 in viral infection of insect vector is still unkown. In this study, RNA interference(RNAi) induced by synthesized ds RNAs was used to investigate the function of Pns6 in N. lugens. Second-instar nymphs of N.lugens were fed with ds RNAs targeting the Pns6 gene(ds Pns6), green fluorescent protein(GFP) gene(ds GFP)or 10% sucrose diet control through a membrane- feeding method, then fed on diseased rice(Oryza sativa)plants for 2 d. At 9 d post- first access to diseased plants, immunofluorescence assay showed that the viruliferous rates of insects treated by ds GFP and 10% sucrose diet control were 27% and 30%, respectively,while the viruliferous rate of insects treated by ds Pns6 was 10%. The q RT- PCR results displayed that inhibiting the expression of Pns6 could also significantly reduced(P<0.05) the expression levels of viral structural proteins P8 and P9. In addition, the transmission rate of insects treated with ds GFP and diet control were 20% and 22%, respectively. In contrast, the transmission rate of insects treated with ds Pns6 was 6%.These results revealed that Pns6 was an important protein essential for RRSV multiplication in N. lugens.Inhibition of Pns6 expression could significantly reduce(P<0.05) the viruliferous rate and block viral multiplication, finally prevent the transmitting ability of insects. The results also confirmed that Pns6 of RRSV could act as a good target for blocking the multiplication of RRSV in its insect vector.
引文
郭年梅,韩庆梅,毛倩卓,等.2012.水稻齿叶矮缩病毒非结构蛋白Pns7形成伸出细胞膜的纤维丝状结构[EBOL].北京:中国科技论文在线,http://www.paper.edu cn/releasepaper/content/201212-472.(Guo N M,Han Q M,Mao Q Z,et al.2012.Nonstructural protein Pns7of Rice ragged stunt virus induces the formation of fila ment structures growing from insect cell surface[J].Bei jing:Sciencepaper Online,http://www.paper.edu.cn/re leasepaper/content/201212-472.)
贾东升,任堂雨,陈红燕,等.2013.白背飞虱体内RNA干扰技术体系的建立[J].福建农林大学学报(自然科学版)42(6):579-583.(Jia D S,Ren T L,Chen H Y,et al2013.Establishment of RNA interference system in white-backed planthopper[J].Journal of Fujian Agricul true and Forestry Univercity(Natural Science Edition)42(6):579-583.)
吕慧涓,邵朝纲,龚祖埙,等.2002.水稻齿叶矮缩病毒S8基因编码的结构蛋白质及其自聚集性质[J].生物化学与生物物理学报,34(5):565-570.(Lv H J,Shao C GGong Z X,et al.2002.Self-aggregation of the structura protein encoded by Rice ragged stunt Oryzavirus genom sequent 8[J].Acta Biochimica et Biophysica Sinica,34(5):565-570.)
谢联辉,林奇英.1980.锯齿叶矮缩病毒在我国水稻上的发现[J].植物病理学报,10(1):59-64.(Xie L H,Lin Q Y1980.The ragged stunt disease,a new record of rice dis ease in China[J].Acta Phytopathologyica Sinica,10(1)59-64.)
郑立敏,刘华敏,陈红燕,等.2014.干扰水稻瘤矮病毒(RGDV)非结构蛋白(Pns12)的表达抑制病毒在介体昆虫培养细胞内的复制[J].农业生物技术学报,22(11)1321-1328.(Zheng L M,Liu H M,Chen H Y,et al2014.Knockdown of nonstructural protein(Pns12)o Rice gall dwarf virus(RGDV)inhibits viral replication in insect vector cells[J].Journal of Agricultural Biotech nology,22(11):1321-1328.)
郑璐平,谢荔岩,连玲丽,等.2008.水稻齿叶矮缩病毒的研究进展[J].中国农业科技导报,10(5):8-12.(Zheng LP,Xie L Y,Lian L L,et al.2008.Research progress on Rice ragged stunt virus(RRSV)[J].Journal of Agricultural Science and Technology,10(5):8-12.)
Attoui H,Mertens P P C,Becnel J,et al.2012.Family reoviri dae,p:541-637[C].In King AMQ,Adams M JCarstens EB,Lefkowits EJ(ed),Virus Ttaxonomy Ninth Report of the International Committee for the Tax onomy of Viruses.Elsevier Academic Press,New York NY.
Chen H,Chen Q,Omura T,et al.2011.Sequential infection of Rice dwarf virus in the internal organs of its insec vector after ingestion of virus[J].Virus Research,160(12):389-394.
Chen H,Zheng L,Mao Q,et al.2014.Development of contin uous cell culture of brown planthopper to trace the early infection process of oryzaviruses in insect vector cell[J].Journal of Virology,88(8):4265-4274.
Hibino H.1996.Biology and epidemiology of rice viruses[J]Annual Review of Phytopathology,34:249-274.
Hibino H,Saleh N,Roechan M.1979.Reovirus-like particle associated with rice ragged stunt diseased rice and in sect vector cells[J].Annals of Phytopathology Society Japan,45:228-239.
Hogenhout S,Ammar D,Whitfield A,et al.2008.Insect vec tor interactions with persistently transmitted viruses[J]Annual Review of Phytopathology,46:327-359.
Jia D,Chen H,Zheng A,et al.2012a.Development of an in sect vector cell culture and RNA interference system to investigate the functional role of fijivirus replication protein[J].Journal of Virology,86(10):5800-5807.
Jia D,Guo N,Chen H,et al.2012b.Assembly of the viro plasm by viral non-structural protein Pns10 is essentia for persistent infection of Rice ragged stunt virus in its in sect vector[J].Journal of General Virology,93(10)2299-2309.
Kawano S,Uyeda I,Shikata E.1984.Particle structure and double stranded RNA of Rice ragged stunt virus[J].Jour nal of the Faculty of Agriculture,Hokkaido University61(4):408-418.
Li Z,Upadhyaya N,Kosltratana W,et al.1996.Genome seg ment 5 of Rice ragged stunt virus encodes a virion protein[J].Journal of General Virology,77(12):3155-3160.
Livak K J,Schmittgen T D.2001.Analysis of relative gen expression data using Real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Shao C,Lu H,Wu J,et al.2004.Nucleicacid binding activity of Pns6 encoded by genome segment 6 of Rice ragged stunt oryzavirus[J].Acta Biochimica et Biophysica Sini ca,36(7):457-466.
Shao C,Wu J,Zhou G,et al.2003.Ectopic expression of th spike protein of Rice ragged stunt oryzavirus in transgen ic rice plants inhibits transmission of the virus to insect[J].Molecular Breeding,1(4):295-301.
Upadhyaya N,Ramm K,Gellady J,et al.1998.Rice ragged stunt oryzavirus genome segment S4 could encode an RNA dependent RNA polymerase and a second protein of unknown function[J].Archives of Virology,143(9)1815-1822.
Upadhyaya N,Ramm K,Gellatly J,et al.1997.Rice ragged stunt oryzavirns genome segments s7 and S10 encod non-structural proteins of Mr 68,025(Pns7)and Mr 32364(Pnsl0)[J].Archives of Virology,142(8):17191726.
Uyeda I,Suga N,Lee S,et a1.1995.Rice ragged stunt Oryzavi rus genome segment 9 encodes a 38600 Mr structura protein[J].Journal of General Virology,76(4):975-978.
Wu J,Du Z,Wang C,et al.2010a.Identification of Pns6,a pu tative movement protein of RRSV,as a silencing sup pressor[J].Virology Journal,7:335.
Wu Z,Wu J,Scott A,et al.2010b.Rice ragged stunt virus seg ment S6-encoded nonstructural protein Pns6 comple ments cell-to-cell movement of Tobacco mosaic virus based chimeric virus[J].Virus Research,152(1-2):176179.
Zheng L,Chen H,Liu H,et al.2015.Assembly of viroplasm by viral nonstructural protein Pns9 is essential for persis tent infection of Rice gall dwarf virus in its insect vecto[J].Virus Research,196:162-169.
Zhou G,Lu X,Lu H,et al.1999.Rice ragged stunt oryzavires Role of the viral spike protein in transmission by the in sect vector[J].Annals of Applied Biology,135(3):573578.
贾东升,任堂雨,陈红燕,等.2013.白背飞虱体内RNA干扰技术体系的建立[J].福建农林大学学报(自然科学版)42(6):579-583.(Jia D S,Ren T L,Chen H Y,et al2013.Establishment of RNA interference system in white-backed planthopper[J].Journal of Fujian Agricul true and Forestry Univercity(Natural Science Edition)42(6):579-583.)
吕慧涓,邵朝纲,龚祖埙,等.2002.水稻齿叶矮缩病毒S8基因编码的结构蛋白质及其自聚集性质[J].生物化学与生物物理学报,34(5):565-570.(Lv H J,Shao C GGong Z X,et al.2002.Self-aggregation of the structura protein encoded by Rice ragged stunt Oryzavirus genom sequent 8[J].Acta Biochimica et Biophysica Sinica,34(5):565-570.)
谢联辉,林奇英.1980.锯齿叶矮缩病毒在我国水稻上的发现[J].植物病理学报,10(1):59-64.(Xie L H,Lin Q Y1980.The ragged stunt disease,a new record of rice dis ease in China[J].Acta Phytopathologyica Sinica,10(1)59-64.)
郑立敏,刘华敏,陈红燕,等.2014.干扰水稻瘤矮病毒(RGDV)非结构蛋白(Pns12)的表达抑制病毒在介体昆虫培养细胞内的复制[J].农业生物技术学报,22(11)1321-1328.(Zheng L M,Liu H M,Chen H Y,et al2014.Knockdown of nonstructural protein(Pns12)o Rice gall dwarf virus(RGDV)inhibits viral replication in insect vector cells[J].Journal of Agricultural Biotech nology,22(11):1321-1328.)
郑璐平,谢荔岩,连玲丽,等.2008.水稻齿叶矮缩病毒的研究进展[J].中国农业科技导报,10(5):8-12.(Zheng LP,Xie L Y,Lian L L,et al.2008.Research progress on Rice ragged stunt virus(RRSV)[J].Journal of Agricultural Science and Technology,10(5):8-12.)
Attoui H,Mertens P P C,Becnel J,et al.2012.Family reoviri dae,p:541-637[C].In King AMQ,Adams M JCarstens EB,Lefkowits EJ(ed),Virus Ttaxonomy Ninth Report of the International Committee for the Tax onomy of Viruses.Elsevier Academic Press,New York NY.
Chen H,Chen Q,Omura T,et al.2011.Sequential infection of Rice dwarf virus in the internal organs of its insec vector after ingestion of virus[J].Virus Research,160(12):389-394.
Chen H,Zheng L,Mao Q,et al.2014.Development of contin uous cell culture of brown planthopper to trace the early infection process of oryzaviruses in insect vector cell[J].Journal of Virology,88(8):4265-4274.
Hibino H.1996.Biology and epidemiology of rice viruses[J]Annual Review of Phytopathology,34:249-274.
Hibino H,Saleh N,Roechan M.1979.Reovirus-like particle associated with rice ragged stunt diseased rice and in sect vector cells[J].Annals of Phytopathology Society Japan,45:228-239.
Hogenhout S,Ammar D,Whitfield A,et al.2008.Insect vec tor interactions with persistently transmitted viruses[J]Annual Review of Phytopathology,46:327-359.
Jia D,Chen H,Zheng A,et al.2012a.Development of an in sect vector cell culture and RNA interference system to investigate the functional role of fijivirus replication protein[J].Journal of Virology,86(10):5800-5807.
Jia D,Guo N,Chen H,et al.2012b.Assembly of the viro plasm by viral non-structural protein Pns10 is essentia for persistent infection of Rice ragged stunt virus in its in sect vector[J].Journal of General Virology,93(10)2299-2309.
Kawano S,Uyeda I,Shikata E.1984.Particle structure and double stranded RNA of Rice ragged stunt virus[J].Jour nal of the Faculty of Agriculture,Hokkaido University61(4):408-418.
Li Z,Upadhyaya N,Kosltratana W,et al.1996.Genome seg ment 5 of Rice ragged stunt virus encodes a virion protein[J].Journal of General Virology,77(12):3155-3160.
Livak K J,Schmittgen T D.2001.Analysis of relative gen expression data using Real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-408.
Shao C,Lu H,Wu J,et al.2004.Nucleicacid binding activity of Pns6 encoded by genome segment 6 of Rice ragged stunt oryzavirus[J].Acta Biochimica et Biophysica Sini ca,36(7):457-466.
Shao C,Wu J,Zhou G,et al.2003.Ectopic expression of th spike protein of Rice ragged stunt oryzavirus in transgen ic rice plants inhibits transmission of the virus to insect[J].Molecular Breeding,1(4):295-301.
Upadhyaya N,Ramm K,Gellady J,et al.1998.Rice ragged stunt oryzavirus genome segment S4 could encode an RNA dependent RNA polymerase and a second protein of unknown function[J].Archives of Virology,143(9)1815-1822.
Upadhyaya N,Ramm K,Gellatly J,et al.1997.Rice ragged stunt oryzavirns genome segments s7 and S10 encod non-structural proteins of Mr 68,025(Pns7)and Mr 32364(Pnsl0)[J].Archives of Virology,142(8):17191726.
Uyeda I,Suga N,Lee S,et a1.1995.Rice ragged stunt Oryzavi rus genome segment 9 encodes a 38600 Mr structura protein[J].Journal of General Virology,76(4):975-978.
Wu J,Du Z,Wang C,et al.2010a.Identification of Pns6,a pu tative movement protein of RRSV,as a silencing sup pressor[J].Virology Journal,7:335.
Wu Z,Wu J,Scott A,et al.2010b.Rice ragged stunt virus seg ment S6-encoded nonstructural protein Pns6 comple ments cell-to-cell movement of Tobacco mosaic virus based chimeric virus[J].Virus Research,152(1-2):176179.
Zheng L,Chen H,Liu H,et al.2015.Assembly of viroplasm by viral nonstructural protein Pns9 is essential for persis tent infection of Rice gall dwarf virus in its insect vecto[J].Virus Research,196:162-169.
Zhou G,Lu X,Lu H,et al.1999.Rice ragged stunt oryzavires Role of the viral spike protein in transmission by the in sect vector[J].Annals of Applied Biology,135(3):573578.