白背飞虱中肠内与SRBSDV P6蛋白互作的介体因子鉴定
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摘要
南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus, SRBSDV)由白背飞虱(Sogatella furcifera)以持久增殖型方式进行传播。该病毒能够在白背飞虱体内大量增殖并使昆虫终生带毒,是因病毒在介体昆虫内能够诱导形成病毒复制工厂-病毒原质所致。SRBSDV病毒原质主要由病毒编码的P5、P6和P9-1 3个非结构蛋白及其介体因子参与形成。其中,P6蛋白分别和P5、P9-1蛋白及其介体因子相互作用,在病毒原质形成过程中发挥重要作用。为了揭示哪些介体因子参与调控SRBSDV在昆虫体内的增殖过程,本实验利用酵母双杂交技术,将SRBSDV P6基因构建到酵母诱饵表达载体pGBKT7上,并转化酵母感受态细胞Y2HGold,Western blot分析显示P6蛋白在酵母感受态细胞内成功表达,表达的P6蛋白对细胞无毒性和自激活活性。本研究以携带SRBSDV的白背飞虱中肠cDNA文库为筛选对象,初探介体昆虫内与SRBSDV P6蛋白相互作用的蛋白。在酵母Y2HGold细胞内经大量筛选和回转验证,最终获得5个可能与P6蛋白互作的介体因子。这些介体因子主要参与基因的转录、蛋白质翻译、蛋白翻译后修饰和蛋白质的合成过程。本实验为深入解析介体因子调控病毒的复制过程和白背飞虱高效传播病毒的分子机制提供了理论基础。
Southern rice black-streaked dwarf virus(SRBSDV) is transmitted by white-backed planthopper(Sogatella furcifera) in a persistent-propagative manner. During SRBSDV infection in its insect vector,viroplasm, putative sites of viral replication, contained the nonstructural viral protein P5, P6, P9-1 and insect vector proteins. The P6 protein is initially expressed, and interacts with P5, P9-1 and insect proteins in insect vector, respectively, which demonstrate that P6 protein is essential for viroplasm formation and viral replication. In order to investigate which insect proteins play an important role during viral replication, yeast two-hybrid experiments was be used to identify insect proteins interacting with SRBSDV P6 protein in the white-backed planthopper midgut. Firstly, SRBSDV P6 gene was constructed into the expression vector(pGBKT7-P6) and transformed into Y2 HGold cell. Western blot showed P6 protein successfully expressed in yeast cell, which was non-toxicity and non-autonomous activation to cells. Then c DNA library plasmids were transformed into Y2 HGold containing bait plasmid pGBKT7-P6. Transformants were selected from high stringency medium SD/-Leu/-Trp/-His/-Ade, and then replicated to SD/-Leu/-Trp/-His/-Ade/X-α-gal medium for further analysis. Colony growth and blue color indicated an interaction between the two-hybrid proteins.Plasmid DNA was isolated from yeast and then transformed into Escherichia coli DH5α. Lastly, cDNA inserts were sequenced to verify the preference of an open reading frame(ORF) and compared the sequence in Blast of NCBI.. The results showed 5 insect proteins, interacting with P6, which were involved in gene transcription, translation, post-translational modification and protein systhesis. Results of this study will be useful in revealing the replication mechanisms of SRBSDV in its insect vector, which will facilitate the development of effective control strategies against SRBSDV disease.
Southern rice black-streaked dwarf virus(SRBSDV) is transmitted by white-backed planthopper(Sogatella furcifera) in a persistent-propagative manner. During SRBSDV infection in its insect vector,viroplasm, putative sites of viral replication, contained the nonstructural viral protein P5, P6, P9-1 and insect vector proteins. The P6 protein is initially expressed, and interacts with P5, P9-1 and insect proteins in insect vector, respectively, which demonstrate that P6 protein is essential for viroplasm formation and viral replication. In order to investigate which insect proteins play an important role during viral replication, yeast two-hybrid experiments was be used to identify insect proteins interacting with SRBSDV P6 protein in the white-backed planthopper midgut. Firstly, SRBSDV P6 gene was constructed into the expression vector(pGBKT7-P6) and transformed into Y2 HGold cell. Western blot showed P6 protein successfully expressed in yeast cell, which was non-toxicity and non-autonomous activation to cells. Then c DNA library plasmids were transformed into Y2 HGold containing bait plasmid pGBKT7-P6. Transformants were selected from high stringency medium SD/-Leu/-Trp/-His/-Ade, and then replicated to SD/-Leu/-Trp/-His/-Ade/X-α-gal medium for further analysis. Colony growth and blue color indicated an interaction between the two-hybrid proteins.Plasmid DNA was isolated from yeast and then transformed into Escherichia coli DH5α. Lastly, cDNA inserts were sequenced to verify the preference of an open reading frame(ORF) and compared the sequence in Blast of NCBI.. The results showed 5 insect proteins, interacting with P6, which were involved in gene transcription, translation, post-translational modification and protein systhesis. Results of this study will be useful in revealing the replication mechanisms of SRBSDV in its insect vector, which will facilitate the development of effective control strategies against SRBSDV disease.
引文
刘万才,刘宇,郭荣.2010.南方水稻黑条矮缩病发生现状及防控对策[J].中国植保导刊,30(3):17-18.(Liu WC,Liu Y,Guo R,et al.2010.A comprehensive review on Southern rice black-streaked dwarf virus:Occurrence and control strategies[J].China Plant Protection,30(3):17-18.)
卢嫣红,张金凤,熊如意,等.2011.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,44(14):2909-2917.(Lu Y H,Zhang J F,Xiong R Y,et al.2011.Identification of an RNA silencing suppressor encoded by Southern rice black-streaked dwarf virus S6[J].Scientia Agricultura Sinica,44(14):2909-2917.)
章松柏.2013.南方水稻黑条矮缩病的监测及其病原病毒Pns6与寄主水稻的互作[D].博士学位论文,福建农林大学,导师:谢联辉,吴祖建.pp.73-101.(Zhang S B.2013.The Surveillance of Southern rice black streaked disease and study on the interaction between Pns6 of the pathogenic virus and host rice plants[D].Thesis for Ph D,Fujian Agriculture and Forestry University,Supervisor:Xie L H,Wu Z J,pp.73-101.)
周国辉,张曙光,邹寿发,等.2010.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,36(1):144-146.(Zhou G H,Zhang S G,Zou S F,et al.2010.Occurrence and damage analysis of a new rice dwarf disease caused by Southern rice black-streaked dwarf virus[J].Plant Protection,36(1):144-146.)
周国辉,温锦君,蔡德江,等.2008.呼肠孤病毒科斐济病毒属一新种:南方水稻黑条矮缩病毒[J].科学通报,53(20):2500-2508.(Zhou G H,Wen J J,Cai D J,et al.2008.Southern rice black-streaked dwarf virus:A new proposed Fijivirus species in the family Reoviridae[J].Chinese Science Bulletin,53(20):3677-3685.)
郑立敏,彭静,陈建斌等.2016.SRBSDV侵染的白背飞虱中肠酵母双杂交cDNA文库的构建和分析[J].农业生物技术学报,24(2):245-251.(Zheng L M,Peng J,Chen JB,et al.2016.Construction and analysis of yeast twohybrid cDNA library of the SRBSDV-infected midgut of white backed-planthopper(Sogatella furcifera)[J].Journal of Agricultural Biotechnology,24(2):245-251.)
Chaturvedi S,Rao A L N.2016.A shift in plant proteome profile for a Bromodomain containing RNA binding Protein(BRP1)in plants infected with Cucumber mosaic virus and its satellite RNA[J].Journal of Proteomics,131:1-7.
Chaturvedi S1,Kalantidis K,Rao AL.2014.A bromodomaincontaining host protein mediates the nuclear importation of a satellite RNA of Cucumber mosaic virus[J].Journal of Virology,88(4):1890-1896.
Dejarnac O,Hafirassou ML,Chazal M,et al.2018.TIM-1ubiquitination mediates dengue virus entry[J].Cell Report.23(6):1779-1793.
Dreher T W,Miller W A.2006.Translational control in positive strand RNA plant viruses[J].Virology,344(1):185-197.
Hoang A T,Zhang H M,Yang J,et al.2011.Identification,characterization,and distribution of Southern rice blackstreaked dwarf virus in Vietnam[J].Plant Disease,95(9):1063-1069.
Jia D,Chen H,Mao Q,et al.2012a.Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus[J].Virus Research,167(2):404-408.
Jia D,Chen H,Zheng A,et al.2012b.Development of an insect vector cell culture and RNA interference system to investigate the functional role of Fijivirus replication protein[J].Journal of Virology,86(10):5800-5807.
Li J,Xue J,Zhang H,et al.2015.Characterization of homologous and heterologous interactions between viroplasm proteins P6 and P9-1 of the Fijivirus Southern rice blackstreaked dwarf virus[J].Archives of Virology,160(2):453-457.
Lv D Q,Liu S W,Zhao J H,et al.2016.Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroidbinding protein[J].Scientific Report,6:35751.
Mao Q,Zheng S,Han Q,et al.2013.New model for the genesis and maturation of viroplasms induced by Fijiviruses in insect vector cells[J].Journal of Virology,87(12):6819-6828.
Matsukura K,Towata T,Sakai J,et al.2013.Dynamics of South?ern rice black-streaked dwarf virus in rice and implication for virus acquisition[J].Phytopathology,103(5):509-12.
Otuka A.2013.Migration of rice planthoppers and their vectored re-emerging and novel rice viruses in East Asia[J].Frontiers in Microbiology,4:309.
Pu L,Xie G,Ji C,et al.2012.Transmission characteristics of Southern rice black-streaked dwarf virus by rice planthoppers[J].Crop Protect,41(6):71-76.
Zhang S B,Du Z G,Yang L,et al.2013.Identification and characterization of the interaction between two viroplasm associated proteins from two different plant-infecting reoviruses and eEF-1A of rice[J].Archives of Virology,158(10):2031-2039.
Song D G,Kim Y S,Jung B C,et al.2013.Parkin induces upregulation of 40S ribosomal protein sa and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells[J].Applied Biochemistry&Biotechnology,171(7):1630.
Wei T,Shimizu T,Hagiwara K,et al.2006.Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes[J].Journal of General Virology,87(Pt2):429-438.
Wei T,Uehara-Ichiki T,Miyazaki N,et al.2009.Association of Rice gall dwarf virus with microtubules is necessary for viral release from cultured insect vector cells[J].Journal of Virology,83(20):10830-10835.
Wise R P,Moscou M J,Bogdanove A J,et al.2007.Transcript profiling in host-pathogen interactions[J].Annual Review of Phytopathology,45:329-369.
Yang C,Guo R,Jie F,et al.2007.Spatial analysis of Arabidop?sis thaliana gene expression in response to Turnip mosa?ic virus infection[J].Molecular Plant-Microbe Interactions,20:358-70.
Zhou G,Xu D,Xu D,et al.2013.Southern rice blackstreaked dwarf virus:A white-backed planthopper-transmitted fijivirus threatening rice production in Asia[J].Frontiers in Microbiology,4:270.
卢嫣红,张金凤,熊如意,等.2011.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,44(14):2909-2917.(Lu Y H,Zhang J F,Xiong R Y,et al.2011.Identification of an RNA silencing suppressor encoded by Southern rice black-streaked dwarf virus S6[J].Scientia Agricultura Sinica,44(14):2909-2917.)
章松柏.2013.南方水稻黑条矮缩病的监测及其病原病毒Pns6与寄主水稻的互作[D].博士学位论文,福建农林大学,导师:谢联辉,吴祖建.pp.73-101.(Zhang S B.2013.The Surveillance of Southern rice black streaked disease and study on the interaction between Pns6 of the pathogenic virus and host rice plants[D].Thesis for Ph D,Fujian Agriculture and Forestry University,Supervisor:Xie L H,Wu Z J,pp.73-101.)
周国辉,张曙光,邹寿发,等.2010.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,36(1):144-146.(Zhou G H,Zhang S G,Zou S F,et al.2010.Occurrence and damage analysis of a new rice dwarf disease caused by Southern rice black-streaked dwarf virus[J].Plant Protection,36(1):144-146.)
周国辉,温锦君,蔡德江,等.2008.呼肠孤病毒科斐济病毒属一新种:南方水稻黑条矮缩病毒[J].科学通报,53(20):2500-2508.(Zhou G H,Wen J J,Cai D J,et al.2008.Southern rice black-streaked dwarf virus:A new proposed Fijivirus species in the family Reoviridae[J].Chinese Science Bulletin,53(20):3677-3685.)
郑立敏,彭静,陈建斌等.2016.SRBSDV侵染的白背飞虱中肠酵母双杂交cDNA文库的构建和分析[J].农业生物技术学报,24(2):245-251.(Zheng L M,Peng J,Chen JB,et al.2016.Construction and analysis of yeast twohybrid cDNA library of the SRBSDV-infected midgut of white backed-planthopper(Sogatella furcifera)[J].Journal of Agricultural Biotechnology,24(2):245-251.)
Chaturvedi S,Rao A L N.2016.A shift in plant proteome profile for a Bromodomain containing RNA binding Protein(BRP1)in plants infected with Cucumber mosaic virus and its satellite RNA[J].Journal of Proteomics,131:1-7.
Chaturvedi S1,Kalantidis K,Rao AL.2014.A bromodomaincontaining host protein mediates the nuclear importation of a satellite RNA of Cucumber mosaic virus[J].Journal of Virology,88(4):1890-1896.
Dejarnac O,Hafirassou ML,Chazal M,et al.2018.TIM-1ubiquitination mediates dengue virus entry[J].Cell Report.23(6):1779-1793.
Dreher T W,Miller W A.2006.Translational control in positive strand RNA plant viruses[J].Virology,344(1):185-197.
Hoang A T,Zhang H M,Yang J,et al.2011.Identification,characterization,and distribution of Southern rice blackstreaked dwarf virus in Vietnam[J].Plant Disease,95(9):1063-1069.
Jia D,Chen H,Mao Q,et al.2012a.Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of Southern rice black-streaked dwarf virus[J].Virus Research,167(2):404-408.
Jia D,Chen H,Zheng A,et al.2012b.Development of an insect vector cell culture and RNA interference system to investigate the functional role of Fijivirus replication protein[J].Journal of Virology,86(10):5800-5807.
Li J,Xue J,Zhang H,et al.2015.Characterization of homologous and heterologous interactions between viroplasm proteins P6 and P9-1 of the Fijivirus Southern rice blackstreaked dwarf virus[J].Archives of Virology,160(2):453-457.
Lv D Q,Liu S W,Zhao J H,et al.2016.Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroidbinding protein[J].Scientific Report,6:35751.
Mao Q,Zheng S,Han Q,et al.2013.New model for the genesis and maturation of viroplasms induced by Fijiviruses in insect vector cells[J].Journal of Virology,87(12):6819-6828.
Matsukura K,Towata T,Sakai J,et al.2013.Dynamics of South?ern rice black-streaked dwarf virus in rice and implication for virus acquisition[J].Phytopathology,103(5):509-12.
Otuka A.2013.Migration of rice planthoppers and their vectored re-emerging and novel rice viruses in East Asia[J].Frontiers in Microbiology,4:309.
Pu L,Xie G,Ji C,et al.2012.Transmission characteristics of Southern rice black-streaked dwarf virus by rice planthoppers[J].Crop Protect,41(6):71-76.
Zhang S B,Du Z G,Yang L,et al.2013.Identification and characterization of the interaction between two viroplasm associated proteins from two different plant-infecting reoviruses and eEF-1A of rice[J].Archives of Virology,158(10):2031-2039.
Song D G,Kim Y S,Jung B C,et al.2013.Parkin induces upregulation of 40S ribosomal protein sa and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells[J].Applied Biochemistry&Biotechnology,171(7):1630.
Wei T,Shimizu T,Hagiwara K,et al.2006.Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes[J].Journal of General Virology,87(Pt2):429-438.
Wei T,Uehara-Ichiki T,Miyazaki N,et al.2009.Association of Rice gall dwarf virus with microtubules is necessary for viral release from cultured insect vector cells[J].Journal of Virology,83(20):10830-10835.
Wise R P,Moscou M J,Bogdanove A J,et al.2007.Transcript profiling in host-pathogen interactions[J].Annual Review of Phytopathology,45:329-369.
Yang C,Guo R,Jie F,et al.2007.Spatial analysis of Arabidop?sis thaliana gene expression in response to Turnip mosa?ic virus infection[J].Molecular Plant-Microbe Interactions,20:358-70.
Zhou G,Xu D,Xu D,et al.2013.Southern rice blackstreaked dwarf virus:A white-backed planthopper-transmitted fijivirus threatening rice production in Asia[J].Frontiers in Microbiology,4:270.
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