马铃薯StTOM1和StTOM3双干扰植物双元表达载体的构建及转化验证
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摘要
病毒病是制约马铃薯生产的重要因素之一。在拟南芥等植物的研究中发现,抑制寄主因子可以显著降低细胞中病毒的累积量,从而缓解病害症状。本研究在获得与拟南芥寄主因子AtTOM1和AtTOM3具有同源性的马铃薯基因StTOM1和StTOM3的基础上,尝试用RNAi方法同时沉默StTOM1和StTOM3。以pUCCRNAi为中间载体,构建同时含StTOM1和StTOM3双基因干扰片段StT1-StT3的质粒pUCStT1-StT3-dRi(±),再将双基因干扰片段StT1-StT3切下并连接到双元载体pBI121上。用农杆菌介导方法,将StT1-StT3片段导入马铃薯中,获得转基因马铃薯小苗,阳性率达到83.6%。RT-PCR检测表明,转StT1-StT3马铃薯中StTOM1基因mRNA的表达水平下调了78%,StTOM3基因下调了81%。StTOM1和StTOM3沉默转基因马铃薯的获得,为将来验证和评价StTOM1和StTOM3是否为马铃薯病毒的寄主因子及在创建抗病毒马铃薯新种质的潜力,奠定了基础。
Viral diseases are important limiting factors for the potato industry.Studies in Arabidopsis thaliana reveals that inhibition of host factors results in reduced accumulation of viruses in the cells and thus relieves the symptom of the infected plants.In this study,two potato genes homologous to the host factor genes AtTOM1 and AtTOM3 of A.thaliana,StTOM1 and StTOM3,were cloned.To obtain StTOM1 and StTOM3 knockdown potato plants,RNAi strategy was employed.Fragments of StTOM1 and StTOM3 were generated by PCR and head-to-head gene fragment of the StTOM1 and StTOM3 in a single construct was obtained with aid of the intermediate vector pUCCRNAi.The RNA interference structure was then introduced into the plant binary vector pBI121 to generate the RNAi construct pBIStT1-StT3-dRi(±).Agrobacterium haboring pBIStT1-StT3-dRi(±) was used to infect potato explants.Transgenic potato explants with an 83.6% positive rate were obtained.Quantitative RT-PCR showed that the StTOM1 mRNA expression was down-regulated by 78% and the StTOM3 by 81%,demonstrating the efficient silencing of both StTOM1 and StTOM3.The availability of StTOM1 and StTOM3 knockdown potato provides basis for future verification of the host factor nature of these two genes to potato viruses and the potential of these genes in creating new anti-viral potato germplasms.
Viral diseases are important limiting factors for the potato industry.Studies in Arabidopsis thaliana reveals that inhibition of host factors results in reduced accumulation of viruses in the cells and thus relieves the symptom of the infected plants.In this study,two potato genes homologous to the host factor genes AtTOM1 and AtTOM3 of A.thaliana,StTOM1 and StTOM3,were cloned.To obtain StTOM1 and StTOM3 knockdown potato plants,RNAi strategy was employed.Fragments of StTOM1 and StTOM3 were generated by PCR and head-to-head gene fragment of the StTOM1 and StTOM3 in a single construct was obtained with aid of the intermediate vector pUCCRNAi.The RNA interference structure was then introduced into the plant binary vector pBI121 to generate the RNAi construct pBIStT1-StT3-dRi(±).Agrobacterium haboring pBIStT1-StT3-dRi(±) was used to infect potato explants.Transgenic potato explants with an 83.6% positive rate were obtained.Quantitative RT-PCR showed that the StTOM1 mRNA expression was down-regulated by 78% and the StTOM3 by 81%,demonstrating the efficient silencing of both StTOM1 and StTOM3.The availability of StTOM1 and StTOM3 knockdown potato provides basis for future verification of the host factor nature of these two genes to potato viruses and the potential of these genes in creating new anti-viral potato germplasms.
引文
Ambesajir A.,Kaushik A.,Kaushik J.J.,and Petros S.T.,2012,RNA interference:A futuristic tool and its therapeutic applications,Saudi Journal of Biological Sciences,19:395-403
Asano M.,Satoh R.,Mochizuki A.,Tsuda S.,Yamanaka T.,Nishiguchi M.,Hirai K.,Meshi T.,Naito S.,and Ishikawa M.,2005,Tobamovirus-resistant tobacco generated by RNA interference directed against host genes,FEBS Letters,579(20):4479-4484
Buck K.W.,1999,Replication of tobacco mosaic virus RNA,Philosophical Transactions of the Royal Society of London series B:Biological Sciences,354(1383):613-627
Chisholm S.T.,Parra M.A.,Anderberg R.J.,and Carrington J.C.,2001,Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus,Plant Physiology,127(4):1667-1675
Decroocq V.,Sicard O.,Alamillo J.M.,Lansac M.,Eyquard J.P.,Garcia J.A.,Candresse T.,Le Gall O.,and Revers F.,2006,Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana,Molecular Plant-Microbe Interactions,19(5):541-549
Fujisaki K.,Ravelo G.B.,Naito S.,and Ishikawa M.,2006,Involvement of THH1,an Arabidopsis thaliana homologue of the TOM1 gene,in tobamovirus multiplication,Journal of General Virology,87(8):2397-2401
Hagiwara Y.,Komoda K.,Yamanaka T.,Tamai A.,Meshi T.,Funada R.,Tsuchiya T.,Naito S.,and Ishikawa M.,2003,Subcellular localization of host and viral proteins associated with tobamovirus RNA replication,The EMBO Journal,22(2):344-353
Huang T.S.,Wei T.,Laliberte J.F.,and Wang A.,2010,A host RNA helicase-like protein,AtRH8,interacts with the potyviral genome-linked protein,VPg,associates with the virus accumulation complex,and is essential for infection,Plant Physiology,152(1):255-266
Kim D.H.,and Rossi J.J.,2009,Overview of gene silencing by RNA interference,Current Protocols in Nucleic Acid Chemistry,16(1):1-10
Meng J.,2005,The cloning and functional identification of a RNA virus host factor gene ToTOM3 from tomato,Dissertation for Ph.D.,South China Agricultural University,Supervisors:Chen B.S.,and Li H.P.,pp.116
Micheli F.,2001,Pectin methylesterases:Cell wall enzymes with important roles in plant physiology,Trends in Plant Science,6(9):414-419
Noueiry A.O.,and Ahlquist P.,2003,Brome mosaic virus RNA replication:Revealing the role of the host in RNA virus replication,Annual Review of Phytopathology,41(1):77-98
Roudet-Tavert G.,Michon T.,Walter J.,Delaunay T.,Redondo E.,and Le Gall O.,2007,Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro,Journal of General Virology,88(3):1029-1033
Tsujimoto Y.,Numaga T.,Ohshima K.,Yano M.A.,Ohsawa R.,Goto D.B.,Naito S.,and Ishikawa M.,2003,Arabidopsis TOBAMOVIRUS MULTIPLICATION(TOM)2 locus encodes a transmembrane protein that interacts with TOM1,The EMBO Journal,22(2):335-343
Yamanaka T.,Imai T.,Satoh R.,Kawashima A.,Takahashi M.,Tomita K.,Kubota K.,Meshi T.,Naito S.,and Ishikawa M.,2002,Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes,Journal of Virology,76(5):2491-2497
Yamanaka T.,Ohta T.,Takahashi M.,Meshi T.,Schmidt R.,Dean C.,Naito S.,and Ishikawa M.,2000,TOM1,an Arabidopsis gene required for efficient multiplication of a tobamovirus,encodes a putative transmembrane protein,Proc.Natl.Acad.Sci.USA,97(18):10107-10112
Yin Y.,Chory J.,and Baulcombe D.,2005,RNAi in transgenic plants,Current Protocols in Molecular Biology,26(6):1-19
Yoshii M.,Nishikiori M.,Tomita K.,Yoshioka N.,Kozuka R.,Naito S.,and Ishikawa M.,2004,The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G,Journal of Virology,78(12):6102-6111
Asano M.,Satoh R.,Mochizuki A.,Tsuda S.,Yamanaka T.,Nishiguchi M.,Hirai K.,Meshi T.,Naito S.,and Ishikawa M.,2005,Tobamovirus-resistant tobacco generated by RNA interference directed against host genes,FEBS Letters,579(20):4479-4484
Buck K.W.,1999,Replication of tobacco mosaic virus RNA,Philosophical Transactions of the Royal Society of London series B:Biological Sciences,354(1383):613-627
Chisholm S.T.,Parra M.A.,Anderberg R.J.,and Carrington J.C.,2001,Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus,Plant Physiology,127(4):1667-1675
Decroocq V.,Sicard O.,Alamillo J.M.,Lansac M.,Eyquard J.P.,Garcia J.A.,Candresse T.,Le Gall O.,and Revers F.,2006,Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana,Molecular Plant-Microbe Interactions,19(5):541-549
Fujisaki K.,Ravelo G.B.,Naito S.,and Ishikawa M.,2006,Involvement of THH1,an Arabidopsis thaliana homologue of the TOM1 gene,in tobamovirus multiplication,Journal of General Virology,87(8):2397-2401
Hagiwara Y.,Komoda K.,Yamanaka T.,Tamai A.,Meshi T.,Funada R.,Tsuchiya T.,Naito S.,and Ishikawa M.,2003,Subcellular localization of host and viral proteins associated with tobamovirus RNA replication,The EMBO Journal,22(2):344-353
Huang T.S.,Wei T.,Laliberte J.F.,and Wang A.,2010,A host RNA helicase-like protein,AtRH8,interacts with the potyviral genome-linked protein,VPg,associates with the virus accumulation complex,and is essential for infection,Plant Physiology,152(1):255-266
Kim D.H.,and Rossi J.J.,2009,Overview of gene silencing by RNA interference,Current Protocols in Nucleic Acid Chemistry,16(1):1-10
Meng J.,2005,The cloning and functional identification of a RNA virus host factor gene ToTOM3 from tomato,Dissertation for Ph.D.,South China Agricultural University,Supervisors:Chen B.S.,and Li H.P.,pp.116
Micheli F.,2001,Pectin methylesterases:Cell wall enzymes with important roles in plant physiology,Trends in Plant Science,6(9):414-419
Noueiry A.O.,and Ahlquist P.,2003,Brome mosaic virus RNA replication:Revealing the role of the host in RNA virus replication,Annual Review of Phytopathology,41(1):77-98
Roudet-Tavert G.,Michon T.,Walter J.,Delaunay T.,Redondo E.,and Le Gall O.,2007,Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro,Journal of General Virology,88(3):1029-1033
Tsujimoto Y.,Numaga T.,Ohshima K.,Yano M.A.,Ohsawa R.,Goto D.B.,Naito S.,and Ishikawa M.,2003,Arabidopsis TOBAMOVIRUS MULTIPLICATION(TOM)2 locus encodes a transmembrane protein that interacts with TOM1,The EMBO Journal,22(2):335-343
Yamanaka T.,Imai T.,Satoh R.,Kawashima A.,Takahashi M.,Tomita K.,Kubota K.,Meshi T.,Naito S.,and Ishikawa M.,2002,Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes,Journal of Virology,76(5):2491-2497
Yamanaka T.,Ohta T.,Takahashi M.,Meshi T.,Schmidt R.,Dean C.,Naito S.,and Ishikawa M.,2000,TOM1,an Arabidopsis gene required for efficient multiplication of a tobamovirus,encodes a putative transmembrane protein,Proc.Natl.Acad.Sci.USA,97(18):10107-10112
Yin Y.,Chory J.,and Baulcombe D.,2005,RNAi in transgenic plants,Current Protocols in Molecular Biology,26(6):1-19
Yoshii M.,Nishikiori M.,Tomita K.,Yoshioka N.,Kozuka R.,Naito S.,and Ishikawa M.,2004,The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G,Journal of Virology,78(12):6102-6111