针对水稻条纹病毒的人工miRNA设计与应用
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摘要
水稻条纹病毒(Rice stripe virus, RSV)引起的水稻条纹叶枯病是最重要的水稻病毒病之一,给我国水稻生产安全造成了巨大威胁。为了创制高抗RSV的水稻新种质,本研究首先根据天然microRNA (miRNA)的结构特点和作用原理,针对水稻条纹叶枯病毒基因组设计了人工miRNA(artificial microRNA, amiRNA),同时克隆了miRNA528基因的前体(pre-miRNA)。应用重叠PCR (over-lapping)技术并以这个水稻天然miRNA前体作为骨架,构建2个水稻转化载体(pAmiR-1和pAmiR-2),其中载体pAmiR-1是针对RSV p3基因；载体pAmiR-2是针对RSV pc3基因。通过农杆菌介导法将2个人工miRNA表达载体导入水稻品种武育粳3号,分子鉴定表明共获得219株转基因水稻植株。利用携带RSV的介体灰飞虱(Laodelphax striatellus, Fallen)对部分转基因株系进行抗病毒鉴定,结果表明共有22个转基因水稻株系表现为高抗。进一步通过实时定量RT-PCR(real-time RT-PCR)分析表明所设计构建的人工miRNA高效大量表达。
The disease caused by rice stripe virus is one of the most important viral diseases, and it is a threat to rice yield in our country. According to the theory of miRNA, several artificial microRNAs (amiRNAs) were designed for down-regulating the gene expression of RSV. The precursor of miRNA528 was cloned and used for construction of 2 amiRNA genes, amiR-1 and amiR-2, by over-lapping PCR. These genes were ligated into plasmid pBI121 respectively, The pAmiR-1 and pAmiR-2 are specific for interfering the expression of p3 and pc3 genes encoded by RSV RNA3. The 2 vectors were transferred into the japonica rice cultivar Wuyujing 3 by Agrobacterium-mediated transformation.219 transgenic rice plants (To) harbouring the amiRNAs were identified by PCR. The T1 transgenic plants were used to evaluate the resistance to rice stripe virus by artificial inoculation. The results showed that 22 plants are highly resistant to RSV. Real-time RT-PCR analysis suggested the transgenic amiRNA could be expressed at high level expressed in transgenic plants.
The disease caused by rice stripe virus is one of the most important viral diseases, and it is a threat to rice yield in our country. According to the theory of miRNA, several artificial microRNAs (amiRNAs) were designed for down-regulating the gene expression of RSV. The precursor of miRNA528 was cloned and used for construction of 2 amiRNA genes, amiR-1 and amiR-2, by over-lapping PCR. These genes were ligated into plasmid pBI121 respectively, The pAmiR-1 and pAmiR-2 are specific for interfering the expression of p3 and pc3 genes encoded by RSV RNA3. The 2 vectors were transferred into the japonica rice cultivar Wuyujing 3 by Agrobacterium-mediated transformation.219 transgenic rice plants (To) harbouring the amiRNAs were identified by PCR. The T1 transgenic plants were used to evaluate the resistance to rice stripe virus by artificial inoculation. The results showed that 22 plants are highly resistant to RSV. Real-time RT-PCR analysis suggested the transgenic amiRNA could be expressed at high level expressed in transgenic plants.
引文
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[3]林奇英,谢联辉,周仲驹等.水稻条纹叶枯病的研究.病毒的分布和损失.福建农业学报[J],1990,19(4)：421-425
[4]Toriyama S. The Association of App lied Biologists (AAB) Descrip tions of Plant Viruses Journal of General Virology[J],1983,26 (9):124-125
[5]Toriyama S. An RNA dependent RNA polymerase associated with the filamentous nucleop roteins of rice stripe virus Journal of General Virology[J],1986,67 (7): 1247-1255
[6]Ishikawa K, Omura T, Tsuchizaki T. Association of double and single-stranded RNAs with each four components of rice stripe virus. [M],1989,35 (3):315-333
[7]Toriyama S, Watanabe Y. Characterization of single and double stranded RNAs in particles of rice stripe virus.Journal of General Virology [J],1989,70 (3):505-511
[8]Hammamatsu C, Toriyama S, Toyoda T. Ambisense coding strategy of the rice stripe virus genome:in vitro translation studies.Journal of General Virology[J], 1993,74(6):1125-1131
[9]Liang D, Ma X Q, Qu Z. Nucleic acid binding p roperty of the gene products of rice stripe virus.Virus Genes [J],2005,31 (2):203-209
[10]Zhang H M, Yang J, Xin X, et al. Genomic analysis of rice stripe virus Zhejiang isolate shows the p resence of an OUT-like domain in the p rotein encoded by RNA1 and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses. Achive of Virology[J],2007,10:1007-1013
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[13]Bucher E, Sijen T, de Haan P. Negative-strand tospoviruses and tenuiviruses carry a gene for a supp ressor of gene silencing at analogous genomic positions. Journal of Virology[J],2003,77 (2):1329-1336
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[15]TakahashiM, Goto C, Ishikawa K. R ice stripe virus 23.9K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells. Archives of Virology [J],2003,148 (11):2167-2179
[16]Koganezawa H. Purification and p roperties of rice stripe virus. Tropical Agriculture Research[J],1977,10:151-154
[17]Kiso A, Yamamoto T. Infection and symp tom development in rice stripe disease, with special reference to disease-specific protein other than virus. Review of Plant Protection Research[J],1973,6:75-100
[18]林含新,林奇田,吴祖建,等.水稻条纹病毒病害特异蛋白的提纯及血清学特性.中国病毒学[J],1999,14(3)：222-229
[19]LauN C, Lim LP, WeinsteinEG. An abundant class of tiny RNAs wim probable regulatory roles in Caenorhabditis elegans.Science[J],2001,294 (5543):858-862
[20]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell[J],1993, 75:843-854
[21]Reinhart BJ, Slack FA, Basson M, Pasquinelli AE, BettingerJC, Rougvie AC, Horvitz HR, Ruvkun G. The 21 nucleotide let-7 RNA regulates C. elegans developmental timing in Caenorhabditis elegans. Nature[J],2000,403 (6772): 901-906
[22]Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl.Identification of Novel Genes Coding for Small Expressed RNAs.Science[J],2001,294:853-858
[23]Lau Nelson C, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science [J],2001,294:858-869
[24]Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN. MicroRNA genes are transcribed by RNA polymerase Ⅱ. EMBO[J],2004,23:4051-4060
[25]Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC. Expression of Arabidopsis MIRNA genes.Plant Physiol[J],2005,138:2145-2154.
[26]Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl Acad.Sci.USA[J],2004,101:12753-12758
[27]Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat.Rev. Mol. Cell Biol[J],2005,6:376-385
[28]Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW,Steward R, Chen X. Methylation as a crucial step in plant microRNA biogenesis. Science[J],2005, 307:932-935
[29]Li J, Yang Z, Yu B, Liu J, Chen X. Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis. Curr. Biol[J].2005,15: 1501-1507.
[30]Bartel D P. MicmRNAs genomics, biogenesis, mechanism, and function. Cell[J],2004,116(2):281-297
[31]Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotechnol[J],2006,24:1420-1428
[32]Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell[J],2002,9:1327-1333
[33]Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O. In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev[J].2004,18: 2237-2242
[34]Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y. Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell[J],2006, 18:1134-1151.
[35]Schwab R, Ossowski S, Riester M, WarthmannN, Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell[J],2006,18: 1121-1133.
[36]Donis-Keller H. Site specific enzymatic cleavage of RNA.Nucleic Acids Res[J], 1979,7:179-192
[37]Ameres SL, Martinez J, Schroeder R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell[J],2007,130:101-112
[38]Long D, Lee R, Williams P, Chan CY, Ambros V, Ding Y. Potent effect of target structure on microRNA function. Nat.Struct. Mol. Biol[J],2007,14:287-294
[39]Ossowski S, Schwab S, Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs.Plant Journal[J],2008,53:674-690
[40]Voinnet O. Non-cell autonomous RNA silencing. FEBS Lett[J].2005,579: 5858-5871
[41]Liu Y, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato. Plant. J Nucleic Acids Res [J],2002,31:777-786
[42]Choi K, Park C, Lee J, Oh M, Noh B,Lee I. Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development. Development. Planta [J],2007,134:1931-1941
[43]Byzova M, Verduyn C, De Brouwer D, De Block M. Transforming petals into sepaloid organs in Arabidopsis and oilseed rape:implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner. Planta[J], 2004,218:379-387
[44]Mathieu J, Warthmann N, Kuttner F, Schmid M. Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr. Biol[J],2007,17:1055-1060.
[45]Prins, M. Broad virus resistance in transgenic plants. Trends Biotechnol[J].2003, 21:373-375
[46]卢扬江,郑康乐.提取水稻DNA的一种简易方法.中国水稻科学[J],1992,(1)：47-48
[47]张恒木,孙焕然,王华弟,陈剑平.水稻条纹病毒分子生物学研究进展.植物保护学报[J],2007,8：436-440
[48]Xiong R Y, Wu J X, Zhou Y J.Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus.Virology [J],2009, 387:29-40
[49]Xiong Ruyi, Wu Jianxiang, Zhou Yijun.Identification of a Movement Protein of the Tenuivirus Rice Stripe Virus.Journal of Virology[J],2008,498:12304-12311
[50]Reinhart BJ, Slack FA, Basson M, Pasquinelli AE, BettingerJC, Rougvie AC, Horvitz HR, Ruvkun G. The 21 nucleotide let-7 RNA regulates C. elegans developmental timing in Caenorhabditis elegans. Nature[J],2000,403(6772): 901-906
[51]Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl.Identification of Novel Genes Coding for Small Expressed RNAs. Science[J],2001,294:853-858
[52]Lau Nelson C, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science[J], 2001,294:858-869
[2]阮义理,金登迪,许如银.水稻条纹叶枯病毒的寄主植物.植物保护[J],1984,10(3)：22-23
[3]林奇英,谢联辉,周仲驹等.水稻条纹叶枯病的研究.病毒的分布和损失.福建农业学报[J],1990,19(4)：421-425
[4]Toriyama S. The Association of App lied Biologists (AAB) Descrip tions of Plant Viruses Journal of General Virology[J],1983,26 (9):124-125
[5]Toriyama S. An RNA dependent RNA polymerase associated with the filamentous nucleop roteins of rice stripe virus Journal of General Virology[J],1986,67 (7): 1247-1255
[6]Ishikawa K, Omura T, Tsuchizaki T. Association of double and single-stranded RNAs with each four components of rice stripe virus. [M],1989,35 (3):315-333
[7]Toriyama S, Watanabe Y. Characterization of single and double stranded RNAs in particles of rice stripe virus.Journal of General Virology [J],1989,70 (3):505-511
[8]Hammamatsu C, Toriyama S, Toyoda T. Ambisense coding strategy of the rice stripe virus genome:in vitro translation studies.Journal of General Virology[J], 1993,74(6):1125-1131
[9]Liang D, Ma X Q, Qu Z. Nucleic acid binding p roperty of the gene products of rice stripe virus.Virus Genes [J],2005,31 (2):203-209
[10]Zhang H M, Yang J, Xin X, et al. Genomic analysis of rice stripe virus Zhejiang isolate shows the p resence of an OUT-like domain in the p rotein encoded by RNA1 and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses. Achive of Virology[J],2007,10:1007-1013
[11]Toriyama S, TakahashiM, Sano Y. Nucleotide sequence of RNA1, the largest genomic segment of rice stripe virus, the p rototype of the tenuiviruses. Journal of General Virology[J],1994,75(12):3569-3579
[12]Liang D, Qu Z, Ma X. Detection and localization of rice stripe virus gene products in vivo. Virus Genes[J],2005,31 (2):211-221
[13]Bucher E, Sijen T, de Haan P. Negative-strand tospoviruses and tenuiviruses carry a gene for a supp ressor of gene silencing at analogous genomic positions. Journal of Virology[J],2003,77 (2):1329-1336
[14]曲志才,沈大棱,徐亚南.水稻条叶枯病毒基因产物在水稻和昆虫体内的Western印迹分析.遗传学报[J],1999,26(5)：512-517
[15]TakahashiM, Goto C, Ishikawa K. R ice stripe virus 23.9K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells. Archives of Virology [J],2003,148 (11):2167-2179
[16]Koganezawa H. Purification and p roperties of rice stripe virus. Tropical Agriculture Research[J],1977,10:151-154
[17]Kiso A, Yamamoto T. Infection and symp tom development in rice stripe disease, with special reference to disease-specific protein other than virus. Review of Plant Protection Research[J],1973,6:75-100
[18]林含新,林奇田,吴祖建,等.水稻条纹病毒病害特异蛋白的提纯及血清学特性.中国病毒学[J],1999,14(3)：222-229
[19]LauN C, Lim LP, WeinsteinEG. An abundant class of tiny RNAs wim probable regulatory roles in Caenorhabditis elegans.Science[J],2001,294 (5543):858-862
[20]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell[J],1993, 75:843-854
[21]Reinhart BJ, Slack FA, Basson M, Pasquinelli AE, BettingerJC, Rougvie AC, Horvitz HR, Ruvkun G. The 21 nucleotide let-7 RNA regulates C. elegans developmental timing in Caenorhabditis elegans. Nature[J],2000,403 (6772): 901-906
[22]Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl.Identification of Novel Genes Coding for Small Expressed RNAs.Science[J],2001,294:853-858
[23]Lau Nelson C, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science [J],2001,294:858-869
[24]Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN. MicroRNA genes are transcribed by RNA polymerase Ⅱ. EMBO[J],2004,23:4051-4060
[25]Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC. Expression of Arabidopsis MIRNA genes.Plant Physiol[J],2005,138:2145-2154.
[26]Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl Acad.Sci.USA[J],2004,101:12753-12758
[27]Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat.Rev. Mol. Cell Biol[J],2005,6:376-385
[28]Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW,Steward R, Chen X. Methylation as a crucial step in plant microRNA biogenesis. Science[J],2005, 307:932-935
[29]Li J, Yang Z, Yu B, Liu J, Chen X. Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis. Curr. Biol[J].2005,15: 1501-1507.
[30]Bartel D P. MicmRNAs genomics, biogenesis, mechanism, and function. Cell[J],2004,116(2):281-297
[31]Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotechnol[J],2006,24:1420-1428
[32]Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell[J],2002,9:1327-1333
[33]Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O. In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev[J].2004,18: 2237-2242
[34]Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y. Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell[J],2006, 18:1134-1151.
[35]Schwab R, Ossowski S, Riester M, WarthmannN, Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell[J],2006,18: 1121-1133.
[36]Donis-Keller H. Site specific enzymatic cleavage of RNA.Nucleic Acids Res[J], 1979,7:179-192
[37]Ameres SL, Martinez J, Schroeder R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell[J],2007,130:101-112
[38]Long D, Lee R, Williams P, Chan CY, Ambros V, Ding Y. Potent effect of target structure on microRNA function. Nat.Struct. Mol. Biol[J],2007,14:287-294
[39]Ossowski S, Schwab S, Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs.Plant Journal[J],2008,53:674-690
[40]Voinnet O. Non-cell autonomous RNA silencing. FEBS Lett[J].2005,579: 5858-5871
[41]Liu Y, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato. Plant. J Nucleic Acids Res [J],2002,31:777-786
[42]Choi K, Park C, Lee J, Oh M, Noh B,Lee I. Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development. Development. Planta [J],2007,134:1931-1941
[43]Byzova M, Verduyn C, De Brouwer D, De Block M. Transforming petals into sepaloid organs in Arabidopsis and oilseed rape:implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner. Planta[J], 2004,218:379-387
[44]Mathieu J, Warthmann N, Kuttner F, Schmid M. Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr. Biol[J],2007,17:1055-1060.
[45]Prins, M. Broad virus resistance in transgenic plants. Trends Biotechnol[J].2003, 21:373-375
[46]卢扬江,郑康乐.提取水稻DNA的一种简易方法.中国水稻科学[J],1992,(1)：47-48
[47]张恒木,孙焕然,王华弟,陈剑平.水稻条纹病毒分子生物学研究进展.植物保护学报[J],2007,8：436-440
[48]Xiong R Y, Wu J X, Zhou Y J.Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus.Virology [J],2009, 387:29-40
[49]Xiong Ruyi, Wu Jianxiang, Zhou Yijun.Identification of a Movement Protein of the Tenuivirus Rice Stripe Virus.Journal of Virology[J],2008,498:12304-12311
[50]Reinhart BJ, Slack FA, Basson M, Pasquinelli AE, BettingerJC, Rougvie AC, Horvitz HR, Ruvkun G. The 21 nucleotide let-7 RNA regulates C. elegans developmental timing in Caenorhabditis elegans. Nature[J],2000,403(6772): 901-906
[51]Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl.Identification of Novel Genes Coding for Small Expressed RNAs. Science[J],2001,294:853-858
[52]Lau Nelson C, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science[J], 2001,294:858-869