PRSV编码基因同源dsRNA的原核表达及其抗番木瓜环斑病毒的研究
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摘要
番木瓜环斑病毒(Papaya Ringspot Virus, PRSV)是属于马铃薯Y病毒(genus Potyvirus, family Potyviridae)的一种植物病毒,为单链正义RNA, PRSV引起的番木瓜环斑病病毒病在全球各生长区给番木瓜种植业造成了严重的损失,病毒编码的复制酶(NIb)、辅助蛋白(HC-Pro)和外壳蛋白(CP)等参与蚜虫介导的传毒、病毒在宿主细胞内复制、积累及细胞间运动等过程,在病毒为害番木瓜的过程中发挥着重要作用。人们在生产实践中积累的控制PRSV的常规方法大多是限制蚜虫介导的传播或销毁病株,仅能在一定程度上控制病毒的蔓延,并不能直接消除病毒,非常被动。番木瓜杂交抗病毒育种因其种内缺乏抗病资源、种间杂交不亲和等原因,一直进展缓慢,尚未得到适合大面积推广应用的高抗品系。基因工程技术克服了番木瓜抗病毒种质资源缺乏的困难,充分利用了来自病毒和其他种属植物的抗病毒基因,为番木瓜抗病毒育种开辟了新的有效途径。但转基因抗病毒番木瓜的抗性谱较窄,能高抗PRSV某一株系的转基因番木瓜品系往往对其它地区的其他PRSV株系无效或只有低度抗病性,且转基因番木瓜植株潜在的生态风险和食品安全问题也饱受争议,限制了基因工程技术的应用。番木瓜生产中迫切需要既适合于大田生产、又行之有效的PRSV防治方法。本研究利用dsRNA介导的RNA沉默抗病毒机制,构建了PRSV编码基因同源dsRNA的原核表达系统,并使用诱导表达的同源dsRNA处理番木瓜植株,研究了利用原核表达的dsRNA防治番木瓜环斑病毒病的可行性。
通过对已报道PRSV株系的NIb基因、HC-Pro基因和CP基因的序列同源性比对,发现这3个编码基因的3’端序列的同源性均较高。本研究通过PCR扩增得到了PRSV HN株系NIb基因、HC-Pro基因和CP基因3’端的N312、N501、N809、H315、H489、 H824、C279、C432、C867等9个核苷酸序列,利用OZ-LIC法分别构建了与N312、N501、 N809、H315、H489、H824、C279、C432、C867同源的发夹RNA编码结构(含有PDK内含子),以M-JM1091acY菌株为宿主菌分别构建了高效的PRSV编码基因同源dsRNA原核表达系统M-Jm109LacY/pSP73-RNAi-N312、M-Jm109LacY/pSP73-RNAi-N501、 M-Jm109LacY/pSP73-RNAi-N809、M-Jm109LacY/pSP73-RNAi-H315、 M-Jm109LacY/pSP73-RNAi-H489、M-Jm109LacY/pSP73-RNAi-H824、 M-Jm109LacY/pSP73-RNAi-C279、M-Jm109LacY/pSP73-RNAi-C432、 M-Jm109LacY/pSP73-RNAi-C867。经IPTG诱导成功利用原核系统表达了dsRNA,并证明dsRNA不被DNase I和RNase A降解,稳定性较好。
本研究分别用PRSV Nib基因、HC-Pro基因、CP基因构建了GFP瞬时植物融合表达载体pNIb-GFP、pHC-GFP、pCP-GFP。对经Trizol法提取的dsRNA和对应GFP瞬时融合表达载体共转化的番木瓜叶片原生质体的共聚焦显微镜观察及通过半定量RT-PCR对其中mRNA表达量的分析结果证实：融合基因NIb-GFP、HC-GFP、CP-GFP的表达均发生了不同程度的下调,说明dsRNA在原生质体中引发了针对同源基因的沉默。
本研究采用高压细胞破碎法处理经IPTG诱导的dsRNA原核表达工程菌细胞制备不同的dsRNA粗制品,利用dsRNA粗制品分别在接种PRSV前对番木瓜植株进行保护性处理、在表现环斑病症状后对番木瓜植株进行治疗性处理,并实施了田间的保护性处理实验。保护性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRNA、 H315-dsRNA、H489-dsRNA、H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组分别表现出了27%、50%、60%、20%、44%、50%、27%、40%、54%的抗病性,ELISA分析和Real-time PCR结果证实实验组番木瓜叶片中的病毒积累受到了不同程度的抑制,Northern杂交实验结果说明其抗性是由RNA沉默引起的抗性。治疗性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRN、H315-dsRNA、H489-dsRNA、 H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组在喷洒后dsRNA后第3天均检测到病毒积累量的小幅下降,第10天左右有部分染病植株叶片出现回绿或新长出的叶片没有症状,但随后病毒积累量又恢复至对照水平,回绿叶片重新花叶,无症状叶片也表现出症状,Northern杂交结果表明病毒积累量的下降是RNA沉默所致。田间保护性实验结果显示,定期喷洒N809-dsRNA粗制品使番木瓜植株获得了稳定抵抗PRSV的能力,在近距离生长的对照植株发病的情况下处理植株仍能保持不发病,长期保持较好长势,效果良好,证明该方法是一种绿色、廉价、便捷、有效的番木瓜环斑病毒防治方法。以上这些研究结果为利用dsRNA引发RNA沉默防治番木瓜环斑病毒的田间应用奠定了基础。
Papaya ringspot virus (PRSV) is a plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV causes severe economic losses in papaya throughout the tropics and subtropics. PRSV disease management practices include quarantine, eradication, avoidance by planting papaya in areas isolated from the virus, continual rogueing of infected plants, use of tolerant lines to lower the economic losses caused by PRSV, crossprotection and transgenic resistance. Development of PRSV-resistant cultivars through conventional breeding met with limited success because of difficulties in overcoming intergeneric reproductive barriers of wild, related species of papaya. In addition, partial loss of tolerance in back-crosses with the commercial papaya parent also limits the usefulness of this approach. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. We provide a strategy to develop effective and stable PRSV-resistant plants via dsRNAs expressed in Escherichia coli.
In this study,9different dsRNA prokaryotic expression vectors carrying different sizes of PRSV-NIb(312,501,809bp), PRSV-HC-Pro(315,489,824bp) and PRSV-CP(279,432,867bp)cDNA were constructed, which were named as, pSP73-RNAi-N312, pSP73-RNAi-N501, pSP73-RNAi-N809, pSP73-RNAi-H315, pSP73-RNAi-H489, pSP73-RNAi-H824, pSP73-RNAi-C279, pSP73-RNAi-C432, pSP73-RNAi-C867respectively. These prokaryotic expression vectors were transformed into the RNaseⅢ-deficient strain M-Jm109LacY, and then were induced with IPTG, respectively. All these E.coli strains deficient for RNaseⅢ could express the predicted sizes of PRSV-NIb-dsRNA, PRSV-HC-Pro-dsRNA and PRSV-CP-dsRNA, respectively.
To monitor the silencing efficiency of these9different bacterially expressed dsRNAs derived from PRSV-Nib, PRSV-HC-Pro and PRSV-CP, we developed an effective transient gene silencing system for using the bacterially expressed dsRNA in papaya leaf protoplasts. Three plant transient expression vectors encoding the PRSV-NIb, PRSV-HC-Pro and PRSV-CP with an N-terminal GFP fusion were constructed respectively, which were named as pNIb-GFP, pHC-GFP and pCP-GFP respectively. Protoplasts were co-transfected using the transient GFP-fusion expression vector and the corresponding bacterially expressed dsRNA. The results of GFP fluorescence intensity using a confocal microscope and semi-quantitative RT-PCR analysis of the PRSV-Nib, PRSV-HC-Pro and PRSV-CP mRNA expression showed that the effective bacterially expressed dsRNA-triggered gene silencing was detected.
To prove whether the bacterial-produced dsRNA could interfere with PRSV infection, we carry out a protective resistance assay and a therapeutic resistance assay. In the protective resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surface before inoculation of papaya leaves with PRSV. Protective treatment experimental results showed dsRNAs derived from the different functional genes of PRSV could all protect papaya plants from virus infection, and the resistance was obviously different due to different vectors. ELISA analysis and Real-time PCR results confirmed that the virus accumulation could be inhibited in different degree by dsRNA. Northern blot showed that the small RNA fragments specifically complementary to the PRSV-Nib, PRSV-HC-Pro and PRSV-CP antisense probe were detected, which suggested that the resistance is an RNA-mediated virus resistance. In the therapeutic resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surfaces after inoculation of papaya leaves with PRSV. ELISA analysis and Real-time PCR results showed that the virus accumulation declined slightly after spraying dsRNA for three days. Field protective experimental results showed that regular spraying the crude product of the bacterially expressed N809-dsRNA could obtain stable resistance to PRSV.
Our work indicated that bacterially expressed double-stranded RNA targeting three viral genes of PRSV could interfere with PRSV infection, which is a potential green and effective approach protecting plants from virus infections compared with the requirements for regenerating PRSV-resistant transgenic plants.
通过对已报道PRSV株系的NIb基因、HC-Pro基因和CP基因的序列同源性比对,发现这3个编码基因的3’端序列的同源性均较高。本研究通过PCR扩增得到了PRSV HN株系NIb基因、HC-Pro基因和CP基因3’端的N312、N501、N809、H315、H489、 H824、C279、C432、C867等9个核苷酸序列,利用OZ-LIC法分别构建了与N312、N501、 N809、H315、H489、H824、C279、C432、C867同源的发夹RNA编码结构(含有PDK内含子),以M-JM1091acY菌株为宿主菌分别构建了高效的PRSV编码基因同源dsRNA原核表达系统M-Jm109LacY/pSP73-RNAi-N312、M-Jm109LacY/pSP73-RNAi-N501、 M-Jm109LacY/pSP73-RNAi-N809、M-Jm109LacY/pSP73-RNAi-H315、 M-Jm109LacY/pSP73-RNAi-H489、M-Jm109LacY/pSP73-RNAi-H824、 M-Jm109LacY/pSP73-RNAi-C279、M-Jm109LacY/pSP73-RNAi-C432、 M-Jm109LacY/pSP73-RNAi-C867。经IPTG诱导成功利用原核系统表达了dsRNA,并证明dsRNA不被DNase I和RNase A降解,稳定性较好。
本研究分别用PRSV Nib基因、HC-Pro基因、CP基因构建了GFP瞬时植物融合表达载体pNIb-GFP、pHC-GFP、pCP-GFP。对经Trizol法提取的dsRNA和对应GFP瞬时融合表达载体共转化的番木瓜叶片原生质体的共聚焦显微镜观察及通过半定量RT-PCR对其中mRNA表达量的分析结果证实：融合基因NIb-GFP、HC-GFP、CP-GFP的表达均发生了不同程度的下调,说明dsRNA在原生质体中引发了针对同源基因的沉默。
本研究采用高压细胞破碎法处理经IPTG诱导的dsRNA原核表达工程菌细胞制备不同的dsRNA粗制品,利用dsRNA粗制品分别在接种PRSV前对番木瓜植株进行保护性处理、在表现环斑病症状后对番木瓜植株进行治疗性处理,并实施了田间的保护性处理实验。保护性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRNA、 H315-dsRNA、H489-dsRNA、H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组分别表现出了27%、50%、60%、20%、44%、50%、27%、40%、54%的抗病性,ELISA分析和Real-time PCR结果证实实验组番木瓜叶片中的病毒积累受到了不同程度的抑制,Northern杂交实验结果说明其抗性是由RNA沉默引起的抗性。治疗性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRN、H315-dsRNA、H489-dsRNA、 H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组在喷洒后dsRNA后第3天均检测到病毒积累量的小幅下降,第10天左右有部分染病植株叶片出现回绿或新长出的叶片没有症状,但随后病毒积累量又恢复至对照水平,回绿叶片重新花叶,无症状叶片也表现出症状,Northern杂交结果表明病毒积累量的下降是RNA沉默所致。田间保护性实验结果显示,定期喷洒N809-dsRNA粗制品使番木瓜植株获得了稳定抵抗PRSV的能力,在近距离生长的对照植株发病的情况下处理植株仍能保持不发病,长期保持较好长势,效果良好,证明该方法是一种绿色、廉价、便捷、有效的番木瓜环斑病毒防治方法。以上这些研究结果为利用dsRNA引发RNA沉默防治番木瓜环斑病毒的田间应用奠定了基础。
Papaya ringspot virus (PRSV) is a plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV causes severe economic losses in papaya throughout the tropics and subtropics. PRSV disease management practices include quarantine, eradication, avoidance by planting papaya in areas isolated from the virus, continual rogueing of infected plants, use of tolerant lines to lower the economic losses caused by PRSV, crossprotection and transgenic resistance. Development of PRSV-resistant cultivars through conventional breeding met with limited success because of difficulties in overcoming intergeneric reproductive barriers of wild, related species of papaya. In addition, partial loss of tolerance in back-crosses with the commercial papaya parent also limits the usefulness of this approach. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. We provide a strategy to develop effective and stable PRSV-resistant plants via dsRNAs expressed in Escherichia coli.
In this study,9different dsRNA prokaryotic expression vectors carrying different sizes of PRSV-NIb(312,501,809bp), PRSV-HC-Pro(315,489,824bp) and PRSV-CP(279,432,867bp)cDNA were constructed, which were named as, pSP73-RNAi-N312, pSP73-RNAi-N501, pSP73-RNAi-N809, pSP73-RNAi-H315, pSP73-RNAi-H489, pSP73-RNAi-H824, pSP73-RNAi-C279, pSP73-RNAi-C432, pSP73-RNAi-C867respectively. These prokaryotic expression vectors were transformed into the RNaseⅢ-deficient strain M-Jm109LacY, and then were induced with IPTG, respectively. All these E.coli strains deficient for RNaseⅢ could express the predicted sizes of PRSV-NIb-dsRNA, PRSV-HC-Pro-dsRNA and PRSV-CP-dsRNA, respectively.
To monitor the silencing efficiency of these9different bacterially expressed dsRNAs derived from PRSV-Nib, PRSV-HC-Pro and PRSV-CP, we developed an effective transient gene silencing system for using the bacterially expressed dsRNA in papaya leaf protoplasts. Three plant transient expression vectors encoding the PRSV-NIb, PRSV-HC-Pro and PRSV-CP with an N-terminal GFP fusion were constructed respectively, which were named as pNIb-GFP, pHC-GFP and pCP-GFP respectively. Protoplasts were co-transfected using the transient GFP-fusion expression vector and the corresponding bacterially expressed dsRNA. The results of GFP fluorescence intensity using a confocal microscope and semi-quantitative RT-PCR analysis of the PRSV-Nib, PRSV-HC-Pro and PRSV-CP mRNA expression showed that the effective bacterially expressed dsRNA-triggered gene silencing was detected.
To prove whether the bacterial-produced dsRNA could interfere with PRSV infection, we carry out a protective resistance assay and a therapeutic resistance assay. In the protective resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surface before inoculation of papaya leaves with PRSV. Protective treatment experimental results showed dsRNAs derived from the different functional genes of PRSV could all protect papaya plants from virus infection, and the resistance was obviously different due to different vectors. ELISA analysis and Real-time PCR results confirmed that the virus accumulation could be inhibited in different degree by dsRNA. Northern blot showed that the small RNA fragments specifically complementary to the PRSV-Nib, PRSV-HC-Pro and PRSV-CP antisense probe were detected, which suggested that the resistance is an RNA-mediated virus resistance. In the therapeutic resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surfaces after inoculation of papaya leaves with PRSV. ELISA analysis and Real-time PCR results showed that the virus accumulation declined slightly after spraying dsRNA for three days. Field protective experimental results showed that regular spraying the crude product of the bacterially expressed N809-dsRNA could obtain stable resistance to PRSV.
Our work indicated that bacterially expressed double-stranded RNA targeting three viral genes of PRSV could interfere with PRSV infection, which is a potential green and effective approach protecting plants from virus infections compared with the requirements for regenerating PRSV-resistant transgenic plants.
引文
1. Akbergenov R, et al. Molecular characterization of geminivirusderived small RNAs in different plant species. Nucleic Acids Res,2006,34:462.
2. Bau H J, et al. Field evaluation transgenic papaya lins carrying the coat protein gene of papaya ringspot virus in Taiwan. Plant Disease, 2004,88(6):594.
3. Baulcombe DC. RNA silencing in plants. Nature,2004,431:356.
4. Bor ja M, et al. Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Mol Plant-Microbe Interact,1999,12:153.
5. Chang L S. Response of papaya cultivars to inoculation with the SMN papaya ringspot virus strain. Fruit Varieties Journal,1996,50(2):80.
6. Chen B, et al. Cucumovirus transmission by the aphid Myzus persicae is determined solely by the coat protein. J Gen Virol,1990,71:939.
7. Chen G, et al. Cloning of the papaya ringspot virus replicase gene and generation of PRSV-resistant papayas through the in troduction of the PRSV replicase gene. Plant Cell Reports,2001,20(3):272.
8. Conover R A, et al. Carif lora-a papaya Ringspot virus-tolerant papaya for south Florida and the Caribbean. Hortscience,1986,21(4):1072.
9. Conover R A, et al. Progress in breading papayas with tolerance to papaya ringspot virus. Proc. Fla. State Hort. Soc,1978,91:182.
10. Daiguan Y, et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Nat lAcad Sci USA,2000,97(11): 5978.
11. Datsenko K A, et al. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA,2000, 97(12):6640.
12. De La Rosa M, et al. Purification and partial characterization of papaya ringspot virus. Phytopathol Z,1983,106:329.
13. Dillon S, et al. Genetic mapping of a PRSV-P resistance gene in "high land papaya" based on inheritance of RAF markers. Euphytica: International Journal of Plant Breeding,2005,145(1-2):11.
14. Dunn J J. RNase III cleavage of single-stranded RNA:Effect of ionic strength on the fidelity of cleavage. J Biol Chem,1976,251 (12):3807.
15. Falk B W, et al.Will transgenic crops generate new viruses and new diseases? Science,1994,163:1395.
16. Ferm in G, et al. Engineered resistance against papaya ringspot virus in Venzuelan transgenic papayas. Plant Disease,2004,88:516.
17. Francisco Tenllado, et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol,2003,3(1):3.
18. Gama M IC. Cruzamento de especies de Carica para resistencia ao virus do mosaico do mamoeiro. Fitopatolog ia B rasilerira. Rev. Soc. Bras Fitopatologia,1985,10(2):318.
19. Gang Wu, et al. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development,2006,133:3539
20. Golemboski DB, et al. Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci U S A.1990 Aug;87(16):6311.
21. Greene A E, et al. F. Recombination between viral RNA and transgenic plant transcripts. Sciencel994,263(5152):1423.
22. Griffiths-Jones S. The microRNA registry. Nucleic Acids Res,2006,32: 109.
23. Guo HS, et al. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell,2005,17:1376.
24. Han J, et al. Kim VN Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell,2006,125:887.
25. Hang H L, Yeh S D, Chiu R J, et al. Effectiveness of cross-protection by mild ringspot virus in Taiwan, Plant Disease,1987,71:491.
26. Ian JM. et al. Ribonuclease revisited:structural in sights into ribonuclease III family enzymes. Curr Opin Struct Biol,2007,17(1): 138.
27. Jain R K, et al. First report of occurrence of papaya ringspot virus infecting papaya in Bangladesh. Plant Disease,2004,88(4):221.
28. Jain R K, et al. Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India. Archives of virology, 2004,149(12):2435.
29. Jarvis T C, et al. Poliovirus RNA recombination:mechanistic studies in the absence of selection. EMBO J,1992,11:3135.
30. Javier F. Palatnik, et al. Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319. Development cell,2007,13:115.
31. Jones-Rhoades MW, et al. MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol,2006,57:19.
32. Kameda T, et al. A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi. Biochem Biophys Res Commun,2004,315:599.
33. KD Kasschau, et al. P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA. Developmental Cell,2003,4:205.
34. Konstantinova P, et al. Trans-inhibition of HIV-I by a long hairpin RNA expressed within the viral genome. Retrovirology,2007 Mar 1;4:15.
35. Ling K, et al. Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene. Biotechnology (N Y).1991 Aug;9(8):752
36. Lawson C. et al. Engineering resistance to mixed virus infection in a commercial potato cultivar:resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Biotechnology (N Y).1990 Feb;8(2):127.
37. Lee Y, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature,2003,425:415.
38. Manshardt R, et al. Papaya breeding for PRSV resistance. Actahorticulture,1995,370:27.
39. MaokaT, et al. Occurrence of the Pstra in of papaya ringspot virus in Japan. Ann Phytopathol Soc. Japan,1995,61:91.
40. Merai Z, et al. Double stranded RNA binding, may be a general plant RNA viral strategy to suppress RNA silencing. J Virol,2006,80(12): 5747.
41. Miller A W, et al. Are there risks associated with transgenic resistance to luteoviruses?. Plant Disease,1997,81(7):700.
42. Moissiard G, et al. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Ara-bidopsis Dicer-like proteins. Proc Natl Acad Sci USA,2006,103:19593.
43. Mourrain P, et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell. 2000 May 26;101(5):533
44. Niu QW, et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. NatBiotech,2006, 24:1420.
45. Oliverira A C, et al. Resistance to papaya ringspot virus in summer squash Cucurbita pepo L. introgressed from an interspecific C. pepo x C. moschata cross. Euphytica:International Jioumal of Plant Breeding, 2003,132 (2):211.
46. Poteete A R, et al. Genetic requirements of phage lambdared-mediated gene replacement in Escherichia coli K-12. J Bacteriol,2000, 182(8): 2336.
47. Pruss G, et al. Plant viral synergism:the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell,1997,9:859.
48. Purcifull D E, et al. Papaya ringspot virus. Discription of plant viruses. CMI/ABB,1985:292.
49. Schwab R, et al. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell,2006,18:1121.
50. Sheen T F, Wang H L, Wang D N. Control of papaya ringspot virus by cross protection and cultivation techniques. Review of Plant Pathology, 1999,78(3):294.
51. Sijen T. et al. Post-transcriptional gene-silencing:RNAs on the attack or on the defense? Bioessays.2000 Jun;22(6):520.
52. Simon-Mateo C, et al. MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism. mVirol,2006,80(5):2429.
53. Singh I P, et al. HPSC-3:a high yielding new papaya hybrid for Tripura. H. A.,1996,67(10):1154.
54. Smith N A, et al. Total silencing by intronspliced hairpin RNAs. Nature, 2000,407:319.
55.SouzaM T Jr, et al. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates. HortScience,2005,40(7):2083.
56. Szittya G, et al. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO,2003,22:633.
57. Talianky M E, et al. Role of cucumovirus capsid protein in long-distance movement within the infected plant. Journal of Virology,1995,69:916.
58. Tenllado F, et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnology, 2003,3:3.
59. Tenllado F, et al. RNA interference as new biotechnological tool for the control of virus disease in plants. Virus Res,2004:85.
60. Tenllado F, et al. Transient Expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing. MPMI,2003,16:49.
61.Tennant P, et al. Line 63-1:a new virus-resistant transgenic papaya. HortScience,2005,40(5):1196.
62. Timmons L, et al. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabdtis elegans. Gene,2001,263(1-2):103.
63. Van den Boogaart T. et al. Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing. Mol Plant Microbe Interact.2001 Feb;14(2):196.
64. Wang Der-nan. Screen of papaya varieties for ringspot virus tolerance. Agric. Res,1982,31(2):162.
65. Wesley S V, et al. Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal,2001, 27 (6):581.
66. Xie Z, et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol,2004,2:642.
67. Xu G. et al. One-step, zero-background ligation-independent cloning intron-containing hairpin RNA constructs for RNAi in plants. New Phytol.2010 Jul;187(1):240
68. Yang L, et al. Serrate is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J,2006,47:841.
69.Yi R, et al. Overexpression of exportin 5 enhancesRNA interference mediated by short hairpin RNAs and microRNAs. RNA,2005,11:220.
70. Zee F. Breeding for papaya ringspot virus tolerancein solo papayas, Carica papaya L. Manoa:University of Hawaii,1985:105.
71. Zhou ZS, et al. Identification of an RNA-silencing suppressor in the genome of Grapevine virus A. J. Gen Virol,2006,87(Pt 8):2387.
72.蔡良琬等.超声波辐射对大分子核糖核酸的影响.生物化学与生物物理学报,1964,4(1)：16.
73.陈健主编.番木瓜品种与栽培彩色图说.中国农业出版社,2002.10.
74.甘德芳等.玉米矮花叶病毒CP基因dsRNA的原核表达与分离.激光生物学报,2011(3)
75.黄冰艳等.转录后沉默(PTGS)及其在作物遗传改良中的应用.中国生物工程杂志,2005,2(5)：1.
76.黄建昌等.番木瓜抗PRSV育种研究进展与展望.福建果树,2006,139：24.
77.黄建昌,等.60Co-γ射线射线与GA3复合处理对番木瓜的遗传诱变效应研究.核农学报,2003(3)：10.
78.黄建昌,等.抗环斑型花叶病毒病番木瓜新品种选育研究初报.西南农业大学学报,2004(1)：25.
79.乐宁等.水稻OsNPR1基因RNA干涉载体的构建及其对水稻的转化.广西农业生物科学,2005,24(4)：269.
80.李小平等.利用RNA干扰技术敲减rlpk2基因的表达可以延缓大豆叶片衰老.科学通报,2005,50(11)：1090.
81.林冠雄,等.我国番木瓜育种研究进展与展望.广东农业科学,2005(4)：22.
82.饶雪琴,等.番木瓜环斑病毒融合基因植物表达载体的构建.华中农业大学学报,2005,24(4)：325.
83.任佩喻,等.番木瓜花叶病毒初步调查研究.植物保护学报,1964,3(4)：432.
84.阮小蕾,等.转PRSV复制酶基因T2番木瓜植株的抗病性测定.华南农业大学学报,2004,25(4)：12.
85.韦金菊等.番木瓜品种对PRSV抗性的苗期鉴定.中国热带农业,2007(4)： 38.
86.魏军亚,等.花粉管通道法介导PRSV-CP基因dsRNA转化番木瓜.西北植物学报,2008,28(11)：2159.
87.肖火根,等.番木瓜环斑病毒株系、分子生物学及其防治研究进展.中国病毒学,1996,11(2)：104.
88.肖艳等.番木瓜环斑型花叶病毒(PRSV)的为害特征及分子生物学.福建果树,2006,138(3)：11.
89.熊华.木瓜蛋白酶应用研究进展.保鲜与加工,2006(1)：7.
90.叶长明,等.番木瓜环斑病毒外壳蛋白基因的构建.植物病理学报,1991,21(3)：161.
91.叶长明,等.转基因番木瓜的抗病性及分子鉴定.遗传,2003,25(2)：181.
92.尹国华等.利用Red重组系统敲除大肠杆菌rnc基因构建dsRNA原核表达体系.山东农业大学学报(自然科学版),2009,40(3)：461.
93.张德咏等.表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染.植物病理学报,2008(6)：304.
94.张帆,等.dsRNA介导的番木瓜环斑病毒(PRSV)的抗病性研究.植物科学学报,2011,29(3)：385.
95.张建波.番木瓜叶肉原生质体分离以及GFP瞬时表达研究.海南大学,2011年.
96.郑海刚等.诱导提取的dsRNA粗提液对黄瓜绿斑驳花叶病毒的抑制作用.福建农林大学学报(自然科学版),2011,40(4)：346.
97.周国辉,等.番木瓜环斑花叶病突变体抗性遗传及RAPD标记.植物病理学报,2001,31(2)：157.
98.周鹏,等.PRSV-CP-SN转基因番木瓜表达与抗病能力的研究.热带作物学报,1996,17(2)：84.
99.朱俊华等.马铃薯Y病毒衣壳蛋白基因片段长度对RNA介导抗病性的影响.中国科学C辑生命科学2004,34(1)：23.
100.朱西儒,等.番木瓜杂交果座籽与发芽及F1代的环斑花叶病调查研究.广西热作科技,1999,71(2)：5.
101.周雪平.RNA沉默及其利用.中国科学C辑生命科学2012,42(1)：1.
2. Bau H J, et al. Field evaluation transgenic papaya lins carrying the coat protein gene of papaya ringspot virus in Taiwan. Plant Disease, 2004,88(6):594.
3. Baulcombe DC. RNA silencing in plants. Nature,2004,431:356.
4. Bor ja M, et al. Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Mol Plant-Microbe Interact,1999,12:153.
5. Chang L S. Response of papaya cultivars to inoculation with the SMN papaya ringspot virus strain. Fruit Varieties Journal,1996,50(2):80.
6. Chen B, et al. Cucumovirus transmission by the aphid Myzus persicae is determined solely by the coat protein. J Gen Virol,1990,71:939.
7. Chen G, et al. Cloning of the papaya ringspot virus replicase gene and generation of PRSV-resistant papayas through the in troduction of the PRSV replicase gene. Plant Cell Reports,2001,20(3):272.
8. Conover R A, et al. Carif lora-a papaya Ringspot virus-tolerant papaya for south Florida and the Caribbean. Hortscience,1986,21(4):1072.
9. Conover R A, et al. Progress in breading papayas with tolerance to papaya ringspot virus. Proc. Fla. State Hort. Soc,1978,91:182.
10. Daiguan Y, et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Nat lAcad Sci USA,2000,97(11): 5978.
11. Datsenko K A, et al. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA,2000, 97(12):6640.
12. De La Rosa M, et al. Purification and partial characterization of papaya ringspot virus. Phytopathol Z,1983,106:329.
13. Dillon S, et al. Genetic mapping of a PRSV-P resistance gene in "high land papaya" based on inheritance of RAF markers. Euphytica: International Journal of Plant Breeding,2005,145(1-2):11.
14. Dunn J J. RNase III cleavage of single-stranded RNA:Effect of ionic strength on the fidelity of cleavage. J Biol Chem,1976,251 (12):3807.
15. Falk B W, et al.Will transgenic crops generate new viruses and new diseases? Science,1994,163:1395.
16. Ferm in G, et al. Engineered resistance against papaya ringspot virus in Venzuelan transgenic papayas. Plant Disease,2004,88:516.
17. Francisco Tenllado, et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol,2003,3(1):3.
18. Gama M IC. Cruzamento de especies de Carica para resistencia ao virus do mosaico do mamoeiro. Fitopatolog ia B rasilerira. Rev. Soc. Bras Fitopatologia,1985,10(2):318.
19. Gang Wu, et al. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development,2006,133:3539
20. Golemboski DB, et al. Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci U S A.1990 Aug;87(16):6311.
21. Greene A E, et al. F. Recombination between viral RNA and transgenic plant transcripts. Sciencel994,263(5152):1423.
22. Griffiths-Jones S. The microRNA registry. Nucleic Acids Res,2006,32: 109.
23. Guo HS, et al. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell,2005,17:1376.
24. Han J, et al. Kim VN Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell,2006,125:887.
25. Hang H L, Yeh S D, Chiu R J, et al. Effectiveness of cross-protection by mild ringspot virus in Taiwan, Plant Disease,1987,71:491.
26. Ian JM. et al. Ribonuclease revisited:structural in sights into ribonuclease III family enzymes. Curr Opin Struct Biol,2007,17(1): 138.
27. Jain R K, et al. First report of occurrence of papaya ringspot virus infecting papaya in Bangladesh. Plant Disease,2004,88(4):221.
28. Jain R K, et al. Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India. Archives of virology, 2004,149(12):2435.
29. Jarvis T C, et al. Poliovirus RNA recombination:mechanistic studies in the absence of selection. EMBO J,1992,11:3135.
30. Javier F. Palatnik, et al. Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319. Development cell,2007,13:115.
31. Jones-Rhoades MW, et al. MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol,2006,57:19.
32. Kameda T, et al. A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi. Biochem Biophys Res Commun,2004,315:599.
33. KD Kasschau, et al. P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA. Developmental Cell,2003,4:205.
34. Konstantinova P, et al. Trans-inhibition of HIV-I by a long hairpin RNA expressed within the viral genome. Retrovirology,2007 Mar 1;4:15.
35. Ling K, et al. Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene. Biotechnology (N Y).1991 Aug;9(8):752
36. Lawson C. et al. Engineering resistance to mixed virus infection in a commercial potato cultivar:resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Biotechnology (N Y).1990 Feb;8(2):127.
37. Lee Y, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature,2003,425:415.
38. Manshardt R, et al. Papaya breeding for PRSV resistance. Actahorticulture,1995,370:27.
39. MaokaT, et al. Occurrence of the Pstra in of papaya ringspot virus in Japan. Ann Phytopathol Soc. Japan,1995,61:91.
40. Merai Z, et al. Double stranded RNA binding, may be a general plant RNA viral strategy to suppress RNA silencing. J Virol,2006,80(12): 5747.
41. Miller A W, et al. Are there risks associated with transgenic resistance to luteoviruses?. Plant Disease,1997,81(7):700.
42. Moissiard G, et al. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Ara-bidopsis Dicer-like proteins. Proc Natl Acad Sci USA,2006,103:19593.
43. Mourrain P, et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell. 2000 May 26;101(5):533
44. Niu QW, et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. NatBiotech,2006, 24:1420.
45. Oliverira A C, et al. Resistance to papaya ringspot virus in summer squash Cucurbita pepo L. introgressed from an interspecific C. pepo x C. moschata cross. Euphytica:International Jioumal of Plant Breeding, 2003,132 (2):211.
46. Poteete A R, et al. Genetic requirements of phage lambdared-mediated gene replacement in Escherichia coli K-12. J Bacteriol,2000, 182(8): 2336.
47. Pruss G, et al. Plant viral synergism:the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell,1997,9:859.
48. Purcifull D E, et al. Papaya ringspot virus. Discription of plant viruses. CMI/ABB,1985:292.
49. Schwab R, et al. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell,2006,18:1121.
50. Sheen T F, Wang H L, Wang D N. Control of papaya ringspot virus by cross protection and cultivation techniques. Review of Plant Pathology, 1999,78(3):294.
51. Sijen T. et al. Post-transcriptional gene-silencing:RNAs on the attack or on the defense? Bioessays.2000 Jun;22(6):520.
52. Simon-Mateo C, et al. MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism. mVirol,2006,80(5):2429.
53. Singh I P, et al. HPSC-3:a high yielding new papaya hybrid for Tripura. H. A.,1996,67(10):1154.
54. Smith N A, et al. Total silencing by intronspliced hairpin RNAs. Nature, 2000,407:319.
55.SouzaM T Jr, et al. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates. HortScience,2005,40(7):2083.
56. Szittya G, et al. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO,2003,22:633.
57. Talianky M E, et al. Role of cucumovirus capsid protein in long-distance movement within the infected plant. Journal of Virology,1995,69:916.
58. Tenllado F, et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnology, 2003,3:3.
59. Tenllado F, et al. RNA interference as new biotechnological tool for the control of virus disease in plants. Virus Res,2004:85.
60. Tenllado F, et al. Transient Expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing. MPMI,2003,16:49.
61.Tennant P, et al. Line 63-1:a new virus-resistant transgenic papaya. HortScience,2005,40(5):1196.
62. Timmons L, et al. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabdtis elegans. Gene,2001,263(1-2):103.
63. Van den Boogaart T. et al. Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing. Mol Plant Microbe Interact.2001 Feb;14(2):196.
64. Wang Der-nan. Screen of papaya varieties for ringspot virus tolerance. Agric. Res,1982,31(2):162.
65. Wesley S V, et al. Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal,2001, 27 (6):581.
66. Xie Z, et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol,2004,2:642.
67. Xu G. et al. One-step, zero-background ligation-independent cloning intron-containing hairpin RNA constructs for RNAi in plants. New Phytol.2010 Jul;187(1):240
68. Yang L, et al. Serrate is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J,2006,47:841.
69.Yi R, et al. Overexpression of exportin 5 enhancesRNA interference mediated by short hairpin RNAs and microRNAs. RNA,2005,11:220.
70. Zee F. Breeding for papaya ringspot virus tolerancein solo papayas, Carica papaya L. Manoa:University of Hawaii,1985:105.
71. Zhou ZS, et al. Identification of an RNA-silencing suppressor in the genome of Grapevine virus A. J. Gen Virol,2006,87(Pt 8):2387.
72.蔡良琬等.超声波辐射对大分子核糖核酸的影响.生物化学与生物物理学报,1964,4(1)：16.
73.陈健主编.番木瓜品种与栽培彩色图说.中国农业出版社,2002.10.
74.甘德芳等.玉米矮花叶病毒CP基因dsRNA的原核表达与分离.激光生物学报,2011(3)
75.黄冰艳等.转录后沉默(PTGS)及其在作物遗传改良中的应用.中国生物工程杂志,2005,2(5)：1.
76.黄建昌等.番木瓜抗PRSV育种研究进展与展望.福建果树,2006,139：24.
77.黄建昌,等.60Co-γ射线射线与GA3复合处理对番木瓜的遗传诱变效应研究.核农学报,2003(3)：10.
78.黄建昌,等.抗环斑型花叶病毒病番木瓜新品种选育研究初报.西南农业大学学报,2004(1)：25.
79.乐宁等.水稻OsNPR1基因RNA干涉载体的构建及其对水稻的转化.广西农业生物科学,2005,24(4)：269.
80.李小平等.利用RNA干扰技术敲减rlpk2基因的表达可以延缓大豆叶片衰老.科学通报,2005,50(11)：1090.
81.林冠雄,等.我国番木瓜育种研究进展与展望.广东农业科学,2005(4)：22.
82.饶雪琴,等.番木瓜环斑病毒融合基因植物表达载体的构建.华中农业大学学报,2005,24(4)：325.
83.任佩喻,等.番木瓜花叶病毒初步调查研究.植物保护学报,1964,3(4)：432.
84.阮小蕾,等.转PRSV复制酶基因T2番木瓜植株的抗病性测定.华南农业大学学报,2004,25(4)：12.
85.韦金菊等.番木瓜品种对PRSV抗性的苗期鉴定.中国热带农业,2007(4)： 38.
86.魏军亚,等.花粉管通道法介导PRSV-CP基因dsRNA转化番木瓜.西北植物学报,2008,28(11)：2159.
87.肖火根,等.番木瓜环斑病毒株系、分子生物学及其防治研究进展.中国病毒学,1996,11(2)：104.
88.肖艳等.番木瓜环斑型花叶病毒(PRSV)的为害特征及分子生物学.福建果树,2006,138(3)：11.
89.熊华.木瓜蛋白酶应用研究进展.保鲜与加工,2006(1)：7.
90.叶长明,等.番木瓜环斑病毒外壳蛋白基因的构建.植物病理学报,1991,21(3)：161.
91.叶长明,等.转基因番木瓜的抗病性及分子鉴定.遗传,2003,25(2)：181.
92.尹国华等.利用Red重组系统敲除大肠杆菌rnc基因构建dsRNA原核表达体系.山东农业大学学报(自然科学版),2009,40(3)：461.
93.张德咏等.表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染.植物病理学报,2008(6)：304.
94.张帆,等.dsRNA介导的番木瓜环斑病毒(PRSV)的抗病性研究.植物科学学报,2011,29(3)：385.
95.张建波.番木瓜叶肉原生质体分离以及GFP瞬时表达研究.海南大学,2011年.
96.郑海刚等.诱导提取的dsRNA粗提液对黄瓜绿斑驳花叶病毒的抑制作用.福建农林大学学报(自然科学版),2011,40(4)：346.
97.周国辉,等.番木瓜环斑花叶病突变体抗性遗传及RAPD标记.植物病理学报,2001,31(2)：157.
98.周鹏,等.PRSV-CP-SN转基因番木瓜表达与抗病能力的研究.热带作物学报,1996,17(2)：84.
99.朱俊华等.马铃薯Y病毒衣壳蛋白基因片段长度对RNA介导抗病性的影响.中国科学C辑生命科学2004,34(1)：23.
100.朱西儒,等.番木瓜杂交果座籽与发芽及F1代的环斑花叶病调查研究.广西热作科技,1999,71(2)：5.
101.周雪平.RNA沉默及其利用.中国科学C辑生命科学2012,42(1)：1.