BYDVs编码的两种抑制子及其作用机制研究
|
摘要
由大麦黄矮病毒(Barley yellow dwarf viruses,BYDVs)引起的小麦黄矮病在世界范围内广泛发生,在我国小麦产区间隙性大发生,威胁小麦的生产。为最终达到防治该病害的目的,各国的科学家们对大麦黄矮病毒的基因表达、进化、传播介体与寄主的相互作用等方面开展了深入的基础研究。筛选鉴定出BYDVs基因组中可能存在的沉默抑制子,可为研究BYDV基因与寄主的互作提供新的证据。
本实验室已经初步鉴定出BYDV-GAV的ORF6和BYDV-GPV的P0在局部侵染中具有沉默抑制子活性。在已构建好的质粒载体中筛选出pGD-GPV-P0、pGD-GPV-CP、pGD-GPV-MP、pBin-GAV-CP、pBin-GAV-ORF1和pBin-GAV-ORF6共6个蛋白基因的重组载体,为增加实验的严谨性,本实验新构建了pGD-GPV-UTR-P0重组载体;其次实验通过重复沉默表型观察证明了GPV的P0和GAV的ORF6在局部侵染中确实具有抑制子活性。随后采用半定量PCR对浸润区GFP的mRNA水平进行了检测,发现强抑制子蛋白HC-Pro与pBin-mGFP5-ER共同浸润本生烟后,浸润区GFP的mRNA水平明显比pBin-GAV-ORF6或pGD-GPV-P0与pBin-mGFP5-ER共浸润叶片后GFP mRNA的积累水平要高,其中接种pGD-GPV-P0与pBin-mGFP5- ER共浸润叶片后GFP mRNA的积累水平比pBin-GAV-ORF6与pBin-mGFP5- ER的积累水平高。而作为RNA定量标准的内参基因EF-1-α的mRNA水平在不同浸润组合的植株中都是相同的。结果表明GAV-ORF6抑制子的活性比GPV-P0弱。GFP蛋白的Western blot和GFP mRNA、siRNA的Northern blot分析结果进一步证实了GPV-P0与GAV-ORF6表现出的抑制子活性的差异。虽然GPV-P0、GAV-ORF6的表达与阳性对照HC-Pro一样,能够增加GFP蛋白的表达量和GFP mRNA的累积量,进而增强GFP荧光,但从实验可以看出GPV-P0这种抑制子的GFP蛋白的表达量以及GFP mRNA的累积量明显高于GAV-ORF6。表明GPV-P0是抑制子而GAV-ORF6是弱抑制子。另外,分别将pGD-GPV-P0、pBin-GAV-ORF6与含GFP的植物表达载体pBin-mGFP5- ER对四叶期转基因烟草16c植株共浸润,并以HC-Pro作为对照。在侵染16天后对非浸润叶片进行了Northern blot检测,也证明GPV的P0、GAV的ORF6与PVY的HC-Pro一样能抑制由GFP正单链RNA引起的系统性基因沉默。
此外,为研究BYDV的基因组结构中基因突变(插入、缺失、重复等)对病毒复制和表达策略的影响、验证病毒的蛋白功能以及确定重要功能结构域,建立BYDVs侵染性cDNA克隆技术平台尤为重要。本文以PAV-06KM14分离物为对象,成功构建了pTCK303-06KM14 (ubi启动子控制下)和pGD-06KM14全长基因组侵染性克隆载体,分别进行基因枪转染小麦幼胚或农杆菌侵染本生烟以获得具有侵染性的植株。目前实验已获得似BYDV症状的植株,侵染活性验证实验正在进行当中。
Wheat yellow dwarf disease caused by barley yellow dwarf viruses (BYDVs) takes place worldwide, and occurs intermittently in china’s main wheat-growing area, threatening wheat production. To achieve the final purpose of preventing and curing this disease, scientists have carried out in-depth researches on gene expression, evolution, and the interaction between transmission vector and host in barley yellow dwarf virus. Screening out silencing suppressors possibly present in BYDV genome can provide new evidence for the study of interaction between BYDV genes and the host.
Through preliminary detection, ORF6 in BYDV-GAV and P0 in BYDV-GPV have been identified that they have silencing suppressor activity. On this basis, experimental materials are optimized firstly, and six recombinant vectors P0, CP, MP of GPV with pGD as vector and CP, ORF1, ORF6 of GAV with pBinPLUS as vector are screened out and a new vector pGD-UTR-P0 of GPV is reconstructed to make the experiment more precise and accurate. Besides, through repeating silence phenotype observation, GPV-P0 and GAV-ORF6 is proved to possess silencing suppressor activity in local infection. Then semi-quantitative PCR is used to detect mRNA level of GFP in infiltrating area, and it is found that GFP mRNA accumulation level when strong suppressor protein HC-Pro and pBin-mGFP5-ER are altogether infiltration Nicotiana benthamiana is higher than pBin-GAV-ORF6 or pGD-GPV-P0 and pBin-mGFP5-ER are altogether infiltrated blades, and GFP mRNA accumulation level infiltrated with pGD-GPV-P0 and pBin-mGFP5-ER is higher than that infiltrated with pBin-GAV-ORF6 and pBin-mGFP5- ER. mRNA level of reference genes EF-1-α, quantitative criteria of RNA, is the same in different infiltrating combination of plants. The result indicates that the activity of suppressor GAV-ORF6 is weaker than GPV-P0. The analysis results from Western blot of GFP protein and Northern blot of GFP mRNA and siRNA further confirm the differences in the suppressor activity expressed by GPV-P0 and GAV-ORF6. Though the expression of GPV- P0 and GAV-ORF6, the same as the positive control, can increase the expression and accumulation of GFP protein, the GFP mRNA accumulation, and further strengthens GFP fluorescence. It can also be seen from the experiment that the expression, accumulation and GFP mRNA accumulation of GFP protein of the suppressor GPV-P0 is obviously higher than that of GAV-ORF6. In addition, pGD-GPV-P0, pBin-GAV-ORF6 and the plant expression vector containing GFP (pBin-mGFP5- ER) are respectively used to infiltrate tobacco 16c plant at four-leaf stage with HC-Pro as a control sample. 16 days after the infiltration, Northern blot test to the non-infection leaves proves that GPV-P0, GAV-ORF6, the same as PVY HC-Pro, can restrain the systemic gene silencing caused by single-strand RNA of GFP.
In addition, to study the effect of gene mutation (insertion, loss, repetition) of BYDV genome structure on virus replication and its expression strategy, verify the protein function of virus, and confirm the important function domains, it is particularly important to establish BYDVs infectious cDNA clone technology platform. This paper takes isolate PAV-06KM14 as the subject, successfully constructs full-length genome infectious clone vectors pTCK303-06KM14(under control of ubi promoter) and pGD-06KM14, and respectively transfects wheat embryo with Particle gun or infect Nicotiana benthamiana with agrobacterium infiltration to obtain the infective plants. Recently plants with similar BYDV symptoms have been received, and the verification test of the infective activity is underway.
本实验室已经初步鉴定出BYDV-GAV的ORF6和BYDV-GPV的P0在局部侵染中具有沉默抑制子活性。在已构建好的质粒载体中筛选出pGD-GPV-P0、pGD-GPV-CP、pGD-GPV-MP、pBin-GAV-CP、pBin-GAV-ORF1和pBin-GAV-ORF6共6个蛋白基因的重组载体,为增加实验的严谨性,本实验新构建了pGD-GPV-UTR-P0重组载体;其次实验通过重复沉默表型观察证明了GPV的P0和GAV的ORF6在局部侵染中确实具有抑制子活性。随后采用半定量PCR对浸润区GFP的mRNA水平进行了检测,发现强抑制子蛋白HC-Pro与pBin-mGFP5-ER共同浸润本生烟后,浸润区GFP的mRNA水平明显比pBin-GAV-ORF6或pGD-GPV-P0与pBin-mGFP5-ER共浸润叶片后GFP mRNA的积累水平要高,其中接种pGD-GPV-P0与pBin-mGFP5- ER共浸润叶片后GFP mRNA的积累水平比pBin-GAV-ORF6与pBin-mGFP5- ER的积累水平高。而作为RNA定量标准的内参基因EF-1-α的mRNA水平在不同浸润组合的植株中都是相同的。结果表明GAV-ORF6抑制子的活性比GPV-P0弱。GFP蛋白的Western blot和GFP mRNA、siRNA的Northern blot分析结果进一步证实了GPV-P0与GAV-ORF6表现出的抑制子活性的差异。虽然GPV-P0、GAV-ORF6的表达与阳性对照HC-Pro一样,能够增加GFP蛋白的表达量和GFP mRNA的累积量,进而增强GFP荧光,但从实验可以看出GPV-P0这种抑制子的GFP蛋白的表达量以及GFP mRNA的累积量明显高于GAV-ORF6。表明GPV-P0是抑制子而GAV-ORF6是弱抑制子。另外,分别将pGD-GPV-P0、pBin-GAV-ORF6与含GFP的植物表达载体pBin-mGFP5- ER对四叶期转基因烟草16c植株共浸润,并以HC-Pro作为对照。在侵染16天后对非浸润叶片进行了Northern blot检测,也证明GPV的P0、GAV的ORF6与PVY的HC-Pro一样能抑制由GFP正单链RNA引起的系统性基因沉默。
此外,为研究BYDV的基因组结构中基因突变(插入、缺失、重复等)对病毒复制和表达策略的影响、验证病毒的蛋白功能以及确定重要功能结构域,建立BYDVs侵染性cDNA克隆技术平台尤为重要。本文以PAV-06KM14分离物为对象,成功构建了pTCK303-06KM14 (ubi启动子控制下)和pGD-06KM14全长基因组侵染性克隆载体,分别进行基因枪转染小麦幼胚或农杆菌侵染本生烟以获得具有侵染性的植株。目前实验已获得似BYDV症状的植株,侵染活性验证实验正在进行当中。
Wheat yellow dwarf disease caused by barley yellow dwarf viruses (BYDVs) takes place worldwide, and occurs intermittently in china’s main wheat-growing area, threatening wheat production. To achieve the final purpose of preventing and curing this disease, scientists have carried out in-depth researches on gene expression, evolution, and the interaction between transmission vector and host in barley yellow dwarf virus. Screening out silencing suppressors possibly present in BYDV genome can provide new evidence for the study of interaction between BYDV genes and the host.
Through preliminary detection, ORF6 in BYDV-GAV and P0 in BYDV-GPV have been identified that they have silencing suppressor activity. On this basis, experimental materials are optimized firstly, and six recombinant vectors P0, CP, MP of GPV with pGD as vector and CP, ORF1, ORF6 of GAV with pBinPLUS as vector are screened out and a new vector pGD-UTR-P0 of GPV is reconstructed to make the experiment more precise and accurate. Besides, through repeating silence phenotype observation, GPV-P0 and GAV-ORF6 is proved to possess silencing suppressor activity in local infection. Then semi-quantitative PCR is used to detect mRNA level of GFP in infiltrating area, and it is found that GFP mRNA accumulation level when strong suppressor protein HC-Pro and pBin-mGFP5-ER are altogether infiltration Nicotiana benthamiana is higher than pBin-GAV-ORF6 or pGD-GPV-P0 and pBin-mGFP5-ER are altogether infiltrated blades, and GFP mRNA accumulation level infiltrated with pGD-GPV-P0 and pBin-mGFP5-ER is higher than that infiltrated with pBin-GAV-ORF6 and pBin-mGFP5- ER. mRNA level of reference genes EF-1-α, quantitative criteria of RNA, is the same in different infiltrating combination of plants. The result indicates that the activity of suppressor GAV-ORF6 is weaker than GPV-P0. The analysis results from Western blot of GFP protein and Northern blot of GFP mRNA and siRNA further confirm the differences in the suppressor activity expressed by GPV-P0 and GAV-ORF6. Though the expression of GPV- P0 and GAV-ORF6, the same as the positive control, can increase the expression and accumulation of GFP protein, the GFP mRNA accumulation, and further strengthens GFP fluorescence. It can also be seen from the experiment that the expression, accumulation and GFP mRNA accumulation of GFP protein of the suppressor GPV-P0 is obviously higher than that of GAV-ORF6. In addition, pGD-GPV-P0, pBin-GAV-ORF6 and the plant expression vector containing GFP (pBin-mGFP5- ER) are respectively used to infiltrate tobacco 16c plant at four-leaf stage with HC-Pro as a control sample. 16 days after the infiltration, Northern blot test to the non-infection leaves proves that GPV-P0, GAV-ORF6, the same as PVY HC-Pro, can restrain the systemic gene silencing caused by single-strand RNA of GFP.
In addition, to study the effect of gene mutation (insertion, loss, repetition) of BYDV genome structure on virus replication and its expression strategy, verify the protein function of virus, and confirm the important function domains, it is particularly important to establish BYDVs infectious cDNA clone technology platform. This paper takes isolate PAV-06KM14 as the subject, successfully constructs full-length genome infectious clone vectors pTCK303-06KM14(under control of ubi promoter) and pGD-06KM14, and respectively transfects wheat embryo with Particle gun or infect Nicotiana benthamiana with agrobacterium infiltration to obtain the infective plants. Recently plants with similar BYDV symptoms have been received, and the verification test of the infective activity is underway.
引文
1.常胜军,王锡锋,李莉,等.大麦黄矮病毒GAV株系通读蛋白基因的序列分析及其在大肠杆菌中的表达.中国农业科学, 2000, 33(4):38-45.
2.成卓敏,周广和.小麦黄矮病毒GPV株系的提纯及血清学研究.病毒学报, 1986,2(3): 275-277.
3.成卓敏,周广和.小麦黄矮病毒GPV株系的提纯及血清学研究.病毒学报, 1986,2(3): 275-277.
4.成卓敏,吴茂森,等.应用基因枪法获得抗大麦黄矮病毒转基因小麦.植物病理学报, 2000,30(2):116-121.
5.崔晓峰,周雪平.(2005). RNA沉默的病毒抑制子.生物化学与生物物理进展, 32 (3):210-216.
6.崔晓峰,周雪平.中国番茄黄化曲叶病毒和烟草曲茎病毒AC2和AC4蛋白为RNA沉默的抑制子.科学通报, 2004,49(23): 2431-2436.
7.董家红.小麦黄花叶病毒侵染性cDNA克隆及细胞侵染体系的建立.[博士论文].中国农业大学,2004.
8.晋治波,王锡锋,常胜军,周广和.大麦黄矮病毒GAV基因组全序列测定及其结构分析.中国科学(C辑), 20033,3(6):505-513.
9.梁新苗.SCMV-BJ全长cDNA克隆的构建及其部分基因与致病性关系的探索. [博士论文].中国农业大学,2005.
10.廖乾生,等.黄瓜花叶病毒辣椒分离物侵染性克隆构建.中国农业科学,2008 41(7):1975-1982.
11.刘福秀,赵芹,等.水稻瘤矮病毒基因组第11片段编码一个RNA沉默抑制子.科学通报,2008,53(1) : 96-103.
12.刘双清.大麦黄矮病毒三种株系的特异性检测及PAV株系的群体遗传变异.[硕士论文].湖北农业大学, 2008.
13.刘涛.利用侵染性cDNA研究甜菜坏死黄脉病毒RNA5与病毒致病性的关系. [博士论文].中国农业大学,2003.
14.牛颜冰,青玲,等.植物病毒抑制子抑制RNA沉默的机制及生物学应用.分子植物育种, 20097, (1):137-142.
15.邵碧英,等.().烟草花叶病毒及其弱毒株基因组的cDNA克隆和序列分析.植物病理学报, 2003,33(4):296-301.
16.孙博.大麦黄矮病毒群体的分子变异. [硕士论文].中国农业科学院,2006.
17.田荣欢,刘迪秋,等.植物病毒基因沉默抑制子研究进展.生物技术通报, 2010 ,(4):11-15.
18.王献兵,张凌娣,李大伟,等.植物病毒编码的RNA沉默抑制因子.中国农业大学学报, 2005,10(4):31-38.
19.吴蓓蕾,王锡锋.植物病毒基因沉默抑制因子研究进展.植物保护, 2007,33(2): 1-6.
20.吴蓓蕾.小麦矮缩病毒分子群体遗传结构与RNAi介导的抗BYDV-GAV转基因小麦研究.[博士论文].中国农业科学院, 2007.
21.席德慧,曹云鹤,等.(2006).甜菜黑色焦枯病毒新疆分离物基因组RNA的序列分析及侵染性cDNA克隆构建.病毒学报,22(4):309-313.
22.席德慧,等.黄瓜花叶病毒卫星RNA XJ s1侵染性cDNA克隆构建及其生物学功能初步研究.微生物学报, 2006,46 (2):219-222.
23.席德慧,林宏辉,等.(2006).黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究.微生物学报,46(2):219-222.
24.熊庆,周雪平.假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子.微生物学报,2007,47 (1): 39-43.
25.薛朝阳,周雪平,等.一种病毒侵染性全长cDNA克隆的快速构建方法.生物化学与生物物理学报, 2000,32(3):270-274.
26.燕飞,宋雪梅,成卓敏.番茄丛矮病毒p19蛋白抑制转录后基因沉默作用机制.病毒学报, 2005,21 (5):403-405.
27.张凌娣,王朝辉,王献兵,等.两种植物病毒编码蛋白的基因沉默抑制子功能鉴定.科学通报, 2005,50(3): 219-224.
28.张文蔚,成卓敏.大麦黄矮病毒GPV株系基因组末端序列的克隆和分析.中国农业科技导报, 2009,11 (1):102-107.
29.赵琨.大麦黄矮病毒LAMP检测及其基因沉默抑制子鉴定.[硕士论文].中国农业科学院, 2010.
30.周广和,张淑香,钱幼亭.小麦黄矮病毒4种株系鉴定与应用.中国农业科学, 1987, 20 (4) :7 - 12.
31.庄木,王晓武,等.转录后基因沉默及植物病毒对它的抑制.生物技术通报, 2002, (6):18-22.1580-1589.
32. Ahlquist, P., French, R., Janda, M., and Loesch-Fries, L. S. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc. Natl. Acad. Sci. USA, 1984, 81: 7066-7070.
33. Aleksnadra,M., DzianPtt., Jozef, J.,et al.Derivation of an inefctious vrial RNA by autolytic cleavage of in vitro transcribed vrial cDNAs. Proc. Natl.Acad. Sci, 1989,USA 86: 4823-4827.
34. Baumberger, N., Tsai, C. H., Lie, M., Havecker, E., and Baulcombe, D. C., The Polerovirus Silencing Suppressor P0 Targets ARGONAUTE Proteins for Degradation. Curr. Biol, 2007 ,17:1609-1614.
35. Beck, D. L., Forster, R. L. S., Bevan, M. W., Boxen, K. A. & Lowe, S. C. Infectious transcripts and nucleotide sequence of cloned cDNA of the potexvirus white clover mosaic virus. Virology, 1990,177 :152-158.
36. Beilei Wu, Alexandra Blanchard-Letort, Yan Liu, Guanghe Zhou, Xifeng Wang, Santiago F. Elena. Dynamics of Molecular Evolution and Phylogeography of Barley yellow dwarf virus-PAV. PLoS ONE 2011,6(2):e16896
37. Bernstein E, Caudy A, Hammond S M, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001,409 : 363-366.
38. Bevan, M. Binary Agrobacterium vectors for plant transformation.Nucleic Acids Research, 1984, 12: 8711-8721.
39. Bortolamiol, D., Pazhouhandeh, M., Marrocco, K., Genschik, P., and Ziegler-Graff, V., The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing. Curr. Biol, 2007, 17:1615-1621.
40. Boscia D, Savino V, Minafra A, Namba S, Elicio V, Castellano MA, Gonsalves D,Martelli GP . Properties of a filamentous virus isolated from grapevines affected by corky bark. Arch Virol, 1993, 130:109-120.
41. Boyer J, Haenni A .Infectious transcripts and cDNA clones of RNA viruses.Virology, 1994,198 :415-426.
42. Brault, V., and Miller, W. A. Translational frame shifting mediated by a viral sequence in plant cells, Proc. Natl. Acad. Sci .USA,1992, 89: 2262.
43. Brigneti G, Voinnet O, Li W X, et al. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana bentha-miana. EMBO J, 1998 ,17:6739-6746.
44. Burton, et al. Virus - induced silencing of a plant celulose synthase gene. Plant Cell, 2000, (12):691-705.
45. Cardozo, T., and Pagano, M., The SCF ubiquitin ligase: insights into a molecular machine. Nat. Rev.Mol. Cell Biol, 2004 ,5:739-751.
46. Chapman E J, Prokhnevsky A I, Gopinath K, et al. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Gen Dev, 2004 ,18 :1179-1186.
47. Chen J, WatanabeY, SakoN, Ohshima K, OkadaY. Complete nucleotide sequence and synthesis of infectious in vitro transcripts from a full-length cDNA clone of a rakkyo strain of tobacco mosaic virus. Arch Virol, 1996, 141: 885-900.
48. Cogoni C, Irelan J T, Schumacher M, et al. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions of DNA methylation. EMBO J., 1996,15(12):3153-3163.
49. Conti M, Milne RG, Luisoni E, Boccardo G. A closterovirus from a stem-pitting diseased grapevine. Phytopathology, 1980 , 70: 394-399.
50. Dagless EM, Shintaku MH, Nelson RS, Foster GD . A CaMV 35S promoter driven cDNA clone of tobacco mosaic virus can infect host plant tissue despite being uninfectious when manually inoculated onto leaves. Arch Virol , 1997, 142: 183-191.
51. Dawson WO, Beck DL, Knorr DA, Grantham GL. cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proc Natl Acad Sci. USA, 1986, 83:1832-1836.
52. Diener TO, Schneider IR. Virus degradation and nucleic acid release in single phase phenol system. Arch Biochem Biophys , 1968, 124:401-412.
53. Ding S W.RNA silencing. Curr Opin Biotechnol, 2000, 11:152 -156.
54. Domier LL, Franklin km, Hunt AG, Rhoads RE, Shaw JG . Infectious in vitro transcripts fromcloned cDNA of a potyvirus, tobacco vein mottling virus. Proc Natl Acad Sci. USA,1989 , 86:3509-3513.
55. Dunn JJ, Studier FW. Complete nucleotide sequence of bacteriophage T7 DNA and the location of T7 genetic elements. J Mol Biol , 1983 , 166:477-535.
56. Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Gen Dev, 2001,15 :188-200.
57. F. Liu, X. Wang, Y. Liu, J. Xie, S. M. Gray, G. Zhou, B. Gao, A Chinese isolate of Barley yellow dwarf virus-PAV represents a third distinct species within the PAV serotype, Archives of Virology, 2007, 127:1365-1373
58. Fakhfakh H, Vilaine F, Makni M, Robaglia C . Cell-free biolistic inoculation of an infectious cDNA clone of potato virus Y. J Gen Virol, 1996,77: 519-523.
59. Flasinski S, Gunasinghe UB, Gonzales RA, Cassidy BG . The cDNA sequence and infectious transcripts of peanut stripe virus. Gene, 1996 , 171:299-300.
60. Froman MA, Dush MK, Martin GR . Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligo-nucleotide primer. Proc Natl Acad Sci .USA , 1988, 85: 8998-9002.
61. Gal-on,A.,Meiri,E.,Huet,H .et al . Particle bombardment drastically increases the infectivity of cloned DNA of Zucchini yellow mosaic potyvirus.J.Gen.Virol, 1995 , 76 :3223-3227 .
62. García J A, Simón-Mateo C. A micropunch against plant viruses. Nat Biotechnol, 2006 , 24 :1358-1359.
63. Gildow, FE. and Gray, MS. The aphid salivary gland basal lamina as a selective barrier associated with vector-specific transmission of barley yellow dwarf luteoviruses. Phytopathology, 1993, 83: 1283.
64. Hamilton, A. J., and Baulcombe, D. C., A novel species of small antisense RNA in post-transcriptional gene silencing. Science ,1999,286: 950-952
65. Hammond S M, Bernstein E, Beach D, et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000 ,404: 293-296.
66. Han, C., Molecular characterization of a Chinese sugar beet-infecting isolate of Beet western yellows virus. Phytopathology,2008, 98(6).
67. Hofgen, R., and Willmitzer, L.,Storage of competent cells for Agrobacterium tumefaciens transformation. Nucleic Acids Res, 1988,16(20): 9877-9877.
68. Holt CA, Beachy RN. In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. Virology, 1991,181:109-117.
69. J. Y. Yoon, B. E. Min, S. H. Choi, and K. H. Ryu. Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cDNA clone of Kyuri green mottle mosaic virus*. Arch Virol , 2001, 146:2085-2096.
70. Jakab G, Droz E, Brigneti G, Baulcombe D, Malnoe P. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J GenVirol,1997,78: 3141-3 145.
71. Johansen E. Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo. Proc Natl Acad Sci. USA, 1996, 93: 12400-12405.
72. Kjemtrup, et al. Gene silencing form plant DNA calried by a geminivirus. Plant J . , 1998, (14):91-100.
73. Kumagai, M. H., Turpen, T. H., Weinzettl, N., Della-Cioppa, G., Turpen, A. M., Donson, J.,et al. Rapid, high-level expression of biologically activeα-trichosanthin in transfected plants by an RNA viral vector. Proc. Natl. Acad. Sci. USA, 1993, 90: 427-430.
74. Li H W , Lucy A P , Guo H S , et al.Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism . EMBO J , 1999, 18 :2683 -2691.
75. Llave C, Kasschau K D, Carrington J C.Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA, 2000, 97:13401-13406.
76. Ma, B., and Nussinov, R.,Trp/Met/Phe hot spots in protein-protein interactions: potential targets in drug design. Curr. Top. Med. Chem, 2007, 7(10): 999-1005.
77. Meshi, T., Ishikawa, M., Motoyoshi, F., Semba, K., and Okada, Y. In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. Proc. Natl. Acad. Sci .USA, 1986 , 83: 5043-5047.
78. Miller, Liu S, Becket R. Barely yellow dwarf virus: Luteotviridate or Tombusviridae. Moleular Plant Pathology, 2002,3(4):177-183.
79. Miller, W. A., Waterhouse, P. M.,Gerlach W. L. Sequence and organization of the barley yellow dwarf virus genomic RNA. Nucleic Acids Research, 1988, 16:6097-6111.
80. Minafra A, Hadidi A . Sensitive detection of grapevine virus A, B, or leafroll- associated III from viruliferous mealybugs and infected tissue by cDNA amplification.J Virol Methods , 1994,47: 175-188.
81. N. L. Robertson, R. French, and T. J. Morris. The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection. Arch Virol, 2000,145:1685-1698.
82. Napoli C , Lemieux C , Jorgensen Reintroduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans .Plant Cell , 1990 ,(2) :279- 289.
83. Niu Q W, Lin S S, Reyes J L, et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol, 2006,24:1420-1428.
84. OkadaY .Molecular assembly of tobacco mosaic virus in vitro. Adv Biophys, 1986,22: 95-149.
85. Oswald J W, Houston B R. A new virus disease of cereals transmitted by aphid. Plant Disease Repoat, 195l ,35:471-475.
86. P. Saldarelli, M. Dell’Orco, and A. Minafra. Infectious cDNA clones of two grapevine viruses. Arch Virol, 2000,145:397-405.
87. Plasterk RHA. RNA Silencing : The genome’s immune system. Science, 2002,296 :1263 -1265.
88. Plasterk, et al. The silence of the Genes . Curr. Opin. Genet, 2000, (10):562-567.
89. Polo S, Ketner G, Levis R, Falgout B . Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast. J Virol, 1997, 71: 5366-5374.
90. Prufer D ,Wipf-Scheibel C ,Richards K .et al. Synthesis of a full-length infectious cDNA clone of Cucurbil aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from otherlutcoviruses.Virol.,1995,214(15):0-8.
91. Puurand U, Valkonen JP, Makinen K, Rabenstein F, SaarmaM. Infectious in vitro transcripts from cloned cDNA of the potato A potyvirus. Virus Res, 1996 , 40:135-140.
92. Quillet L, Guilley H, Jonard G, Richards K. In vitro synthesis of biologically active beet necrotic yellow vein virus RNA. Virology ,1989, 172: 293-301.
93. Ratcliff, et al. A similarity between viral defense and Gene Silencing in Plants . Science, 1997, (276):1558-1560.
94. Rochow W. F. Biological properties of four isolates of BYSV.Phytopathol,1969,59:
95. Rochow W. F.Comparative diagnosis of BYDV by serological and aphid transmission tests. Plantdisease Report, 1979, 63:426-430.
96. Romano N, Macino G .Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol , 1992, (6):3343 -3353.
97. Ruiz, et al.Initiation and maintenance of Virus - induced gene silencing. Plant Cell, 1998, (10):937-946.
98. S.P.DINESH-KUMAR, VERONIQUE BRAULT, W .ALLEN MILLER.Precise Mapping and in vitro Translation of a Trifunctional Subgenomic RNA of Barley Yellow Dwarf Virus.Virology, 1992 ,187 :711-722.
99. Sabine Lamprecht and Wilhelm Jelkmann. Infectious cDNA clone used to identify strawberry mild yellow edge-associated potexvirus as causal agent of the disease. Journal of General Virology, 1997,78 : 2347-2353.
100. Shi B, Ding S, Symons RH . Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus. J Gen Virol, 1997, 78 : 1181-1185.
101. Tellier R, Buch J, Emerson SU, Purcell RH..Amplification of the full-length hepatitis
102. Tomari Y, Zamore P D. Perspective: machines for RNAi. Genes Dev., 2005, 19(5):517-529.
103. Ueng P P. , Vincent J R , Kawata E E , et al. Nucleotide sequence analysis of the genomes of the MAV - PS1 and P– PAV isolates of barley yellow dwarf virus. J . Gen , Virol. , 1992, 73:487-492.
104. Vieira J, Messing J. New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene,1991, 100:189-194.
105. Voinnet O. Induction and suppression of RNA silencing: insights from viral infections. Nature, 2005 , 6: 206-220.
106. Voinnet, O., Vain, P., Angell, S., and Baulcombe, D. C. Systemic spread of sequence-specifictransgene RNA degradation is initiated by localised introduction of ectopic promoterless DNA. Cell, 1998,95(2): 177-187.
107. Waterhouse P M, Graham M W, Wang M B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA .Proc. Natl. Acad. Sci. USA, 1998, 95(23):13959-13964.
108. Xiang, H., Shang, Q., Han, C., Li, D., and Yu, J. Detection of Turnip yellows virus in eight cruciferous crops in Mainland China. Phytopathology ,2009,99(6): s144.
109. Zamore P D , Tuschl T , Sharp P A , et al. RNAi :double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals . Cell , 2000, 101:25 -33.
2.成卓敏,周广和.小麦黄矮病毒GPV株系的提纯及血清学研究.病毒学报, 1986,2(3): 275-277.
3.成卓敏,周广和.小麦黄矮病毒GPV株系的提纯及血清学研究.病毒学报, 1986,2(3): 275-277.
4.成卓敏,吴茂森,等.应用基因枪法获得抗大麦黄矮病毒转基因小麦.植物病理学报, 2000,30(2):116-121.
5.崔晓峰,周雪平.(2005). RNA沉默的病毒抑制子.生物化学与生物物理进展, 32 (3):210-216.
6.崔晓峰,周雪平.中国番茄黄化曲叶病毒和烟草曲茎病毒AC2和AC4蛋白为RNA沉默的抑制子.科学通报, 2004,49(23): 2431-2436.
7.董家红.小麦黄花叶病毒侵染性cDNA克隆及细胞侵染体系的建立.[博士论文].中国农业大学,2004.
8.晋治波,王锡锋,常胜军,周广和.大麦黄矮病毒GAV基因组全序列测定及其结构分析.中国科学(C辑), 20033,3(6):505-513.
9.梁新苗.SCMV-BJ全长cDNA克隆的构建及其部分基因与致病性关系的探索. [博士论文].中国农业大学,2005.
10.廖乾生,等.黄瓜花叶病毒辣椒分离物侵染性克隆构建.中国农业科学,2008 41(7):1975-1982.
11.刘福秀,赵芹,等.水稻瘤矮病毒基因组第11片段编码一个RNA沉默抑制子.科学通报,2008,53(1) : 96-103.
12.刘双清.大麦黄矮病毒三种株系的特异性检测及PAV株系的群体遗传变异.[硕士论文].湖北农业大学, 2008.
13.刘涛.利用侵染性cDNA研究甜菜坏死黄脉病毒RNA5与病毒致病性的关系. [博士论文].中国农业大学,2003.
14.牛颜冰,青玲,等.植物病毒抑制子抑制RNA沉默的机制及生物学应用.分子植物育种, 20097, (1):137-142.
15.邵碧英,等.().烟草花叶病毒及其弱毒株基因组的cDNA克隆和序列分析.植物病理学报, 2003,33(4):296-301.
16.孙博.大麦黄矮病毒群体的分子变异. [硕士论文].中国农业科学院,2006.
17.田荣欢,刘迪秋,等.植物病毒基因沉默抑制子研究进展.生物技术通报, 2010 ,(4):11-15.
18.王献兵,张凌娣,李大伟,等.植物病毒编码的RNA沉默抑制因子.中国农业大学学报, 2005,10(4):31-38.
19.吴蓓蕾,王锡锋.植物病毒基因沉默抑制因子研究进展.植物保护, 2007,33(2): 1-6.
20.吴蓓蕾.小麦矮缩病毒分子群体遗传结构与RNAi介导的抗BYDV-GAV转基因小麦研究.[博士论文].中国农业科学院, 2007.
21.席德慧,曹云鹤,等.(2006).甜菜黑色焦枯病毒新疆分离物基因组RNA的序列分析及侵染性cDNA克隆构建.病毒学报,22(4):309-313.
22.席德慧,等.黄瓜花叶病毒卫星RNA XJ s1侵染性cDNA克隆构建及其生物学功能初步研究.微生物学报, 2006,46 (2):219-222.
23.席德慧,林宏辉,等.(2006).黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究.微生物学报,46(2):219-222.
24.熊庆,周雪平.假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子.微生物学报,2007,47 (1): 39-43.
25.薛朝阳,周雪平,等.一种病毒侵染性全长cDNA克隆的快速构建方法.生物化学与生物物理学报, 2000,32(3):270-274.
26.燕飞,宋雪梅,成卓敏.番茄丛矮病毒p19蛋白抑制转录后基因沉默作用机制.病毒学报, 2005,21 (5):403-405.
27.张凌娣,王朝辉,王献兵,等.两种植物病毒编码蛋白的基因沉默抑制子功能鉴定.科学通报, 2005,50(3): 219-224.
28.张文蔚,成卓敏.大麦黄矮病毒GPV株系基因组末端序列的克隆和分析.中国农业科技导报, 2009,11 (1):102-107.
29.赵琨.大麦黄矮病毒LAMP检测及其基因沉默抑制子鉴定.[硕士论文].中国农业科学院, 2010.
30.周广和,张淑香,钱幼亭.小麦黄矮病毒4种株系鉴定与应用.中国农业科学, 1987, 20 (4) :7 - 12.
31.庄木,王晓武,等.转录后基因沉默及植物病毒对它的抑制.生物技术通报, 2002, (6):18-22.1580-1589.
32. Ahlquist, P., French, R., Janda, M., and Loesch-Fries, L. S. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc. Natl. Acad. Sci. USA, 1984, 81: 7066-7070.
33. Aleksnadra,M., DzianPtt., Jozef, J.,et al.Derivation of an inefctious vrial RNA by autolytic cleavage of in vitro transcribed vrial cDNAs. Proc. Natl.Acad. Sci, 1989,USA 86: 4823-4827.
34. Baumberger, N., Tsai, C. H., Lie, M., Havecker, E., and Baulcombe, D. C., The Polerovirus Silencing Suppressor P0 Targets ARGONAUTE Proteins for Degradation. Curr. Biol, 2007 ,17:1609-1614.
35. Beck, D. L., Forster, R. L. S., Bevan, M. W., Boxen, K. A. & Lowe, S. C. Infectious transcripts and nucleotide sequence of cloned cDNA of the potexvirus white clover mosaic virus. Virology, 1990,177 :152-158.
36. Beilei Wu, Alexandra Blanchard-Letort, Yan Liu, Guanghe Zhou, Xifeng Wang, Santiago F. Elena. Dynamics of Molecular Evolution and Phylogeography of Barley yellow dwarf virus-PAV. PLoS ONE 2011,6(2):e16896
37. Bernstein E, Caudy A, Hammond S M, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001,409 : 363-366.
38. Bevan, M. Binary Agrobacterium vectors for plant transformation.Nucleic Acids Research, 1984, 12: 8711-8721.
39. Bortolamiol, D., Pazhouhandeh, M., Marrocco, K., Genschik, P., and Ziegler-Graff, V., The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing. Curr. Biol, 2007, 17:1615-1621.
40. Boscia D, Savino V, Minafra A, Namba S, Elicio V, Castellano MA, Gonsalves D,Martelli GP . Properties of a filamentous virus isolated from grapevines affected by corky bark. Arch Virol, 1993, 130:109-120.
41. Boyer J, Haenni A .Infectious transcripts and cDNA clones of RNA viruses.Virology, 1994,198 :415-426.
42. Brault, V., and Miller, W. A. Translational frame shifting mediated by a viral sequence in plant cells, Proc. Natl. Acad. Sci .USA,1992, 89: 2262.
43. Brigneti G, Voinnet O, Li W X, et al. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana bentha-miana. EMBO J, 1998 ,17:6739-6746.
44. Burton, et al. Virus - induced silencing of a plant celulose synthase gene. Plant Cell, 2000, (12):691-705.
45. Cardozo, T., and Pagano, M., The SCF ubiquitin ligase: insights into a molecular machine. Nat. Rev.Mol. Cell Biol, 2004 ,5:739-751.
46. Chapman E J, Prokhnevsky A I, Gopinath K, et al. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Gen Dev, 2004 ,18 :1179-1186.
47. Chen J, WatanabeY, SakoN, Ohshima K, OkadaY. Complete nucleotide sequence and synthesis of infectious in vitro transcripts from a full-length cDNA clone of a rakkyo strain of tobacco mosaic virus. Arch Virol, 1996, 141: 885-900.
48. Cogoni C, Irelan J T, Schumacher M, et al. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions of DNA methylation. EMBO J., 1996,15(12):3153-3163.
49. Conti M, Milne RG, Luisoni E, Boccardo G. A closterovirus from a stem-pitting diseased grapevine. Phytopathology, 1980 , 70: 394-399.
50. Dagless EM, Shintaku MH, Nelson RS, Foster GD . A CaMV 35S promoter driven cDNA clone of tobacco mosaic virus can infect host plant tissue despite being uninfectious when manually inoculated onto leaves. Arch Virol , 1997, 142: 183-191.
51. Dawson WO, Beck DL, Knorr DA, Grantham GL. cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proc Natl Acad Sci. USA, 1986, 83:1832-1836.
52. Diener TO, Schneider IR. Virus degradation and nucleic acid release in single phase phenol system. Arch Biochem Biophys , 1968, 124:401-412.
53. Ding S W.RNA silencing. Curr Opin Biotechnol, 2000, 11:152 -156.
54. Domier LL, Franklin km, Hunt AG, Rhoads RE, Shaw JG . Infectious in vitro transcripts fromcloned cDNA of a potyvirus, tobacco vein mottling virus. Proc Natl Acad Sci. USA,1989 , 86:3509-3513.
55. Dunn JJ, Studier FW. Complete nucleotide sequence of bacteriophage T7 DNA and the location of T7 genetic elements. J Mol Biol , 1983 , 166:477-535.
56. Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Gen Dev, 2001,15 :188-200.
57. F. Liu, X. Wang, Y. Liu, J. Xie, S. M. Gray, G. Zhou, B. Gao, A Chinese isolate of Barley yellow dwarf virus-PAV represents a third distinct species within the PAV serotype, Archives of Virology, 2007, 127:1365-1373
58. Fakhfakh H, Vilaine F, Makni M, Robaglia C . Cell-free biolistic inoculation of an infectious cDNA clone of potato virus Y. J Gen Virol, 1996,77: 519-523.
59. Flasinski S, Gunasinghe UB, Gonzales RA, Cassidy BG . The cDNA sequence and infectious transcripts of peanut stripe virus. Gene, 1996 , 171:299-300.
60. Froman MA, Dush MK, Martin GR . Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligo-nucleotide primer. Proc Natl Acad Sci .USA , 1988, 85: 8998-9002.
61. Gal-on,A.,Meiri,E.,Huet,H .et al . Particle bombardment drastically increases the infectivity of cloned DNA of Zucchini yellow mosaic potyvirus.J.Gen.Virol, 1995 , 76 :3223-3227 .
62. García J A, Simón-Mateo C. A micropunch against plant viruses. Nat Biotechnol, 2006 , 24 :1358-1359.
63. Gildow, FE. and Gray, MS. The aphid salivary gland basal lamina as a selective barrier associated with vector-specific transmission of barley yellow dwarf luteoviruses. Phytopathology, 1993, 83: 1283.
64. Hamilton, A. J., and Baulcombe, D. C., A novel species of small antisense RNA in post-transcriptional gene silencing. Science ,1999,286: 950-952
65. Hammond S M, Bernstein E, Beach D, et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000 ,404: 293-296.
66. Han, C., Molecular characterization of a Chinese sugar beet-infecting isolate of Beet western yellows virus. Phytopathology,2008, 98(6).
67. Hofgen, R., and Willmitzer, L.,Storage of competent cells for Agrobacterium tumefaciens transformation. Nucleic Acids Res, 1988,16(20): 9877-9877.
68. Holt CA, Beachy RN. In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. Virology, 1991,181:109-117.
69. J. Y. Yoon, B. E. Min, S. H. Choi, and K. H. Ryu. Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cDNA clone of Kyuri green mottle mosaic virus*. Arch Virol , 2001, 146:2085-2096.
70. Jakab G, Droz E, Brigneti G, Baulcombe D, Malnoe P. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J GenVirol,1997,78: 3141-3 145.
71. Johansen E. Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo. Proc Natl Acad Sci. USA, 1996, 93: 12400-12405.
72. Kjemtrup, et al. Gene silencing form plant DNA calried by a geminivirus. Plant J . , 1998, (14):91-100.
73. Kumagai, M. H., Turpen, T. H., Weinzettl, N., Della-Cioppa, G., Turpen, A. M., Donson, J.,et al. Rapid, high-level expression of biologically activeα-trichosanthin in transfected plants by an RNA viral vector. Proc. Natl. Acad. Sci. USA, 1993, 90: 427-430.
74. Li H W , Lucy A P , Guo H S , et al.Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism . EMBO J , 1999, 18 :2683 -2691.
75. Llave C, Kasschau K D, Carrington J C.Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA, 2000, 97:13401-13406.
76. Ma, B., and Nussinov, R.,Trp/Met/Phe hot spots in protein-protein interactions: potential targets in drug design. Curr. Top. Med. Chem, 2007, 7(10): 999-1005.
77. Meshi, T., Ishikawa, M., Motoyoshi, F., Semba, K., and Okada, Y. In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. Proc. Natl. Acad. Sci .USA, 1986 , 83: 5043-5047.
78. Miller, Liu S, Becket R. Barely yellow dwarf virus: Luteotviridate or Tombusviridae. Moleular Plant Pathology, 2002,3(4):177-183.
79. Miller, W. A., Waterhouse, P. M.,Gerlach W. L. Sequence and organization of the barley yellow dwarf virus genomic RNA. Nucleic Acids Research, 1988, 16:6097-6111.
80. Minafra A, Hadidi A . Sensitive detection of grapevine virus A, B, or leafroll- associated III from viruliferous mealybugs and infected tissue by cDNA amplification.J Virol Methods , 1994,47: 175-188.
81. N. L. Robertson, R. French, and T. J. Morris. The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection. Arch Virol, 2000,145:1685-1698.
82. Napoli C , Lemieux C , Jorgensen Reintroduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans .Plant Cell , 1990 ,(2) :279- 289.
83. Niu Q W, Lin S S, Reyes J L, et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol, 2006,24:1420-1428.
84. OkadaY .Molecular assembly of tobacco mosaic virus in vitro. Adv Biophys, 1986,22: 95-149.
85. Oswald J W, Houston B R. A new virus disease of cereals transmitted by aphid. Plant Disease Repoat, 195l ,35:471-475.
86. P. Saldarelli, M. Dell’Orco, and A. Minafra. Infectious cDNA clones of two grapevine viruses. Arch Virol, 2000,145:397-405.
87. Plasterk RHA. RNA Silencing : The genome’s immune system. Science, 2002,296 :1263 -1265.
88. Plasterk, et al. The silence of the Genes . Curr. Opin. Genet, 2000, (10):562-567.
89. Polo S, Ketner G, Levis R, Falgout B . Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast. J Virol, 1997, 71: 5366-5374.
90. Prufer D ,Wipf-Scheibel C ,Richards K .et al. Synthesis of a full-length infectious cDNA clone of Cucurbil aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from otherlutcoviruses.Virol.,1995,214(15):0-8.
91. Puurand U, Valkonen JP, Makinen K, Rabenstein F, SaarmaM. Infectious in vitro transcripts from cloned cDNA of the potato A potyvirus. Virus Res, 1996 , 40:135-140.
92. Quillet L, Guilley H, Jonard G, Richards K. In vitro synthesis of biologically active beet necrotic yellow vein virus RNA. Virology ,1989, 172: 293-301.
93. Ratcliff, et al. A similarity between viral defense and Gene Silencing in Plants . Science, 1997, (276):1558-1560.
94. Rochow W. F. Biological properties of four isolates of BYSV.Phytopathol,1969,59:
95. Rochow W. F.Comparative diagnosis of BYDV by serological and aphid transmission tests. Plantdisease Report, 1979, 63:426-430.
96. Romano N, Macino G .Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol , 1992, (6):3343 -3353.
97. Ruiz, et al.Initiation and maintenance of Virus - induced gene silencing. Plant Cell, 1998, (10):937-946.
98. S.P.DINESH-KUMAR, VERONIQUE BRAULT, W .ALLEN MILLER.Precise Mapping and in vitro Translation of a Trifunctional Subgenomic RNA of Barley Yellow Dwarf Virus.Virology, 1992 ,187 :711-722.
99. Sabine Lamprecht and Wilhelm Jelkmann. Infectious cDNA clone used to identify strawberry mild yellow edge-associated potexvirus as causal agent of the disease. Journal of General Virology, 1997,78 : 2347-2353.
100. Shi B, Ding S, Symons RH . Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus. J Gen Virol, 1997, 78 : 1181-1185.
101. Tellier R, Buch J, Emerson SU, Purcell RH..Amplification of the full-length hepatitis
102. Tomari Y, Zamore P D. Perspective: machines for RNAi. Genes Dev., 2005, 19(5):517-529.
103. Ueng P P. , Vincent J R , Kawata E E , et al. Nucleotide sequence analysis of the genomes of the MAV - PS1 and P– PAV isolates of barley yellow dwarf virus. J . Gen , Virol. , 1992, 73:487-492.
104. Vieira J, Messing J. New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene,1991, 100:189-194.
105. Voinnet O. Induction and suppression of RNA silencing: insights from viral infections. Nature, 2005 , 6: 206-220.
106. Voinnet, O., Vain, P., Angell, S., and Baulcombe, D. C. Systemic spread of sequence-specifictransgene RNA degradation is initiated by localised introduction of ectopic promoterless DNA. Cell, 1998,95(2): 177-187.
107. Waterhouse P M, Graham M W, Wang M B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA .Proc. Natl. Acad. Sci. USA, 1998, 95(23):13959-13964.
108. Xiang, H., Shang, Q., Han, C., Li, D., and Yu, J. Detection of Turnip yellows virus in eight cruciferous crops in Mainland China. Phytopathology ,2009,99(6): s144.
109. Zamore P D , Tuschl T , Sharp P A , et al. RNAi :double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals . Cell , 2000, 101:25 -33.