摘要
柑橘产业是我国南方农村的重要支柱产业,而每年由柑橘衰退病病毒(Citrus tristeza virus, CTV)造成的经济损失巨大。柑橘衰退病病毒属于正义单链RNA病毒。由于利用RNAi技术进行抗植物RNA病毒已有大量成功的报道,所以应用RNAi原理进行抗CTV育种可能成为解决CTV防治的有效方法。
RNA病毒侵染植物时,一般表达沉默抑制因子使宿主植物细胞中RNAi机制钝化从而达到成功侵染植物的目的。因此阻断病毒的沉默抑制因子的表达将有助于植物抵抗病毒的侵染,从而达到抗病毒的效果。本试验将CTV中三个沉默抑制因子p25、p20和p23作为RNAi靶标序列,目的是要获得对柑橘CTV有广谱抗性的柑橘品种。
(1)RNAi载体的构建、转化和转基因植株的验证
根据CTV基因组RNA中的沉默抑制因子p25、p20和p23的序列保守区域设计引物,经RT-PCR成功克隆相关基因片段。利用重叠延伸PCR拼接法(Gene Splicing by Overlap Extension, SOE)拼接PCR产物,获得两条多基因大片段P2520、P252023。
为了便于克隆操作,在正向片段下游引物末端加上限制性内切酶BglⅡ识别序列；在巢式PCR上游引物末端加上KpnⅠ识别序列,下游引物加上BglⅡ识别序列,经TA克隆得到含两个沉默抑制因子pe5和p20基因片段的反向重复序列载体pTZ57R-HP2520。应用同样的方法得到含三个沉默抑制因子pe5,p20和p23基因片段的反向重复序列载体pTZ57R-HP252023。
载体pTZ57R-HP2520与pCAMBIA-2301G经SacⅠ和BamHⅠ双酶切,胶回收目的片段后用T4-DNA连接酶连接两个片段,然后转入大肠杆菌DH5α中,提取质粒后用Sad和BamHⅠ双酶切检测目的片段。然后将此质粒转化入农杆菌LBA4404菌株。
含有质粒pCAMBIA2301-HP2520的农杆菌被用来与锦橙上胚轴共培养获得转基因植株。本试验共获得了抗性不定芽20个,嫁接后再生成活7株,利用T-DNA区域内的GUS基因特异引物PCR检测6株为阳性,同时用NPTⅡ基因特异引物PCR检测7株为阳性。
应用同样的方法获得RNAi双元质粒表达载体pCAMBIA2301-HP252023,农杆菌介导转化锦橙上胚轴后诱导获得抗性不定芽1个,嫁接再生成活1株,经特异引物检测发现GUS基因和NPTⅡ基因均为阳性。
(2) pCAMBIA2301-HP20转基因酸橙植株的评价
分两步对本实验室已经构建好的针对沉默抑制因子p20的单基因片段RNAi (pCAMBIA2301-HP20)转基因酸橙植株进行评价。首先,8株转基因植株经过GUS组织化学染色鉴定和针对p20基因的特异引物PCR检测,证实所有的转基因植株都成功的转入了目的片段。其次,将转基因植株嫁接备份后做攻毒试验,即对其中5株转基因植株嫁接接种CTV强毒系TRL514。分别在接毒30d、50d和60d后用RT-PCR检测CTV存在情况,结果发现对照植株和其中两个转基因植株中的检查结果呈阳性,其余三株转基因植株迄今为止没有检测出CTV病毒RNA。不过,这三株未检测到CTV的转基因植株还需要再次进行攻毒试验,因此,它们是否真正对CTV具有持续稳定的抗性还需要进一步的研究。
Citrus industry is an important pillar industry in South China areas. However, the industry has been threatened by Citrus tristeza virus (CTV), a positive-sense single-stranded RNA virus. Since many examples of using RNAi to control plant RNA viruses have been reported, it is possible to RNAi to control CTV in citrus.
When plant RNA viruses infect plants, in order to survive, they usually inhibit host's RNAi mechanism by expressing gene-silencing suppressor genes. Our rationale is that we would be able to fight against plant viruses by inactivating the expression of their gene-silencing suppressors. In this study, three suppressor factor genes p25, p20 and p23 of CTV were targeted by RNA interference.
(1) Construction of RNAi vectors, transformation and verification of transgenic plants
PCR primers were designed according to the conserved sequences of CTV genomic RNA that encode the three suppressor genes p25, p20 and p23, and RT-PCR was performed to amplify the target sequences. Sequence overlap extension (SOE) method was used to join the amplified products to form inverted repeats. In this way, two multi-gene fragments P2520 and P252023 were obtained.
To facilitate the following cloning steps, restriction site BglⅡwas added to the down stream sense primer, and KpnⅠand BglⅡrestriction sites were also added to the two nested primers, the upstream primer and the downstream primer, respectively. After TA cloning, the pTZ57R-HP2520 construct, in which HP2520 represents the inverted repeat fragment sequences of CTV's p25 and p20 genes, was obtained. In similar way, the multi-gene construct containing the three suppressor genes was obtained.
The pTZ57R-HP2520 and pCAMBIA-2301G were both digested by SacⅠand BamHⅠrestriction enzymes. The purified HP2520 and pCAMBIA-2301G were then ligated and transformed into E. coli DH5a. The transformation was verified by double digestion of the plasmid with SacⅠand BamHⅠextracted from the transformed cells. The plasmid was then transferred into Agrobacterium strain LBA4404.
Transgenic plants were obtained by co-culturing the epicotyls of a sweet orange cultivar Jingcheng with the pCAMBIA2301-HP2520-containing Agrobacterium. Consequently,20 kanamicin resistant adventitious buds were regenerated and 7 of them survived the following grafting operation. PCR was performed using GUS and NPTⅡgene-specific primers to detect the transgenes in the transgenic plants. The results showed that six plants were GUS gene positive while all 7 were NPTⅡgene positive.
The RNAi binary expression plasmid vector pCAMBIA2301-HP252023 was also obtained by same method. Transgenic plants were obtained but only 1 survived after grafting. PCR result demonstrated that the transgenic plant contained both GUS and NPTⅡgenes.
(2) Evaluation of pCAMBIA2301-HP20 transgenic sour orange plants:
Seven transgenic sour orange plants containg pCAMBIA2301-HP20, which were obtained previously in the lab, were evaluated. First, GUS staining and PCR amplification of p20 fragment were performed to validate the transgenes. The results showed all of them are true transgenic plants. Second, wild type sour orange plants (as control) and five transgenic plants were inoculated with TRL514, a CTV severe strain, to test and compare their anti-CTV activity. CTV virus RNA detection was performed by RT-PCR 30d,50d and 60d after inoculation. The results showed that CTV RNA was found in control and two transgenic plants but not in the rest three transgenic plants. Therefore, the three CTV RNA undetected transgenic plants may exhibit anti-CTV activity. Further inoculation and study are needed to verify their anti-CTV activity.
引文
[1]中国柑橘学会编著.中国柑橘产业[M].北京：中国农业出版社,2008,18-19.
[2]Bar-Joseph M, Marcus R, Lee R F. The continuous challenge of Citrus tristeza virus control [J]. Annual Review of Phytopathol,1989,27:291-316.
[3]周常勇,赵学源,蒋元晖.柑桔衰退病在甜橙苗木各部分分布的全年分析[J].云南农业大学学报,1993,3：241-242.
[4]周常勇.我国柑橘衰退病的发生概况与展望[C].第一次全国植物病毒与病毒病防治研究学术讨论会论文集,中国农业科技出版社,北京,1997,182-187.
[5]Bar-Joseph M, Garnsey S M, Gonsalves D. The closteroviruses:a distinct group of elongated plant viruses [J]. Adv Virus Res,1979,25,93-168.
[6]Muharam A, Whittle A M. Stem pitting strains of Citrus tristeza virus in Indonesia [C], llth IOCV Conference, Riverside,1991:150-155.
[7]Garnsey S M, Gumpf D J, Roistacher C N, Civerolo E L, Lee R F, Yokomi R K, Bar-Joseph M. Toward a standardized evaluation of the biological properties of Citrus tristeza virus [J]. Phytophylactica,1987,19(2):151-157.
[8]Albiach-Marti M R, Mawassi M, Gowda S, Satyanarayana T, Hilf M E, Shanker S, Almira E C, Vives M C, Lopez C, Guerri J, Flores R, Moreno P, Garnsey S M, Dawson W O. Sequences of Citrus tristeza virus separated in time and space are essentially identical [J]. Journal of Virology,2000,74(15):6856-6865.
[9]Mawassi M, Mietkiewska E, Gofman R, Yang G, Bar-Joseph M. Unusual sequence relationship between two isolates of Citrus tristeza virus [J]. Journal of General Virology,1996,77:2359-2364.
[10]Gomes C, Nagata T, Waldir D J, Carlos N, Georgios P, Darren M. Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease [J]. Virology Journal,2008, 5(9):1-7.
[11]Yang Z N, Mathews D M, Dodds J A, Mirkov T E. Molecular characterization of an isolate of Citrus tristeza virus that causes severe symptoms in sweet orange [J]. Virus Genes,1999,19(2):131-142.
[12]Pappu H R, Karasev A V, Anderson E J, Pappu S S, Hilf M E, Febres V J, Eckloff R M, McCffery M, Boyko V, Gowda S. Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome [J]. Virology,1994,199: 35-46.
[13]Karasev A V, Boyko V P, Gowda S, Nikolaeva O V, Hilf M E, Koonin E V, Niblett C L, Cline K, Gumpf D J, Lee R F. Complete sequence of the Citrus tristeza virus RNA genome [J]. Virology,1995,208:511-520.
[14]Gowda S, Tatineni S, Folimonova S Y, Hilf M E, Dawson W O. Accumulation of a 5' proximal subgenomic RNA of Citrus tristeza is correlated with encapsidation by the minor coat protein [J]. Virology,2009,389:122-131.
[15]Rast A T B. MII-16, an artifical symptomless mutant of Tobacco mosaic virus for seedling inoculation of tomato crops [J]. Netherlands Journal of Plant Pathology,1972, 78:110-112.
[16]Yeh S D, gonsalves D. Evalution of induced mutants of papaya ring spot virus for control by cross protection [J]. Phytopathology,1984,74:1086-1091.
[17]Muller G W, Targon M L P N, Machardo M A.30 year of preimmunized 'Pera' sweet orange in the citriculture of Sao Paulo state [C], In:14th Conference of the International Organization of Citrus Virus, Riverside,1998,118.
[18]Sasaki A. Control of Hassaku dwarf by preimmunization with mild strain [J]. Review of Plant Protection Research,1979,12:80-87.
[19]Van Vuuren S P, Van der Vyver. Comparison of South African pre-immunizing Citrus tristeza virus isolates with foreign isolates in three grapefruit selections [C]. In:14th Conference of the International Organization of Citrus Virus, Riverside,2000,50-56.
[20]Fulton W. Practices and precautions in the use of cross protection for virus disease control [J]. Annual Review Phytopathology,1986,24:67-81.
[21]Baulcombe D C. Mechanisms of pathogen-dirived resistance to viruses in transgenic plants [J]. Plant Cell,1996,8:1833-1844.
[22]Brlansky R H, Lee R F. Numbers of inclusion bodies produced by mild and severe strains of Citrus tristeza virus in seven citrus hosts [J]. Plant Disease,1990,74(4): 297-299.
[23]Permar T A, Garnsey S M, Gumpf D J, Lee R F. A monoclonal antibody that discriminates strains of Citrus tristeza virus [J]. Phytopathology,1990,80(3):224-228.
[24]Moreno P, Guerri J, Ballester-Olmos J F, Albiach R, Martinez M E. Separation and interference of strains from a Citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis [J]. Plant Pathology,1993,42(1): 35-41.
[25]Gillings M, Broadbent P, Indsto J, Lee R. Characterisation of isolates and strains of Citrus tristeza closterovirus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction [J]. Journal of Virological Methods,1993,44: 305-317.
[26]宋震,邹敏,徐小峰等.柑橘衰退病毒柚分离株的p25/Hinf 1 RFLP分析[J].植物保护,2005,31(4)：28-31.
[27]Garnsey S M, Permar T A, Cambra M, Henderson C T. Direct Tissue Blot Immunoassay (DTBIA) for Detection of Citrus Tristeza Virus (CTV) [C]. In 11th Conference of the International Organization of Citrus Virus, Riverside,1993,39-50.
[28]Saponarim M, Manjunath K, Yokomi R K. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan) [J]. Journal of Virological Methods,2008,147(1):43-53.
[29]Ochoa F M, Cevik B, Febres V J, Niblett C L, Lee-Proc R F. Molecular characterization of Florida Citrus tristeza virus isolates with potential use in mild strain cross protection [C]. In:14th Conference of the International Organization of Citrus Virus, Riverside,2000,94-102.
[30]Yang Z N, Ye X R, Molina J, Roose M L, Mirkov T E. Sequence analysis of a 282-kilobase region surrounding the Citrus Tristeza virus resistance gene(Ctv) locus in Poncirus trifoliata L. Raf. [J]. Plant Physiol,2001,131(2):482-492.
[31]Deng Z Q, Tao Y L, Chang S H, Ling P, Yu C, Chen C, Gmitter F G, Zhang B. Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates [J]. Theoretical and Applied Genetecs,2001,102:1177-1184.
[32]Sanford J C, Johnson S A. The concept of parasite-derived resistance deriving resistance genes from the parasites own genome [J]. Journal of Theoretical Biology, 1985,113(2):395-405.
[33]Abel P P, Nelson R S, De B, Hoffmann N, Rogers S G, Fraley R T, Beachy R N. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene [J]. Scinece,1986,232(4751):738-743.
[34]Dasgupta I, Malathi V G, Mukherjee S K. Genetic engineering for virus resistance [J]. Current Science,2003,84(3):341-354.
[35]Baulcombe D C. Mechanisms of pathogen-derived resistance to viruses in transgenic plants [J]. Plant Cell,1996,8:1833-1844.
[36]Azevedo F A, Mourao Filho F A A, Mendes B M J, Almeida W A B, Sshinor E H, Pio R, Barbosa J M, Guidetti-Gonzalez S, Carrer H, Lam E. Genetic transformation of Rangpur lime (Citrus limonia osbeck) with the bO (bacterio-opsin) gene and its initial evaluation for phytophthora nicotianae resistance [J]. Plant Molecular Biology Reporter,2006,24:185-196.
[37]Gutierrez M A, Luth D, Moore G A. Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of Citrus tristeza virus [J]. Plant Cell Rep,1997,16: 745-753.
[38]Piestun D, Batuman O, Che X, Gofman R, Filatov V, Zypman S, Gafny R, Bar-Joseph M. Truncated verions of the Citrus tristeza virus (CTV) replicase and basta resistance genes incorporated in transgenic Troyer citrange [D]. Proceedings of The First International Symposium On Citrus Biotechnology, International Society For Horticulturae, Israel,1998.
[39]Ghorbel R, Dominguez A, Navarro L, Penna L. High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of Citrus tristeza virus [J]. Tree Physilo,2000,20(17):1193-1189.
[40]Dominguez A, Fagoaga C, Navarro L, Moreno P, Pena L. Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes [J]. Mol Genet Genomics,2002,267:54-56.
[41]Dominguez A, Guerri J, Cambra M, Navarro L, Moreno P Pena L. Efficient production of transgenic citrus plants expressing the coat protein gene of Citrus tristeza virus [J]. Plant Cell Rep,2000,19:427-433.
[42]Dominguez A, Mendoza A H, Guerri J, Cambra M, Navarro L, Moreno P, Pena L. Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (citrus aurantifolia (Christ.) Swing) plants expressing its p25 coat protein gene [J]. Molecular Breeding,2002,10:1380-1389.
[43]Ghorbel R, Lopez C, Fagoaga C, Moreno P, Navarro L, Flores R, Pena L. Transgenic citrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-like symptoms [J]. Mol Plant Pathol,2001,2:27-36.
[44]Febres V J, Niblett C L, Lee R F, Moore G A. Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes [J]. Plant Cell Rep,2003,21(5):421-428.
[45]Febres V J, Lee R F, Moore G A. Transgenic resistance to Citrus tristeza virus in grapefruit [J]. Plant Cell Report,2008,27:93-104.
[46]Ananthakrishnan G, Orbovic V, Pasquali G. Trnasfer of Citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation [J]. In Vitro Cellular & Developmental Biology-plant,2007,43:593-601.
[47]Fagoaga C, Lopez C, Moreno P, Navarro L, Flores R, Pena L. Viral-like symptoms induced by the ectopic expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain [J]. Mol Plant Microbe Interact,2005,18(5):435-445.
[48]Fagoaga C, Lopez C, Mendoza A H, Moreno P, Navarro L, Flores R, Pena L. Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime [J]. Plant Mol Biol, 2006,60(2):153-165.
[49]Lopez C, Cervera M, Fagoaga C, Moreno P, Vavarro L, Flores R, Pena L. Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime [J]. Mol Plant Pathol, 2010,11(1):33-41.
[50]Batuman O, Mawassi M, Bar-Joseph M. Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3'UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus [J]. Virus Genes,2006,33(3):319-327.
[51]Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature,1998,391:806-811.
[52]Baulcombe D. RNA silencing in plants [J]. Nature,2004,431:356-363.
[53]GroΒhans H, Filipowicz W. The expanding world of small RNAs [J]. Nature,2008,451: 414-416.
[54]Rusk N. When microRNAs activate translation [J]. Nature Methods,2008,5(2): 122-123.
[55]Chuck G, Candela H, Hake S. Big impacts by small RNAs in plant development [J]. Current Opinion In Plant Biology,2009,12(1):81-86.
[56]Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto Y Y, Sieburth L, Voinnet O. Widespread translational inhibition by plant miRNAs and siRNAs [J]. Science,2008,320(5880):1185-1190.
[57]Berstein E, Caudy A A, Hammond S M, Hannon G J. Role for a bidentate ribonuclease in the initiation spet of RNA interference [J]. Nature,2001,409:363-366.
[58]Yu J Y, DeRuiter S L, Tuner D L. RNA interference by expression of short-interfering RNAs and hairpin RNA in mammalian cells [J]. PNAS,2002,99 (9):6047-6052.
[59]Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development [J]. Nature Cell Biology,2000,2: 70-75.
[60]马琳琳,傅松滨,李璞.RNA干涉,国外医学遗传学分册[J].2003,26(4)：189-194.
[61]Covey S N, Al-Kaff N S, Langara A, Turner D S, Plants combat infection by gene silencing [J]. Nature,1997,385(6619):781-782.
[62]Carlo C, Giuseppe M. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase [J]. Nature,1999,399:166-169.
[63]Vaucheret H. Identification of a general Silencer of 19S and 35S promoters in a transgenic tobacco plant:90bp of homology in the promoter sequence are sufficient for trans-inactivation [J]. Plant Genetics and Breeding,1993,316(12):1471-1483.
[64]Chicas A, Macino G Characteristics of post-transcriptional gene silencing [J]. EMBO Reports,2001,2(11):992-996.
[65]Volpe T A, Kidner C, Hall I M, Teng G, Grewal S I S, Martienssen R A. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi [J]. Science, 2002,297(5588):1833-1837.
[66]Mosher R A, Schwach F, Studholme D, Baulcombe D. PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis [J]. PNAS,2008,105(8):364-370.
[67]Hannon G J. RNA interference [J]. Nature,2002,418:244-251.
[68]Kasschau K D, Xie Z X, Allen E, Llave C, Chapman E J, Krizan K A, Carrington J C. Pl/Hc-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function [J]. Developmental Cell,2003,4:205-217.
[69]Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001,411(6836):494-498.
[70]Donz 0, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase [J]. Nucleic Acids Research,2002,30(10):1-4.
[71]Myers J W, Jones J T, Meyer T, Ferrel J E. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for genesilencing [J]. Nature Biotechnology,2003, 21:324-328.
[72]Yang D, Buchholz F, Huang Z D, Goga A, Chen C Y, Brodsky F M, Bishop M. Short RNA duplexes produced by hydrolysis with Escherichia coli RNaselll mediated effective RNA interference in mammalian cells [J]. PNAS,2002,99 (15):9942-9947.
[73]Castanotto D, Li H, Rossi J J. Functional siRNA expression from transfected PCR products [J]. RNA,2002,8:1454-1460.
[74]Shinagawa T, Ishii S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymeraseII promoter [J]. Genes Development,2003, 17:1340-1345.
[75]Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002,20(5):446-448.
[76]Lee N S, Taikoh D, Gerhard B, Li H, Ming-jie L, Ali E, Paul S, John R. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells [J]. Nature Biotechnology,2002,20(5):500-505.
[77]Paul C P, Good P D, Winer I,Engelke D. Effective expression of small interfering RNA in human cells [J]. Nature Biotechnology,2002,20(5):505-508.
[78]魏玲,刘萱,曹诚.siRNA抑制HIV-1基因表达的研究[J].生物化学与生物物理进展,2006,33(10)：1007-1013.
[79]赵鹏,郭永智,傅震,尤永平,陈云祥,陆小明,陆艾林,刘宁,孙天胜,朱兵.靶向人端粒酶逆转录酶小片段干涉RNA表达载体的构建和表达[J].中华神经医学杂志,2007,6(3)：265-269.
[80]Liu Q, Singh S P, Green A G. High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing [J]. Plant Physiology,2002, 129:1-12.
[81]Wesley S V, Helliwell C A, Smith N A, Wang M B, Rouse D, Liu Q, Gooding P S, Singh S P, Abbott D, Stoutjesdijk P A, Robinson S P, Gleave A, Green A G, Waterhouse P. Construct design for efficient efective and high-throughput gene silencing in plants [J]. The Plant Journal,2001,27(6):581-590.
[82]Angelique G, Sachidanandam R, Hannon G J, Carmell M. A germline-specific class of small RNAs binds mammalian Piwi proteins [J]. Nature,2006,422:199-202.
[83]Kim V N. Small RNAs just got bigger:Piwi-interacting RNAs (piRNAs) in mammalian teste [J]. Genes Devlopment,2006,20(15):1993-1997.
[84]Grivna S T, Beyret E, Wang Z, Lin H. A novel class of small RNAs in mouse spermatogenic cells [J]. Genes Development,2006,20(13):1709-1714.
[85]Nishida K M, Saito K, Mori T, Kawamura Y, Nagami-Okada T, Inagaki S, Siomi H, Siomi M C. Gene silencing mechanisms mediated by Aubergine piRNA complexes in Drosophila male gonad [J]. RNA,2007,13(11):1911-1922.
[86]Nagy P, Arndt-jovin D J, Jovin T M. Small interfering RNAs suppress expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbBl-overexpressing cells [J]. Experimental Cell Research,2003,285(1): 39-49.
[87]符生苗.RNA干扰及其技术在临床医学中的应用前景[J].中国热带医学,2006,6(7)：1271-1273.
[88]Bertrand J R, Pottier M, Vekris A, Opolon P, Maksimenko A, Malvy C. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo [J]. Biochemical and Biophysical Research Communications,2002,296(4):1000-1004.
[89]Palauqui J C, Elmayan T, Pollien J M, Vaucheret H. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions [J] The EMBO Journal,1997,16(15):4738-4745.
[90]Fraser A G, Kamath R S, Zipperlen P, Martinez-campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C.elegans chromosome 1 by systematic RNA interference [J]. Nature,2000,408:325-330.
[91]Gonczy P, Echeverri C, Oegema K, Coulson A, Jones S J, Copley R R, Duperon J, Oegema J, Brehm M, Cassin E, Hannak E, Kirkham M, Pichler S, Flohrs K, Goessen A, Leidel S, Alleaume A M, Martin C, Ozlu N, Bork P, Hyman A A. Functional genomic analysis of cell division in C.elegans using RNAi of genes on chromosomeⅢ [J]. Nature,2000,408(6810):331-336.
[92]Lee S, Kim J, Han J J, Han M J, An G. Functional analyses of the flowering time gene OsMADS50, the putative SUPRESSOR OF OVEREXPRESSION Of CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice [J]. Plant Journal,2004, 38(5):754-764.
[93]Gupta R, Julie T T, Sokolov L N, Johnson S A, Luan S. A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis [J]. The Plant Cell,2002, 14(10):2495-2507.
[94]Kazumaru M, Yukihiro I, Akiko S, Nori K. OsHAP3 genes regulate chloroplast biogenesis in rice [J]. Plant Journal,2003,36(4):532-540.
[95]Subramanian S, Graham M Y, Yu O, Graham T L. RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and enhanced susceptibility to Phytophthora sojae [J]. Plant Physiology,2005,137(4): 1345-1353.
[96]Kusaba M. RNA interference in crop plants [J]. Current Opinion In Biotechnology, 2004,15:139-143.
[97]Davuluri G R, Tuinen A V, Fraser P D, Manfredonia A, Newman R, Burgess D, Brummell D A, King S R, Palys J, Uhlig J, Bramley P M, Pennings H M J, Bowler C. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes [J]. Nature Biotechnology,2005,23(7):890-895.
[98]Liu Q, Singh S, Green A. Genetic modification of cotton seed oil using inverted-repeat gene-silencing techniques [J]. Biochemical Society Transaction,2000,28(6):927-929.
[99]Ogita S, Uefuji H, Morimoto M, Sano H. Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties [J]. Plant Molecular Biology,2004,54(6): 931-941.
[100]Knight S W, Bass B L. A role for the RNaselll enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans [J]. Science,2001,293(5538): 2269-2271.
[101]Olsen P H, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation [J]. Developmental Biology,1999,216(2):671-680.
[102]Palatnik J F, Allen E, Wu X, Schommer C, Schwab R, Carrington J C, Weigel D. Control of leaf morphogenesis by microRNAs [J]. Nature,2003,425:257-263.
[103]Guo H S, Fei J F, Xie Q. A chemical-regulated inducible RNAi system in plant [J]. The Plant Journal,2003,34:383-392.
[104]Lin S L, Chuong C M, Ying S Y. A novel Mrna-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells [J]. Biochemical and Biophysical Research Communications,2001,281(3):639-644.
[105]Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) [J]. Oncogene,2002,21(37): 5716-5724.
[106]Kapadia S B, Brideau A A, Chisari F V. Interference of hepatitis C virus RNA replication by short interfering RNAs [J]. PNAS,2003,100(4):2014-2018.
[107]Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference [J]. J Immunol,2002,169(9):5196-5201.
[108]Zhao M M, An D R, Zhao J, Huang G H, Chen J Y. Transiently expressed short hairpin RNA targeting 126KD protein of Tobacco mosaic virus interferes with virus infection [J]. Acta Biochim Biophys Sin,2006,38(1):22-88.
[109]Tenllado F, Diaz-ruiz J R. Double-stranded RNA-mediated interference with plant virus infection [J]. Journal of Virology,2001,75(24):12288-12297.
[110]Takahashi S, Komatsu K, Kagiwada S, Ozeki J, Mori T, Hirata H, Yamaji Y, Ugaki M, Namba S. The efficiency of interference of Potato virus x infection depends on the target gene [J]. Virus Research,2006,116:214-217.
[111]Pinto Y M, Kok R A, Baulcombe D C. Resistance to rice yellow mottle virus (RYMV) in cultivated african rice varieties containing PYMV transgenes [J]. Nature Biotechnology,1999,17(7):702-707.
[112]Kalantidis K, Psaradakis S, Tabler M, Tsagris M. The occurrence of CMV-Specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus [J]. Molecular Plant-microbe Interactions,2002, 15(8):826-833.
[113]Lucioli A, Noris E, Brunetti A, Tavazza R, Ruzza V, Castillo A G, Bejarano E R, Accotto G P, Tavazza M, Tomato yellow leaf curl Sardinia virus rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-Mediated transgene silencing is activated [J]. Journal of Virology, 2003,77(12):6785-6798.
[114]Wang M B, Abbott D, Waterhouse P M. A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus [J]. Molecular Plant Pathology,2000,1(6):347-356.
[115]Tenllado F, Martinez-Garcia B, Vargas M, Diaz-Ruiz J R. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus intections [J], BMC Biotechnology,2003,3(3):1-11.
[116]Csorba T, Bovi A, Dalmay T, Burgyan J. The p122 subunit of tobacco mosaic virus replicase is a potent silencing suppressor and compromises both small interfering RNA-and MicroRNA-Mediated Pathways [J]. Journal of Virology,2007,81(21): 11768-11780.
[117]Mette M F, Matzke A, Matzke M. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing [J]. Curr Biol, 2001,11(14):1119-1123.
[118]Kasschau K D, Carrington J C, A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing [J]. Cell,1998,95(4):461-470.
[119]Merai Z, Kerenyi Z, Molnar A, Barta E, Valoczi A, Bisztray G, Havelda Z, Burgyan J, Silhavy D. Aureusvirus P14 is an efficient RNA silencing suppressor that binds double-stranded RNAs without size specificity [J]. Journal of Virology,2005,79(11): 7217-7226.
[120]Mallory A C, Reinhart B J, Bartel D, Vance V B, Bowman L H. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco [J]. PNAS,2002,99(23):15228-15233.
[121]Chapman E J, Prokhnevsky A I, Gopinath K, Dolja V V, Carrington J C. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step [J]. Genes and Development,2004,18:1179-1186.
[122]Endres M W, Gregory B D, Gao Z, Foreman A W, Mlotshwa S, Ge X, Pruss G J, Ecker J R, Bowman L H, Vance V. Two plant viral suppressors of silencing require the Ethylene-inducible host transcription factor RAV2 to block RNA silencing [J]. Plos Pathogens,2010,6(1):1-12.
[123]Lu R, Folimonov A, Shintaku M, Li W X, Falk B W, Dawson W O, Ding S W. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome [J]. PNAS, 2004,101(44):15742-15747.
[124]Sekiya M E, Lawrence S D, Mccaffery M, Cline K. Molecular cloning and nucleotide sequencing of the coat protein gene of Citrus tristeza virus [J]. Journal of General Virology,1991,72(5):1013-1020.
[125]Pappu H, Pappu S, Niblett C, Lee R, Civerolo E. Comparative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates [J]. Virus Genes, 1993,7(3):255-264.
[126]Gowda S, Satyanarayana T, Davis C L, Navas-Castillo J, Albiach-Marti M R, Mawassi M, Valkov N, Bar-Joseph M, Moreno P, Dawson W O. The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies [J]. Viorology,2000,274: 246-254.
[127]Satyanarayana T, Gowda, Ayllon M A, Albiach-Marti M R, Rabindran S, Dawson W O. The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation [J]. Journal of Virology,2002,76(2):473-483.
[128]Horton R M, Hunt H D, Ho S N, Pullen J K, Pease L R. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension [J]. Gene, 1989,77(1):61-68.
[129]Warrens A N, Jones M D, Lechler R I. Splicing by overlap extension by PCR using asymmetric amplification:an improved technique for the generation of hybrid proteins of immunological interest [J]. Gene,1997,186(1):29-35.
[130]Lebedenko E N, Birikh K R, Plutalov O V, Berlin Y A. Method of artificial DNA splicing by directed ligation(SDL) [J]. Nucleic acids Research,1991,19(24): 6757-6761.
[131]Yao J L, Wu J H, Gleave A P, Morris B A M. Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos [J].1996, Plant Science,113(2):175-183.
[132]Pena L, Cervera M, Ghorbel R, DomoAnguez A, Fagoaga C, Juarez J, Pina J A, Navarro L. Transgenic citrus [M]. In:Jaiwal PK, Singh R P (eds) Plant genetic engineering, Vol 3:Improvement of commercial plants, Houston, SciTech publishing, 2003,261-282.
[133]Hidaka T, Omura M, Ugaki M, Tomiyama M, Kato A, Ohshima M, Motoyoshi F. Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells [J]. Japan Journal Breed,1990,40(2):199-207.
[134]Gmitter F G, Moore G A. Plant regeneration from undeveloped ovules and embryogenic calli of citrus:embryo production, germination, and plant survival [J]. Plant Cell,1986, 6:139-147.
[135]Moore G A, Jacono C C, Neidigh J L, Lawrence S D, Cline K. Agrobacterium-mediated transformation of citrus stem segments and regeneration of transgenic plants [J]. Plant Cell Reports,11(5):238-247.
[136]Pena L, Cervera M, Juarez J, Navarro A, Pina J A, Duran-vila N, Navarro L. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants [J]. Plant Genetics and Breeding,1995,14(10):616-619.
[137]Bond J E, Roose M L. Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange [J]. Plant Cell Reports,1998,18(3): 229-234.
[138]Cervera M, Pina J A, Juarez J, Navarro L, Pena L. A broad exploration of a transgenic population of citrus:stability of gene expression and phenotype [J]. Theoretical and Applied Genetics,2000,100(5):670-677.
[139]Tzfira T, Citovsky V. Agrobacterium-mediated genetic transformation of plants:biology and biotechnology [J]. Curr Opin Biotechnol,2006,17:147-154.
[140]Cervera M, Pina J A, Juarez J, Navarro L, Pena L. Agrobacterium-mediated transformation of citrange:factors affecting transformation and regeneration [J]. Plant Cell Reports,1998,18(3):271-278.
[141]贺红,韩美丽,李耿光.农杆菌介导转化法构建转CTV-cp的枳壳植株[J].中国中药杂志,2001,26(1)：21-23.
[142]Costa M G C, Otoni W C, Moore G A. An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (macf.) and production of transgenic plants containing carotenoid biosynthetic genes [J]. Plant Cell Reports,2002, 21(4):365-373.
[143]Costa M G C, Alves V S, Lani E R G, Mosquim P R, Carvalho C R, Otoni W C. Morphogenic gradients of adventitious bud and shoot regeneration in epicotyl explants of citrus [J]. Scientia Horticulturane,2004,100:63-67.
[144]Batuman O, Mawassi M, Bar-Joseph M. Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3'UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus [J]. Virus Genes,2006,33(3):319-327.
[145]Kamachi S, Mochizuki A, Nishiguchi M, Tabei Y. Transgenic Nicotiana benthamiana plants resistant to cucumber green mottle mosaic virus based on RNA silencing [J]. Plant Cell Rep,2007,26(8):1283-1288.
[146]Zhai Z Y, Sooksa-nguan T, Vatamaniuk O K. Establishing RNA interference as a Reverse-genetic approach for gene functional analysis in protoplasts [J]. Plant Physiology,2009,149(2):642-652.
[147]Lindbo J A, Dougherty W G Untranslatable transcripts of the Tobacco etch virus coat protein gene sequence can interfere with Tobacco etch virus-replication in transgenic plants and protoplasts [J]. Virology,1992,189(2):725-733.
[148]Mueller E, Gilbert J, Davenport G, Brigneti G, Baulcombe D C. Homology-dependent resistance:transgenic virus resistance in plants related to homology-dependent gene silencing [J]. Plant Journal,1995,7(6):1001-1013.
[149]Jones A L, Johansen I E, Bean S J, Bach I, Maule A J. Specificity of resistance to pea seed-borne Mosaic potyvirus in transgenic peas expressing the viral replicase (Nlb) gene [J]. Journal of General Virology,1998,79(12):3129-3137.
[150]Holzberg S, Brosio P, Gross C, Pogue G P. Barley stripe mosaic virus-induced gene silencing in a monocot plant [J]. Plant Journal,2002,30 (3):315-327.
[151]Wesley S V, Helliwell C A, Smith N A, Wang M B, Rouse D T, Liu Q, Gooding P S, Singh S P, Abbott D, Stoutjesdijk P A, Robinson S P, Gleave A P, Green G A, Waterhouse P M. Construct design for efficient, effective and high-throughput gene silencing in plants [J]. The Plant Journal,2001,27(6):581-590.
[152]Sayaka H, Shin-ichiro O, Eri A, Hiroaki K. The effects of spacer sequences on silencing efficiency of plant RNAi vectors [J]. Plant Cell Reports,2007,26(5):651-659.
[153]Wesley S V, Liu Q, WieloPolska A, Ellacott G, Smith N, Singh S, Helliwell C. Custom knock-outs with Hairpin RNA-mediated gene silencing [J]. Methods Mol Biol,2003, 236:273-286.
[154]Pena L, Cervera M, Juarez J, Ortega C, Pina J A, Duran-vila N, Navarro L. High-efficiency Agrobacterium-mediated transformation and regeneration of citrus [J]. Plant Science,1995,104(2):183-191.
[155]Dominguez A, Cervera M, Perez R M, Romero J, Fagoaga C, Cubero J, Lopez M M, Juarez J A, Navarro L, Pena L. Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies [J]. Molecular Breeding,2004,14(2):171-183.
[156]Roy G, Sudarshana M R, Ullman D E, Ding S W, Dandekar A M, Falk B W. Chimeric cDNA sequences from Citrus tristeza virus confer RNA silencing-mediated resistance in transgenic Nicotiana benthamiana plants [J]. Phytopathology,2006,96(8):819-827.
[157]Lopez C, Cervera M, Fagoaga C, Moreno P, Navarro L, Flores R, Pena L. Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime [J]. Molecular Plant Pathology,2010, 11(1):33-44.
