利用Red重组系统构建dsRNA原核表达体系抗烟草花叶病毒的研究
|
摘要
利用DNA重组技术,构建病毒基因发夹结构的RNA(hpRNA)(转录后均形成dsRNA)的植物表达载体,转化植物,获取抗病毒转基因植物是利用基因工程技术控制植物病毒病害的最有效策略。这一策略通常称为RNA介导的病毒抗性(RNA-mediated virus resistance)。RNA介导的病毒抗性的本质是一种转录后的基因沉默,也称为RNA干扰(RNA interference,RNAi)。与其他植物抗病毒基因工程策略相比,RNA介导的病毒抗性表型近乎免疫、抗性持久;且由于转基因植株中不存在有功能的病毒基因或蛋白,也不存在转基因mRNA的积累,因而不存在发生互补、异源包壳、协生和重组的风险,具有较高的生物安全性。尽管如此,但由于公众出于对转基因植物的生态风险和食品安全性的考虑,抗病毒转基因植物的应用仍然受到极大地限制。新近的研究表明,通过细菌原核表达dsRNA(HT115菌株,RNase III缺失体)同样能够干扰植物病毒的侵染,且与获得抗病毒转基因植物相比具有更高的安全性。
RNase III广泛存在于生物体中,它在dsRNA的加工中处于中心地位、参与RNA的降解、RNA沉默(负责产生microRNA或者siRNA)和许多其他的细胞生物活性。大肠杆菌中的RNase III由rnc基因编码。本研究利用Red重组系统,敲除了大肠杆菌JM109(DE3)和HMS174(DE3)PlysS菌株的rnc基因,构建和优化了dsRNA原核表达体系。并利用HT115原核表达系统表达了TMV不同功能基因区域的dsRNA,对不同功能基因dsRNA介导的病毒抗性进行了比较。研究结果有助于将原核表达dsRNA抗病毒体系进一步发展成为一种环境友好、高效便捷的控制植物病毒的新方法,解决目前植物病毒危害日益严重的问题。具体结果如下:
1、原核表达dsRNA抗病毒体系的构建
(1)突变体的构建:利用Red重组系统,设计引物RNaseIII50-5和RNaseIII50-3,分别以pKD3和pKD4质粒为模板,使用KOD-plus高保真酶进行PCR,扩增带有氯霉素抗性和卡那霉素抗性基因的打靶线性DNA片段,将JM109(DE3)和HMS174(DE3)PlysS菌株rnc基因进行敲除,获得缺失RNase III的突变体M-JM109和M-HMS174。在M-JM109突变体的基础上,设计引物LacY-5和LacY-3对M-JM109突变体LacY基因进行敲除,获得LacY基因缺失体M-JM109lacY。
(2)原核表达载体的构建:以TMV CP基因为目的基因,以质粒pBI121上120 bp的葡萄糖苷酸酶基因部分序列为发夹结构的“环”,设计引物TMVCPII-5和TMVCPII-3,扩增TMV CP基因,分别对扩增的PCR产物和L4440质粒进行PstI和SalI双酶切,构建LCP480载体;设计引物TMVCPI-5和TMVCPI-3、TMVCPII-5和TMVCPII-3、GUSII-5和GUSII-3,构建pGEM-CP480载体;设计引物CPI-5和CPI-3、CPII-5和CPII-3、GUSI-5和GUSI-3,构建pET-CP480载体。
(3)原核表达体系的构建:将构建成功的原核表达载体,转入构建的RNase III突变体和HT115菌株中,获得原核表达体系。将构建好的原核表达体系,经IPTG诱导表达,均可提取长度约为660 bp或者480 bp的dsRNA,表明所构建的RNase III缺失体和HT115菌株具有相同特性,均可以用于dsRNA的诱导表达。对不同的原核表达体系表达的dsRNA进行荧光定量PCR分析,结果表明:M-JM109或者M-JM109lacY菌株dsRNA的表达量明显高于其他菌株,载体pGEM-CP480为适宜生产dsRNA的表达载体,因此M-JM109/pGEM-CP480或者M-JM109lacY/pGEM-CP480为最优化的原核表达体系,其相对表达量分别为6.50+ 0.69和7.28+0.56。
(4)抗病性检测:对来源于不同原核表达载体表达的dsRNA进行抗病性鉴定,结果表明,来源于不同原核表达载体表达的dsRNA抗病效果基本一致,都能达到50%左右的抗病率,说明不同的表达载体生产的dsRNA对TMV的防治并没有区别。抗病植株的Northern blot分析表明,抗病植株能够产生siRNA,而野生型对照植株则检测不到siRNA,外源的dsRNA能够防治植物病毒的侵染,这种抗病性为RNA介导的抗病性。
2、原核表达TMV不同功能基因dsRNA介导的抗病性比较研究
(1) TMV不同功能基因dsRNA表达载体的构建:Trizol法提取TMV总RNA,设计引物TMV RP-5和TMV RP-3,TMV MP-5和TMV MP-3,TMV RNA-5和TMV RNA-3,采用RT-PCR技术分别克隆了TMV复制酶基因、运动蛋白基因和54 kDa RNA聚合酶基因的部分序列,采用PCR技术亚克隆三个基因480 bp片段,反向重复插入pGEM-GUS载体,构建三个不同基因的dsRNA表达载体,分别为pGEM-RP480、pGEM-MP480和pGEM-RNA480。将构建好的dsRNA表达载体转入HT115菌株中,经IPTG诱导,均可表达480 bp的dsRNA,证明所构建的dsRNA表达载体正确。
(2) TMV不同功能基因dsRNA介导的抗病性比较:用原核表达的TMV复制酶基因、运动蛋白基因、54 kDa RNA聚合酶基因和衣壳蛋白基因的dsRNA处理烟草植株,进行抗病性鉴定。初步的检测结果表明,原核表达TMV不同功能基因的dsRNA均能保护植物抵抗TMV病毒的侵染,但介导的抗病性存在着差异。其中来源于运动蛋白基因的dsRNA介导的抗病效果最好,66%左右的处理植株表现为抗病;来源于衣壳蛋白基因的dsRNA介导的抗病效果较好,48%的处理植株表现为抗病;而来源于54 kDa RNA聚合酶基因和复制酶基因的dsRNA介导的抗病效果较差,表现为抗病的处理植株的比例分别为40%和34%。
Recombinant DNA technology offers an effective way to obtain virus resistant plants. This technology is often named as an RNA-mediated virus resistance. The essence of RNA-mediated virus resistance is post transcriptional gene silencing (PTGS), also known as RNA interference (RNAi). Compared with other biotechnological approaches in antiviral transgenic engineering, RMVR is highly efficient (almost immunity) and long-resistant duration. Because there is no functional viral gene or protein in transgenic plants and the mRNA of transgenic plants will not be accumulated in them, they have no increasingly raised concern for complementation, heterologous encapsidation, synergy, recombination, and they have higher biosafety. The transgenic plants were limited due to they have potential ecological effects and food safety. Recent studies showed that the bacterial produced dsRNA (often is the HT115 strain that deficient for RNase III) could also interfere with virus infection. Compared to acquiring transgenic plants, using dsRNA transcripts provided by this strategy for RNAi has higher biosafety.
RNase III enzymes occur ubiquitously in different organisms. They have now been shown to occupy a central position in mediating dsRNA-dependent processes, including RNA maturation, RNA decay, gene silencing (responsible for generating microRNAs or siRNA), and a range of other cellular activities. RNase III was encoded by the rnc gene in E. coli. In this work, using Red-mediated recombination, we generated the RNase III-defective E. coli strains M-JM109, M-JM09lacY and M-HMS174 for producing great quantities of dsRNA. This work explores the best vector/host combinations for high output of dsRNA. And we constructed the different dsRNAs vectors derived from the different functional genes of TMV, and transformed them into E. coli HT115. Induced by IPTG, we extracted different dsRNAs and analyzed their resistance to TMV. These results will make dsRNA prokaryotic expression system develop into an environment-friendly, effective, and simple strategy to control the infection of plant virus. The results were as follows:
1. Construction of a dsRNA prokaryotic expression system
(1) Construction of mutants: Using the Red recombination system, we designed primers RNaseIII50-5 and RNaseIII50-3, and amplified the chloramphenicol resistance gene and kanamycin resistance gene with 50 bp homologous sequences with the rnc gene by using pKD3 and pKD4 as templates, respectively. For convenience, we named the rnc gene mutants of these strains M-JM109 and M-HMS174, respectively. Based on the M-JM109, we designed primers LacY-5 and LacY-3 and knocked out the LacY gene of M-JM109, and achieved the M-JM109lacY deficient for the LacY gene.
(2) Construction of the prokaryotic expression vectors: We amplified the fragment consisted of a 480 bp cDNA presenting the entire coding region of the TMV CP gene and a 120 bp spacer (often called“loop”in hpRNA) representing sequences of the bacterial glucoronidase gene and designed primers TMVCPII-5 and TMVCPII-3. We constructed vector LCP480 by inserting a 480 bp TMV CP gene digested with PstI and SalI into the multicloning sites of plasmid L4440 digested with the same restriction endonuclease sites. We constructed vector pGEM-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers TMVCPI-5 and TMVCPI-3, TMVCPII-5 and TMVCPII-3, GUSII-5 and GUSII-3. We constructed vector pET-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers CPI-5 and CPI-3, CPII-5 and CPII-3, GUSI-5 and GUSI-3.
(3) Construction of the prokaryotic expression systems: The prokaryotic expression systems were constructed after transforming the prokaryotic expression vectors into the different E. coli strains deficient for RNase III and HT115. Induced by IPTG, the prokaryotic expression systems could all produce 660 bp or 480 bp dsRNAs. These mutants proved to be efficient in producing dsRNA by lack of dsRNA-specific RNases, just as the previously reported strain HT115 does. To compare the dsRNA produced by different prokaryotic expression systems, we performed quantitative real-time (qRT)-PCR. The results showed that M-JM109 or M-JM109lacY could produce more dsRNA than other strains, and vector pGEM-CP480 was the best choice for dsRNA production. So the M-JM109/pGEM-CP480 and M-JM109lacY/pGEM-CP480 were the best choices for dsRNA production, the relative amount of dsRNAs were 6.50+ 0.69 and 7.28+0.56, respectively.
(4) Resistance analysis: To prove whether the bacterial-produced dsRNA could interfere with TMV infection, we carry out the resistance analysis. The results showed that 50% of the tested plants were resistant, these vectors showed on great difference in resistant to TMV infection. We detected the siRNA signals in the resistant tobacco but not in the wild-type tobacco. All showed that exogenous dsRNA could protect plants from virus infection and strongly support that resistance to TMV is an RNA-mediated virus resistance.
2. Resistance comparative studies of the prokaryotic expressed-dsRNAs derived from the different functional genes of TMV
(1) Construction of the prokaryotic expressed-dsRNAs vectors derived from the different functional genes of TMV: Total RNA was extracted from infected tobacco by using Trizol. We cloned TMV replicase gene (RP), movement protein gene (MP) and 54 kDa RNA polymerase gene by RT-PCR with primers TMV RP-5 and TMV RP-3, TMV MP-5 and TMV MP-3, TMV RNA-5 and TMV RNA-3, respectively. We sub-cloned a 480 bp cDNA of the three genes and inverted inserted vector pGEM-GUS and achieve three dsRNA expression vectors, which were named as pGEM-RP480, pGEM-MP480 and pGEM-RNA480, respectively. All correctly constructed vectors were transformed into E. coli strain HT115. Induced by IPTG, all vectors could produce 480 bp dsRNA and proved the correctness of the constructed dsRNA vectors.
(2) Resistance comparative studies of the different dsRNAs derived from the different functional genes of TMV: For comparison of resistance, we carry out the resistance analysis by using the dsRNAs derived from TMV replicase gene, movement protein gene and 54 kDa RNA polymerase gene. All results showed that dsRNAs derived from the different functional genes of TMV could all protect plants from virus infection, and the resistance was obviously different due to different vectors. The resistance conferred by dsRNA derived from the TMV movement protein was the best, and 66% tested plants were resistant; the resistance conferred by dsRNA derived from the TMV coat protein is better, and 48 % tested plants were resistant; the resistance conferred by dsRNA derived from the TMV 54 kDa RNA polymerase gene and replicase gene is worse, and the percents of resistant plants are 40% and 34%, respectively.
RNase III广泛存在于生物体中,它在dsRNA的加工中处于中心地位、参与RNA的降解、RNA沉默(负责产生microRNA或者siRNA)和许多其他的细胞生物活性。大肠杆菌中的RNase III由rnc基因编码。本研究利用Red重组系统,敲除了大肠杆菌JM109(DE3)和HMS174(DE3)PlysS菌株的rnc基因,构建和优化了dsRNA原核表达体系。并利用HT115原核表达系统表达了TMV不同功能基因区域的dsRNA,对不同功能基因dsRNA介导的病毒抗性进行了比较。研究结果有助于将原核表达dsRNA抗病毒体系进一步发展成为一种环境友好、高效便捷的控制植物病毒的新方法,解决目前植物病毒危害日益严重的问题。具体结果如下:
1、原核表达dsRNA抗病毒体系的构建
(1)突变体的构建:利用Red重组系统,设计引物RNaseIII50-5和RNaseIII50-3,分别以pKD3和pKD4质粒为模板,使用KOD-plus高保真酶进行PCR,扩增带有氯霉素抗性和卡那霉素抗性基因的打靶线性DNA片段,将JM109(DE3)和HMS174(DE3)PlysS菌株rnc基因进行敲除,获得缺失RNase III的突变体M-JM109和M-HMS174。在M-JM109突变体的基础上,设计引物LacY-5和LacY-3对M-JM109突变体LacY基因进行敲除,获得LacY基因缺失体M-JM109lacY。
(2)原核表达载体的构建:以TMV CP基因为目的基因,以质粒pBI121上120 bp的葡萄糖苷酸酶基因部分序列为发夹结构的“环”,设计引物TMVCPII-5和TMVCPII-3,扩增TMV CP基因,分别对扩增的PCR产物和L4440质粒进行PstI和SalI双酶切,构建LCP480载体;设计引物TMVCPI-5和TMVCPI-3、TMVCPII-5和TMVCPII-3、GUSII-5和GUSII-3,构建pGEM-CP480载体;设计引物CPI-5和CPI-3、CPII-5和CPII-3、GUSI-5和GUSI-3,构建pET-CP480载体。
(3)原核表达体系的构建:将构建成功的原核表达载体,转入构建的RNase III突变体和HT115菌株中,获得原核表达体系。将构建好的原核表达体系,经IPTG诱导表达,均可提取长度约为660 bp或者480 bp的dsRNA,表明所构建的RNase III缺失体和HT115菌株具有相同特性,均可以用于dsRNA的诱导表达。对不同的原核表达体系表达的dsRNA进行荧光定量PCR分析,结果表明:M-JM109或者M-JM109lacY菌株dsRNA的表达量明显高于其他菌株,载体pGEM-CP480为适宜生产dsRNA的表达载体,因此M-JM109/pGEM-CP480或者M-JM109lacY/pGEM-CP480为最优化的原核表达体系,其相对表达量分别为6.50+ 0.69和7.28+0.56。
(4)抗病性检测:对来源于不同原核表达载体表达的dsRNA进行抗病性鉴定,结果表明,来源于不同原核表达载体表达的dsRNA抗病效果基本一致,都能达到50%左右的抗病率,说明不同的表达载体生产的dsRNA对TMV的防治并没有区别。抗病植株的Northern blot分析表明,抗病植株能够产生siRNA,而野生型对照植株则检测不到siRNA,外源的dsRNA能够防治植物病毒的侵染,这种抗病性为RNA介导的抗病性。
2、原核表达TMV不同功能基因dsRNA介导的抗病性比较研究
(1) TMV不同功能基因dsRNA表达载体的构建:Trizol法提取TMV总RNA,设计引物TMV RP-5和TMV RP-3,TMV MP-5和TMV MP-3,TMV RNA-5和TMV RNA-3,采用RT-PCR技术分别克隆了TMV复制酶基因、运动蛋白基因和54 kDa RNA聚合酶基因的部分序列,采用PCR技术亚克隆三个基因480 bp片段,反向重复插入pGEM-GUS载体,构建三个不同基因的dsRNA表达载体,分别为pGEM-RP480、pGEM-MP480和pGEM-RNA480。将构建好的dsRNA表达载体转入HT115菌株中,经IPTG诱导,均可表达480 bp的dsRNA,证明所构建的dsRNA表达载体正确。
(2) TMV不同功能基因dsRNA介导的抗病性比较:用原核表达的TMV复制酶基因、运动蛋白基因、54 kDa RNA聚合酶基因和衣壳蛋白基因的dsRNA处理烟草植株,进行抗病性鉴定。初步的检测结果表明,原核表达TMV不同功能基因的dsRNA均能保护植物抵抗TMV病毒的侵染,但介导的抗病性存在着差异。其中来源于运动蛋白基因的dsRNA介导的抗病效果最好,66%左右的处理植株表现为抗病;来源于衣壳蛋白基因的dsRNA介导的抗病效果较好,48%的处理植株表现为抗病;而来源于54 kDa RNA聚合酶基因和复制酶基因的dsRNA介导的抗病效果较差,表现为抗病的处理植株的比例分别为40%和34%。
Recombinant DNA technology offers an effective way to obtain virus resistant plants. This technology is often named as an RNA-mediated virus resistance. The essence of RNA-mediated virus resistance is post transcriptional gene silencing (PTGS), also known as RNA interference (RNAi). Compared with other biotechnological approaches in antiviral transgenic engineering, RMVR is highly efficient (almost immunity) and long-resistant duration. Because there is no functional viral gene or protein in transgenic plants and the mRNA of transgenic plants will not be accumulated in them, they have no increasingly raised concern for complementation, heterologous encapsidation, synergy, recombination, and they have higher biosafety. The transgenic plants were limited due to they have potential ecological effects and food safety. Recent studies showed that the bacterial produced dsRNA (often is the HT115 strain that deficient for RNase III) could also interfere with virus infection. Compared to acquiring transgenic plants, using dsRNA transcripts provided by this strategy for RNAi has higher biosafety.
RNase III enzymes occur ubiquitously in different organisms. They have now been shown to occupy a central position in mediating dsRNA-dependent processes, including RNA maturation, RNA decay, gene silencing (responsible for generating microRNAs or siRNA), and a range of other cellular activities. RNase III was encoded by the rnc gene in E. coli. In this work, using Red-mediated recombination, we generated the RNase III-defective E. coli strains M-JM109, M-JM09lacY and M-HMS174 for producing great quantities of dsRNA. This work explores the best vector/host combinations for high output of dsRNA. And we constructed the different dsRNAs vectors derived from the different functional genes of TMV, and transformed them into E. coli HT115. Induced by IPTG, we extracted different dsRNAs and analyzed their resistance to TMV. These results will make dsRNA prokaryotic expression system develop into an environment-friendly, effective, and simple strategy to control the infection of plant virus. The results were as follows:
1. Construction of a dsRNA prokaryotic expression system
(1) Construction of mutants: Using the Red recombination system, we designed primers RNaseIII50-5 and RNaseIII50-3, and amplified the chloramphenicol resistance gene and kanamycin resistance gene with 50 bp homologous sequences with the rnc gene by using pKD3 and pKD4 as templates, respectively. For convenience, we named the rnc gene mutants of these strains M-JM109 and M-HMS174, respectively. Based on the M-JM109, we designed primers LacY-5 and LacY-3 and knocked out the LacY gene of M-JM109, and achieved the M-JM109lacY deficient for the LacY gene.
(2) Construction of the prokaryotic expression vectors: We amplified the fragment consisted of a 480 bp cDNA presenting the entire coding region of the TMV CP gene and a 120 bp spacer (often called“loop”in hpRNA) representing sequences of the bacterial glucoronidase gene and designed primers TMVCPII-5 and TMVCPII-3. We constructed vector LCP480 by inserting a 480 bp TMV CP gene digested with PstI and SalI into the multicloning sites of plasmid L4440 digested with the same restriction endonuclease sites. We constructed vector pGEM-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers TMVCPI-5 and TMVCPI-3, TMVCPII-5 and TMVCPII-3, GUSII-5 and GUSII-3. We constructed vector pET-CP480 with inverted repeat of the 480 bp TMV CP gene by using primers CPI-5 and CPI-3, CPII-5 and CPII-3, GUSI-5 and GUSI-3.
(3) Construction of the prokaryotic expression systems: The prokaryotic expression systems were constructed after transforming the prokaryotic expression vectors into the different E. coli strains deficient for RNase III and HT115. Induced by IPTG, the prokaryotic expression systems could all produce 660 bp or 480 bp dsRNAs. These mutants proved to be efficient in producing dsRNA by lack of dsRNA-specific RNases, just as the previously reported strain HT115 does. To compare the dsRNA produced by different prokaryotic expression systems, we performed quantitative real-time (qRT)-PCR. The results showed that M-JM109 or M-JM109lacY could produce more dsRNA than other strains, and vector pGEM-CP480 was the best choice for dsRNA production. So the M-JM109/pGEM-CP480 and M-JM109lacY/pGEM-CP480 were the best choices for dsRNA production, the relative amount of dsRNAs were 6.50+ 0.69 and 7.28+0.56, respectively.
(4) Resistance analysis: To prove whether the bacterial-produced dsRNA could interfere with TMV infection, we carry out the resistance analysis. The results showed that 50% of the tested plants were resistant, these vectors showed on great difference in resistant to TMV infection. We detected the siRNA signals in the resistant tobacco but not in the wild-type tobacco. All showed that exogenous dsRNA could protect plants from virus infection and strongly support that resistance to TMV is an RNA-mediated virus resistance.
2. Resistance comparative studies of the prokaryotic expressed-dsRNAs derived from the different functional genes of TMV
(1) Construction of the prokaryotic expressed-dsRNAs vectors derived from the different functional genes of TMV: Total RNA was extracted from infected tobacco by using Trizol. We cloned TMV replicase gene (RP), movement protein gene (MP) and 54 kDa RNA polymerase gene by RT-PCR with primers TMV RP-5 and TMV RP-3, TMV MP-5 and TMV MP-3, TMV RNA-5 and TMV RNA-3, respectively. We sub-cloned a 480 bp cDNA of the three genes and inverted inserted vector pGEM-GUS and achieve three dsRNA expression vectors, which were named as pGEM-RP480, pGEM-MP480 and pGEM-RNA480, respectively. All correctly constructed vectors were transformed into E. coli strain HT115. Induced by IPTG, all vectors could produce 480 bp dsRNA and proved the correctness of the constructed dsRNA vectors.
(2) Resistance comparative studies of the different dsRNAs derived from the different functional genes of TMV: For comparison of resistance, we carry out the resistance analysis by using the dsRNAs derived from TMV replicase gene, movement protein gene and 54 kDa RNA polymerase gene. All results showed that dsRNAs derived from the different functional genes of TMV could all protect plants from virus infection, and the resistance was obviously different due to different vectors. The resistance conferred by dsRNA derived from the TMV movement protein was the best, and 66% tested plants were resistant; the resistance conferred by dsRNA derived from the TMV coat protein is better, and 48 % tested plants were resistant; the resistance conferred by dsRNA derived from the TMV 54 kDa RNA polymerase gene and replicase gene is worse, and the percents of resistant plants are 40% and 34%, respectively.
引文
[1]安利忻,刘一飞,陈章良,李毅(2001) TMV 54K基因的3个突变体介导抗病性的研究.植物学报, 43:395-398
[2]白庆荣,朱俊华,刘晓玲,朱常香,宋云枝,温孚江(2005)利用RNA介导的抗病性获得抗2种病毒的转基因烟草.植物病理学报, 35:148-154
[3]白光兴,孙志伟,黄莺,俞炜源(2005)利用Red重组系统对大肠杆菌clpP基因的敲除.中国生物化学与分子生物学报, 21:35-38
[4]郭兴启,温孚江,朱常香(2000)转基因植物中RNA介导地病毒抗性研究进展.生命科学, 12:166-169
[5]韩聪,张惟材,游松,黄留玉(2004)大肠杆菌ptsG基因敲除及其缺陷株生长特性研究.生物工程学报, 16-20
[6]侯松旺,陈新文,王汉中,胡志红(2003)一种以PCR产物直接构建同源重组杆状病毒的方法.中国科学(C辑), 33:169-174
[7]贾力,吴茂森,张文蔚,肖红成,卓敏(2001)大麦黄矮病毒单双价外壳蛋白基因植物表达载体构建及小麦遗传转化.生物技术学报, 9:23-27
[8]李鹏,宋云枝,刘晓玲,朱常香,温孚江(2007)马铃薯Y病毒CP基因5’端和3’端反向重复结构介导的抗病性研究.植物病理学报, 37:69-76
[9]李世访,郑银英,成卓敏(2001)利用酵母的PacI基因获得抗类病毒转基因植物.植物保护, 27: 35-37
[10]刘晓玲,宋云枝,刘红梅,温孚江,朱常香,白庆荣(2005) PVX 25 kD运动蛋白基因和外壳蛋白基因介导的抗病性研究.作物学报, 31:827-832
[11]王恒梁,冯尔玲,史兆兴,姚潇,苏国富,黄留玉(2002) Red系统快速敲除痢疾杆菌asd基因.军事医学科学院刊, 26:161-164
[12]张德咏,朱春晖,成飞雪,何明远,张战泓,刘勇(2008)表达dsRNA的细菌粗体液可抑制黄瓜花叶病毒对烟草的侵染.植物病理学报, 38:304-311
[13]张建林,邬开朗,张雪,朱应,李雁,赵伟光,吴建国(2004) siRNA表达载体的构建及其表达.中国病毒学, 19: 510-513
[14]张明,邵宁生(2002)RNA阻抑和基因沉默.军事科学院院刊, 26:61-65
[15]张荣意,刘智昕(2002)植物抗病毒基因工程的策略和机制.热带农业科学, 22:68-73
[16]张雪,温延益(2008) Red重组系统用于大肠杆菌基因修饰研究进展.中国生物工程杂志, 28: 89-93
[17]张中华,候永泰(2004) siRNA制备技术的研究进展.生命科学, 16:231-235
[18]赵明敏,安德荣,黄广华,何祖华,陈江野(2006)瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染.植物病理学报36:35-40
[19]赵明敏,杨向东,鲁红学,张长青(2007) siRNA对烟草原生质体中TMV RNA积累的干扰作用研究.植物病理学报37:284-288
[20]朱俊华,朱常香,温孚江,宋云枝(2004)正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究.植物病理学报, 34:133-140
[21]朱俊华,朱晓平,温孚江,白庆荣,朱常香,宋云枝(2004)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, 34:23-30
[22]朱常香,刘红梅,宋云枝,温孚江(2005) RNA介导的病毒抗性在转基因烟草中的烟草分析.遗传学报, 32:94-103
[23]朱常香,宋云枝,张松,郭兴启,温孚江(2001)抗芜菁花叶病毒转基因大白菜的培育.植物病理学报, 31:257-264
[24]竺晓平,朱常香,宋云枝,温孚江,刘红梅,李向东(2006) CP基因3’端短片段介导的对马铃薯Y病毒的抗性.中国农业科学, 39:1153-1158
[25] Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986)Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743
[26] Amarasinghe AK, Calin-Jageman I, Harmouch A, Sun W, Nicholson AW (2001) Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage. Methods Enzymol 342:143-158
[27] Andrew F, Xu S, Mary KM, Steven A K, Samuel E D, Craig CM (1998) Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391:806-811
[28] Apirion D, Gitelman DR (1980) Decay of RNA in RNA processing mutants of Escherichia coli. Mol Gen Genet 177:339-343
[29] Apirion D, Miczak A (1993) RNA processing in prokaryotic cells. Bioessays 15:113-120
[30] Aristarkhov A, Mikulskis A, Belasco JG, Lin EC (1996) Translation of the adhE transcript to produce ethanol dehydrogenase requires RNase III cleavage in Escherichiacoli. J Bacteriol 178: 4327-4332
[31] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol DOI 10.1038/msb4100050
[32] Bao S, Cagan R (2006) Fast cloning inverted repeats for RNA interference. RNA 12:2020-2024
[33] Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297
[34] Bass BL (2000) Double-stranded RNA as a template for gene silencing. Cell 101:235-238
[35] Baulcombe DC (2004) RNA silencing in plants. Nature 431:356-363
[36] Brantl S (2002) Antisense-RNA regulation and RNA interference. Biochim Biophys Acta.1575:15-25
[37] Beran RK, Simons RW (2001) Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation. Mol Microbiol 39:112-125
[38] Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363-366
[39] Blaszczyk J, Tropea JE, Bubunenko M, Routzahn KM, Waugh DS, Court DL, Ji X (2001) Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure 9:1225-1236
[40] Blencowe BJ, Bowman JA, McCracken S, Rosonina E (1999) SR-related proteins and the processing of messenger RNA precursors. Biochem Cell Biol 77: 277-291
[41] Bouché, N, Lauressergues D, Gasciolli V, Vaucheret H (2006) An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J 25:3347-3356
[42] Capecchi MR. (1994) Gene Targeting. Sclentific American 270:34
[43] Capecchi MR (1998) Altering the genome by homologous recombination. Science 244:1288-1292
[44] Chanfreau G, Rotondo G, Legrain P, Jacquier A (1998) Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1. EMBO J 17:3726-3737
[45] Chanfreau G, Legrain P, Jacquier A (1998) Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. J Mol Biol 284:975-988
[46] Chang KY, Ramos A (2005) The double-stranded RNA-binding motif, a versatilemacromolecular docking platform. FEBS J 272:2109-2117
[47] Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287-293
[48] Cherepanov PP, Wackernagel, W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9-14
[49] Cogni C, Romano N, Macino G (1994) Suppression of gene expression by homologous transgenes. Antonie Van Leeuwehoek 65:205-209
[50] Cooper B, Lapidot M, Heick JA, Dodds JA, Beachy RN (1995) A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 206:307-313
[51] Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC (2000) An RNA dependent RNA polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543-553
[52] Dasgupta S, Fernandez L, Kameyama L, Inada T, Nakamura Y, Pappas A, Court DL (1998) Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III - the effect of dsRNA binding on gene expression. Mol Microbiol 28:629-640
[53] Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640-6645
[54] Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, Saigo K (2003) Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eIF2C translation initiation factors. Curr Biol 13:41-46
[55] Drider D, Condon C (2004) The continuing story of endoribonucleaseIII. J Mol Microbiol Biotechnol 8:195-200
[56] Duan CG, Wang CH, Fang RX, Guo HS (2008) Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82:11084-11095
[57] Dunn JJ (1976) RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fidelity of cleavage. J Biol Chem 251:3807-3814
[58] Dunn JJ, Studier FW (1973) T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease III. Proc Natl Acad Sci USA 70:3296-3300
[59] Dykstra CC, Prasher D, Kushner SR (1984) Physical and biochemical analysis of thecloned recB and recC genes of Escherichia coli K-12. J Bacteriol 157:21-27
[60] Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: Short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457-467
[61] Elbashir SM, Harborth J, Weber K, Tuschl T(2002)Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199-213
[62] Elbashir SM, Lendeckel W, Tuschl T (2000) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188-200
[63] Elela SA, Igel H, Ares MJ (1996) RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. Cell 85:115-124
[64] Filippov V, Solovyev V, Filippova M, Gill SS (2000) A novel type of RNase III family proteins in eukaryotes. Gene 245: 213-221
[65] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811
[66] Fortin KR, Nicholson RH, Nicholson AW (2002) Mouse ribonuclease Ill cDNA structure, expression analysis, and chromosomal location. BMC Genomics 3:26
[67] Golemboski DB, Lomonossoff GP, Zaitlin M (1990) Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci USA 87:6311-6315
[68] Goto K, Kanazawa A, Kusaba M, Masuta C (2003) A simple and rapid method to detect plant siRNAs using nonradioactive probes. Plant Mol Biol Rep 21:51-58
[69] Gregory RI, Yan K, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432:235-240
[70] Guo HS, Xie Q, Fei JF, Chua NH (2005) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17:1376-1386
[71] Guo S, Kemphues KJ (1995) Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611-620
[72] Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950-952
[73] Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296
[74] Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18:3016-3027
[75] Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887-901
[76] Huntzinger E, Boisset S, Saveanu C, Benito Y, Geissmann T, Namane A, Lina G, Etienne J, Ehresmann B, Ehresmann C, Jacquier A, Vandenesch F, Romby P (2005) Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J 24:824-835
[77] Jasin M, Elledge SJ, Davis RW, Berg P (1990) Gene targeting at the human CD4 locus by epitope addition. Genes Dev 4:157-166
[78] Jiang F, Ye XC, Liu X, Fincher L, McKearin D, Liu Q (2005) Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev 19:1674-1679
[79] Jung S, Rajewsky K, Radbruch A (1993) Shutdown of class switch recombination by deletion of a switch region control element. Science 259: 984-987
[80] Kato C, Ohmiya R, Mizuno T (1998) A rapid method for disrupting genes in the Escherichia coli genome. Biosci Biotechnol Biochem 62:1826-1829
[81] Ketting RF, Fischer SEJ, Bernsterin E, Sijen T, Hannon GJ, Plasterk RH (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15:2654-2659
[82] Kindler P, Keil TU, Hofschneider PH (1973) Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli. Mol Gen Genet 126:53-69
[83] Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58:401-465
[84] Krautz-Peterson G, Skelly PJ (2008) Schistosoma mansoni: The dicer gene and its expression. Exp Parasitol 118:122-128
[85] Kreuze JF, Savenkov EI, Cuellar W, Li X, Valkonen JP (2005) Viral Class 1 RNase III Involved in Suppression of RNA Silencing. J Virol 79:7227-7238
[86] Kuzminov A (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol Mol Biol Rev 63:751-813
[87] Lam YH , Wong YS ,Wang B, Wong RNS, Yeung HW, Shaw PC (1996) Use of trichosanthin to reduce infection by turnip mosaic virus. Plant Sci 114:111-117
[88] Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer NE (1990)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) 8:127-134
[89] Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, R?dmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415-419
[90] Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ, Carthew RW (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69-81
[91] Liu HM, Zhu CX, Zhu XP, Guo XQ, Song YZ, Wen FJ (2007) A link between PVYN CP gene-mediated virus resistance and transgene arrangement. J Phytopathol 155:676-682
[92] Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods 25:402-408
[93] Lomonossoff GP, (1995) Pathogen derived resistance to plant viruses. Ann Rev Phytopathol 33:323-343
[94] Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58:401-465
[95] Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN (1993) A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant J. 4:959-970
[96] Lipardi C, Wei Q, Paterson BM (2001) RNAi as random degradative PCR:siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107: 297-307
[97] Lodge JK, Kaniewski WK, Tumer NE (1993) Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein. Proc Natl Acad Sci USA 90:7089-7093
[98] Lucas GB (1975) Diseases of tobacco, 3rd ed. Biological Consulting Associates, Raleigh, NC
[99] Macrae IJ, Doudna JA (2007) Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr Opin in Struct Biol 17:138-145
[100] Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311:195-
[101] Malyshenko S, Kondakova OA, Nazarova JV, Kaplan IB, Taliansky ME, Atabekov JG (1993) Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. J Gen Virol 74:1149-1156
[102] Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348-352
[103] McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737-747
[104] Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431: 343-349
[105] Minea R, Swenson S, Costa F, Chen TC, Markland FS (2005) Development of a novel recombinant disintegrin, contortrostatin, as an effective anti-tumor and anti-angiogenic agent. Pathophysiol Haemost Thromb 34:177-188
[106] Mlotshwa S, Pruss GJ, Vance V (2008) Small RNAs in viral infection and host defense. Trends Plant Sci 13:375-382
[107] Moissiard G, Voinnet O (2006) RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci USA103:19593-19598
[108] Molnár A, Csorba T, Lakatos L, Várallyayé, Lacomme C, Burgyán J (2005) Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs. J Virol 79:7812-7818
[109] Murphy KC (1998)Use of bacteriophageλrecombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063-2071
[110] Murphy KC, Campellone KG, Poteete AR (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246:321-330
[111] Muyrers JP, Zhang Y, Testa G, Stewat AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555-1557
[112] Nakayashiki I (2005) RNA silencing in fungi: Mechanisms and applications. FEBS Lett 579:5950-5957
[113] Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous gene in trans. Plant Cell 2:279-289
[114] Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH(2006)Expression 103of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotech 24:1420-1428
[115] Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton AJ (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res 35:e60
[116] Pertzev AV, Nicholson AW (2006) Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III. Nucleic Acids Res 34:3708-3721
[117] Phillip D Zamore, Thomas Tuschl, Phillip A Sharp, and David P. Bartel. (2000) RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25-33
[118] Plasterk RH (2002) RNA silencing: the genome's immune system. Science 296: 1263-1265
[119] Pinto YM, Kok RA, Baulcombe DC (1999) Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes. Nature Biotechnol 7:702-707
[120] Posfai G, Kolisnychenko V, Bereczki Z, Blattner FR (1999) Marker less gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27: 4409-4415
[121] Poteete AR, Fenton AC (2000) Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12. J Bacteriol 182:2336-2340
[122] Poteete AR, Fentom AC, Murphy KC (1999) Roles of RuvC and RecG in phageλRed-mediated recombination. J Bacteriol 181:5402-5408
[123] Prins M, Laimer M, Noris E, Schubert J, Wassenegger M, and Tepfer M (2008) Strategies for antiviral resistance in transgenic plants. Mol Plant Pathol 9:73-83
[124] Przemyslaw L, Carol EJ, Edward K, Andrew JG, John AW (2003) Coat protein-mediated resistance to turnip mosaic virus in oilseed rape (Brassica napus ) Mol Breed11:83-94
[125] Qu LH, Henras A, Lu YJ, Zhou H, Zhou WX, Zhu YQ, Zhao J, Henry Y, Caizergues-Ferrer M, Bachellerie JP (1999) Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast. Mol Cell Biol 19:1144-1158
[126] Régnier P, Grunberg-Manago M (1989) Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decayof the downstream mRNA. J Mol Biol 210:293-302
[127] Reyret JM, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infection and Immun 66: 4011-4017
[128] Robertson HD, Webster RE, Zinder ND (1968) Purification and properties of ribonucleaseIII from Escherichia coli. J Biol Chem 243:82-91
[129] Roca AI, Cox MM (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol 56:129-223
[130] Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343-3353
[131] Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, NY
[132] Sano T, Nagayama A, Ogawa T, Ishida I, Okada Y (1997) Transgenic potato expressing a double- stranded RNA-specific ribonuclease is resistant to potato spindle tuber viroid. Nature Biotechnol 15:1290-1294
[133] Sasaki T, Shimizu N (2007) Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins. Gene 396:312-320
[134] Schwartzberg PL, Goff SP, Robertson EJ (1989) Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246:799-803
[135] Sedivy JM, Sharp PA (1989) Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci USA 86:227-231
[136] Serra-Moreno R, Acosta S, Hernalsteens JP, Jofre J, Muniesa M (2006) Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes. BMC Mol Biol 7:31
[137] Sijen T, Kooter JM (2000)Post-transcriptional gene-silencing: RNAs on the attack or on the defense? Bioessays 22:520-531
[138] Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476
[139] Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320
[140] Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specificgene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121-1133
[141] Sonoda S, Mori M, Nishiguchi M (1999) Homology-dependet virus resistance in transgenic plants with the coat protein gene of sweet potato feathery mottle potyvirus:target specificity and transgene methylation. Phytopathology 89:385-391
[142] Suzuki M, Masuta C, Takanami Y, Kuwata S (1993) Resistance against cucumber mosaic virus in plants expressing the viral replicon. FEBS Lett 379:26-30
[143] Szittya G, Silhavy D, Molnár A, Havelda Z, Lovasá, Lakatos L, BánfalviZ, BurgyánJ (2003) Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22:633-640
[144] Takiff HE, Chen SM, Court DL (1989) Genetic analysis of the rnc operon of Escherichia coli. J Bacteriol 171:2581-2590
[145] Tavladoraki P, Benvenuto E, Trinca S, Martinis DD, Cattaneo A, Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366:469-472
[146] Tenllado F, Díaz-Ruíz JR (2001) Double-stranded RNA-mediated interference with plant virus infection. J Virol 75:12288-12297
[147] Tenllado F, Llave C, Díaz-Ruíz JR (2004) RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res 102:85-96
[148] Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR (2003) Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol 3:3
[149] Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103-112
[150] Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395:854
[151] Torres RM, Kühn R (1997) Laboratory protocols for conditional gene targeting. Oxford: Oxford University Press, 1997
[152] Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191-3197
[153] Van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR (1990) Flavonoid genes in Petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291-229
[154] Vargas M, Martínez-García B, Díaz-Ruíz JR, Tenllado F (2008) Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids. Virol J 5:42
[155] Vogler H, Akbergenov R, Shivaprasad PV, Dang V, Fasler M, Kwon MO, Zhanybekova S, Hohn T, Heinlein M (2007) Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein. J Virol 81:10379-10388
[156] Voss A, Niersbach M, Hain R, Hirsch, HJ, Liao YC, Kreuzaler F, Fischer R (1995) Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Mol Breed 1:39-50
[157] Watanabe Y, Ogawa T, Takahashi H, Ishida I, Takeuchi Y, Yamamoto M, Okada Y (1995) Resistance against multiple plant viruses in plants mediated by a double stranded-RNA specific ribonuclease. FEBS Lett 372:165-168
[158] Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959-13964
[159] Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for effcient, effective and high-throughput gene silencing in plants. Plant J. 27:581-590
[160] Westphal H, Crouch RJ (1975) Cleavage of adenovirus messenger RNA and of 28S and 18S ribosomal RNA by RNase III. Proc Natl Acad Sci USA 72:3077-3081
[161] Wilson RB, Davis D, Mitchell AP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181: 1868-1874
[162] Wu H, Yang RK, Butcher S, Kang S, Chanfreau G, and Feigon J (2001) A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNaseIII. EMBO J 20:7240-7249
[163] Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119
[164] Yeom KH, Lee Y, Han J, Suh MR, Kim VN (2006) Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing. Nucleic Acids Res 34:4622-4629
[165] Yie Y, Zhao F, Zhao SZ, Liu YZ, Liu YL, Tien P (1992) High resistance to cucumber mosaic virus conferred by satellite RNA and coat protein in transgenic commercial tobacco cultivar G-140. Mol Plant Microbe Interact 5:460-465
[166] Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011-3016
[167] Ying SY, Chang DC, Miller JD, Lin SL (2006) MicroRNA Protocols. Perspectives. Methods Mol Biol. 342:351-358
[168] Yi R, Doehle BP, Qin Y, Macara IG, Cullen B (2005) Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 11:220-226
[169] Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97:5978-5983
[170] Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33
[171] Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 21:5875-5885
[172] Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W (2004) Single processing center models for human Dicer and bacterial RNase III. Cell 118:57-68
[173] Zhu CX, Song YZ, Yin GH, Wen FJ (2008) Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus and Cucumber mosaic virus. J Phytopathol 157:101-107
[174] Ziegler-Graff V, Guilford PJ, Baulcombe DC (1991) Tobacco rattle virus RNA-129K gene product potentates viral movement and also affects symptom induction in tobacco. Virology 182:145-155
[2]白庆荣,朱俊华,刘晓玲,朱常香,宋云枝,温孚江(2005)利用RNA介导的抗病性获得抗2种病毒的转基因烟草.植物病理学报, 35:148-154
[3]白光兴,孙志伟,黄莺,俞炜源(2005)利用Red重组系统对大肠杆菌clpP基因的敲除.中国生物化学与分子生物学报, 21:35-38
[4]郭兴启,温孚江,朱常香(2000)转基因植物中RNA介导地病毒抗性研究进展.生命科学, 12:166-169
[5]韩聪,张惟材,游松,黄留玉(2004)大肠杆菌ptsG基因敲除及其缺陷株生长特性研究.生物工程学报, 16-20
[6]侯松旺,陈新文,王汉中,胡志红(2003)一种以PCR产物直接构建同源重组杆状病毒的方法.中国科学(C辑), 33:169-174
[7]贾力,吴茂森,张文蔚,肖红成,卓敏(2001)大麦黄矮病毒单双价外壳蛋白基因植物表达载体构建及小麦遗传转化.生物技术学报, 9:23-27
[8]李鹏,宋云枝,刘晓玲,朱常香,温孚江(2007)马铃薯Y病毒CP基因5’端和3’端反向重复结构介导的抗病性研究.植物病理学报, 37:69-76
[9]李世访,郑银英,成卓敏(2001)利用酵母的PacI基因获得抗类病毒转基因植物.植物保护, 27: 35-37
[10]刘晓玲,宋云枝,刘红梅,温孚江,朱常香,白庆荣(2005) PVX 25 kD运动蛋白基因和外壳蛋白基因介导的抗病性研究.作物学报, 31:827-832
[11]王恒梁,冯尔玲,史兆兴,姚潇,苏国富,黄留玉(2002) Red系统快速敲除痢疾杆菌asd基因.军事医学科学院刊, 26:161-164
[12]张德咏,朱春晖,成飞雪,何明远,张战泓,刘勇(2008)表达dsRNA的细菌粗体液可抑制黄瓜花叶病毒对烟草的侵染.植物病理学报, 38:304-311
[13]张建林,邬开朗,张雪,朱应,李雁,赵伟光,吴建国(2004) siRNA表达载体的构建及其表达.中国病毒学, 19: 510-513
[14]张明,邵宁生(2002)RNA阻抑和基因沉默.军事科学院院刊, 26:61-65
[15]张荣意,刘智昕(2002)植物抗病毒基因工程的策略和机制.热带农业科学, 22:68-73
[16]张雪,温延益(2008) Red重组系统用于大肠杆菌基因修饰研究进展.中国生物工程杂志, 28: 89-93
[17]张中华,候永泰(2004) siRNA制备技术的研究进展.生命科学, 16:231-235
[18]赵明敏,安德荣,黄广华,何祖华,陈江野(2006)瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染.植物病理学报36:35-40
[19]赵明敏,杨向东,鲁红学,张长青(2007) siRNA对烟草原生质体中TMV RNA积累的干扰作用研究.植物病理学报37:284-288
[20]朱俊华,朱常香,温孚江,宋云枝(2004)正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究.植物病理学报, 34:133-140
[21]朱俊华,朱晓平,温孚江,白庆荣,朱常香,宋云枝(2004)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, 34:23-30
[22]朱常香,刘红梅,宋云枝,温孚江(2005) RNA介导的病毒抗性在转基因烟草中的烟草分析.遗传学报, 32:94-103
[23]朱常香,宋云枝,张松,郭兴启,温孚江(2001)抗芜菁花叶病毒转基因大白菜的培育.植物病理学报, 31:257-264
[24]竺晓平,朱常香,宋云枝,温孚江,刘红梅,李向东(2006) CP基因3’端短片段介导的对马铃薯Y病毒的抗性.中国农业科学, 39:1153-1158
[25] Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986)Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743
[26] Amarasinghe AK, Calin-Jageman I, Harmouch A, Sun W, Nicholson AW (2001) Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage. Methods Enzymol 342:143-158
[27] Andrew F, Xu S, Mary KM, Steven A K, Samuel E D, Craig CM (1998) Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391:806-811
[28] Apirion D, Gitelman DR (1980) Decay of RNA in RNA processing mutants of Escherichia coli. Mol Gen Genet 177:339-343
[29] Apirion D, Miczak A (1993) RNA processing in prokaryotic cells. Bioessays 15:113-120
[30] Aristarkhov A, Mikulskis A, Belasco JG, Lin EC (1996) Translation of the adhE transcript to produce ethanol dehydrogenase requires RNase III cleavage in Escherichiacoli. J Bacteriol 178: 4327-4332
[31] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol DOI 10.1038/msb4100050
[32] Bao S, Cagan R (2006) Fast cloning inverted repeats for RNA interference. RNA 12:2020-2024
[33] Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297
[34] Bass BL (2000) Double-stranded RNA as a template for gene silencing. Cell 101:235-238
[35] Baulcombe DC (2004) RNA silencing in plants. Nature 431:356-363
[36] Brantl S (2002) Antisense-RNA regulation and RNA interference. Biochim Biophys Acta.1575:15-25
[37] Beran RK, Simons RW (2001) Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation. Mol Microbiol 39:112-125
[38] Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363-366
[39] Blaszczyk J, Tropea JE, Bubunenko M, Routzahn KM, Waugh DS, Court DL, Ji X (2001) Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure 9:1225-1236
[40] Blencowe BJ, Bowman JA, McCracken S, Rosonina E (1999) SR-related proteins and the processing of messenger RNA precursors. Biochem Cell Biol 77: 277-291
[41] Bouché, N, Lauressergues D, Gasciolli V, Vaucheret H (2006) An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J 25:3347-3356
[42] Capecchi MR. (1994) Gene Targeting. Sclentific American 270:34
[43] Capecchi MR (1998) Altering the genome by homologous recombination. Science 244:1288-1292
[44] Chanfreau G, Rotondo G, Legrain P, Jacquier A (1998) Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1. EMBO J 17:3726-3737
[45] Chanfreau G, Legrain P, Jacquier A (1998) Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. J Mol Biol 284:975-988
[46] Chang KY, Ramos A (2005) The double-stranded RNA-binding motif, a versatilemacromolecular docking platform. FEBS J 272:2109-2117
[47] Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287-293
[48] Cherepanov PP, Wackernagel, W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9-14
[49] Cogni C, Romano N, Macino G (1994) Suppression of gene expression by homologous transgenes. Antonie Van Leeuwehoek 65:205-209
[50] Cooper B, Lapidot M, Heick JA, Dodds JA, Beachy RN (1995) A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 206:307-313
[51] Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC (2000) An RNA dependent RNA polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543-553
[52] Dasgupta S, Fernandez L, Kameyama L, Inada T, Nakamura Y, Pappas A, Court DL (1998) Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III - the effect of dsRNA binding on gene expression. Mol Microbiol 28:629-640
[53] Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640-6645
[54] Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, Saigo K (2003) Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eIF2C translation initiation factors. Curr Biol 13:41-46
[55] Drider D, Condon C (2004) The continuing story of endoribonucleaseIII. J Mol Microbiol Biotechnol 8:195-200
[56] Duan CG, Wang CH, Fang RX, Guo HS (2008) Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82:11084-11095
[57] Dunn JJ (1976) RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fidelity of cleavage. J Biol Chem 251:3807-3814
[58] Dunn JJ, Studier FW (1973) T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease III. Proc Natl Acad Sci USA 70:3296-3300
[59] Dykstra CC, Prasher D, Kushner SR (1984) Physical and biochemical analysis of thecloned recB and recC genes of Escherichia coli K-12. J Bacteriol 157:21-27
[60] Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: Short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457-467
[61] Elbashir SM, Harborth J, Weber K, Tuschl T(2002)Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199-213
[62] Elbashir SM, Lendeckel W, Tuschl T (2000) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188-200
[63] Elela SA, Igel H, Ares MJ (1996) RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. Cell 85:115-124
[64] Filippov V, Solovyev V, Filippova M, Gill SS (2000) A novel type of RNase III family proteins in eukaryotes. Gene 245: 213-221
[65] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811
[66] Fortin KR, Nicholson RH, Nicholson AW (2002) Mouse ribonuclease Ill cDNA structure, expression analysis, and chromosomal location. BMC Genomics 3:26
[67] Golemboski DB, Lomonossoff GP, Zaitlin M (1990) Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci USA 87:6311-6315
[68] Goto K, Kanazawa A, Kusaba M, Masuta C (2003) A simple and rapid method to detect plant siRNAs using nonradioactive probes. Plant Mol Biol Rep 21:51-58
[69] Gregory RI, Yan K, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432:235-240
[70] Guo HS, Xie Q, Fei JF, Chua NH (2005) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17:1376-1386
[71] Guo S, Kemphues KJ (1995) Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611-620
[72] Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950-952
[73] Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296
[74] Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18:3016-3027
[75] Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887-901
[76] Huntzinger E, Boisset S, Saveanu C, Benito Y, Geissmann T, Namane A, Lina G, Etienne J, Ehresmann B, Ehresmann C, Jacquier A, Vandenesch F, Romby P (2005) Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J 24:824-835
[77] Jasin M, Elledge SJ, Davis RW, Berg P (1990) Gene targeting at the human CD4 locus by epitope addition. Genes Dev 4:157-166
[78] Jiang F, Ye XC, Liu X, Fincher L, McKearin D, Liu Q (2005) Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev 19:1674-1679
[79] Jung S, Rajewsky K, Radbruch A (1993) Shutdown of class switch recombination by deletion of a switch region control element. Science 259: 984-987
[80] Kato C, Ohmiya R, Mizuno T (1998) A rapid method for disrupting genes in the Escherichia coli genome. Biosci Biotechnol Biochem 62:1826-1829
[81] Ketting RF, Fischer SEJ, Bernsterin E, Sijen T, Hannon GJ, Plasterk RH (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15:2654-2659
[82] Kindler P, Keil TU, Hofschneider PH (1973) Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli. Mol Gen Genet 126:53-69
[83] Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58:401-465
[84] Krautz-Peterson G, Skelly PJ (2008) Schistosoma mansoni: The dicer gene and its expression. Exp Parasitol 118:122-128
[85] Kreuze JF, Savenkov EI, Cuellar W, Li X, Valkonen JP (2005) Viral Class 1 RNase III Involved in Suppression of RNA Silencing. J Virol 79:7227-7238
[86] Kuzminov A (1999) Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol Mol Biol Rev 63:751-813
[87] Lam YH , Wong YS ,Wang B, Wong RNS, Yeung HW, Shaw PC (1996) Use of trichosanthin to reduce infection by turnip mosaic virus. Plant Sci 114:111-117
[88] Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer NE (1990)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) 8:127-134
[89] Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, R?dmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415-419
[90] Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ, Carthew RW (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69-81
[91] Liu HM, Zhu CX, Zhu XP, Guo XQ, Song YZ, Wen FJ (2007) A link between PVYN CP gene-mediated virus resistance and transgene arrangement. J Phytopathol 155:676-682
[92] Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods 25:402-408
[93] Lomonossoff GP, (1995) Pathogen derived resistance to plant viruses. Ann Rev Phytopathol 33:323-343
[94] Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58:401-465
[95] Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN (1993) A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant J. 4:959-970
[96] Lipardi C, Wei Q, Paterson BM (2001) RNAi as random degradative PCR:siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107: 297-307
[97] Lodge JK, Kaniewski WK, Tumer NE (1993) Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein. Proc Natl Acad Sci USA 90:7089-7093
[98] Lucas GB (1975) Diseases of tobacco, 3rd ed. Biological Consulting Associates, Raleigh, NC
[99] Macrae IJ, Doudna JA (2007) Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr Opin in Struct Biol 17:138-145
[100] Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD, Doudna JA (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311:195-
[101] Malyshenko S, Kondakova OA, Nazarova JV, Kaplan IB, Taliansky ME, Atabekov JG (1993) Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. J Gen Virol 74:1149-1156
[102] Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348-352
[103] McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737-747
[104] Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431: 343-349
[105] Minea R, Swenson S, Costa F, Chen TC, Markland FS (2005) Development of a novel recombinant disintegrin, contortrostatin, as an effective anti-tumor and anti-angiogenic agent. Pathophysiol Haemost Thromb 34:177-188
[106] Mlotshwa S, Pruss GJ, Vance V (2008) Small RNAs in viral infection and host defense. Trends Plant Sci 13:375-382
[107] Moissiard G, Voinnet O (2006) RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci USA103:19593-19598
[108] Molnár A, Csorba T, Lakatos L, Várallyayé, Lacomme C, Burgyán J (2005) Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs. J Virol 79:7812-7818
[109] Murphy KC (1998)Use of bacteriophageλrecombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063-2071
[110] Murphy KC, Campellone KG, Poteete AR (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246:321-330
[111] Muyrers JP, Zhang Y, Testa G, Stewat AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555-1557
[112] Nakayashiki I (2005) RNA silencing in fungi: Mechanisms and applications. FEBS Lett 579:5950-5957
[113] Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous gene in trans. Plant Cell 2:279-289
[114] Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH(2006)Expression 103of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotech 24:1420-1428
[115] Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton AJ (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res 35:e60
[116] Pertzev AV, Nicholson AW (2006) Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III. Nucleic Acids Res 34:3708-3721
[117] Phillip D Zamore, Thomas Tuschl, Phillip A Sharp, and David P. Bartel. (2000) RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25-33
[118] Plasterk RH (2002) RNA silencing: the genome's immune system. Science 296: 1263-1265
[119] Pinto YM, Kok RA, Baulcombe DC (1999) Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes. Nature Biotechnol 7:702-707
[120] Posfai G, Kolisnychenko V, Bereczki Z, Blattner FR (1999) Marker less gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27: 4409-4415
[121] Poteete AR, Fenton AC (2000) Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12. J Bacteriol 182:2336-2340
[122] Poteete AR, Fentom AC, Murphy KC (1999) Roles of RuvC and RecG in phageλRed-mediated recombination. J Bacteriol 181:5402-5408
[123] Prins M, Laimer M, Noris E, Schubert J, Wassenegger M, and Tepfer M (2008) Strategies for antiviral resistance in transgenic plants. Mol Plant Pathol 9:73-83
[124] Przemyslaw L, Carol EJ, Edward K, Andrew JG, John AW (2003) Coat protein-mediated resistance to turnip mosaic virus in oilseed rape (Brassica napus ) Mol Breed11:83-94
[125] Qu LH, Henras A, Lu YJ, Zhou H, Zhou WX, Zhu YQ, Zhao J, Henry Y, Caizergues-Ferrer M, Bachellerie JP (1999) Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast. Mol Cell Biol 19:1144-1158
[126] Régnier P, Grunberg-Manago M (1989) Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decayof the downstream mRNA. J Mol Biol 210:293-302
[127] Reyret JM, Pelicic V, Gicquel B, Rappuoli R (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infection and Immun 66: 4011-4017
[128] Robertson HD, Webster RE, Zinder ND (1968) Purification and properties of ribonucleaseIII from Escherichia coli. J Biol Chem 243:82-91
[129] Roca AI, Cox MM (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol 56:129-223
[130] Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343-3353
[131] Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, NY
[132] Sano T, Nagayama A, Ogawa T, Ishida I, Okada Y (1997) Transgenic potato expressing a double- stranded RNA-specific ribonuclease is resistant to potato spindle tuber viroid. Nature Biotechnol 15:1290-1294
[133] Sasaki T, Shimizu N (2007) Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins. Gene 396:312-320
[134] Schwartzberg PL, Goff SP, Robertson EJ (1989) Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246:799-803
[135] Sedivy JM, Sharp PA (1989) Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci USA 86:227-231
[136] Serra-Moreno R, Acosta S, Hernalsteens JP, Jofre J, Muniesa M (2006) Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes. BMC Mol Biol 7:31
[137] Sijen T, Kooter JM (2000)Post-transcriptional gene-silencing: RNAs on the attack or on the defense? Bioessays 22:520-531
[138] Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476
[139] Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320
[140] Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specificgene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121-1133
[141] Sonoda S, Mori M, Nishiguchi M (1999) Homology-dependet virus resistance in transgenic plants with the coat protein gene of sweet potato feathery mottle potyvirus:target specificity and transgene methylation. Phytopathology 89:385-391
[142] Suzuki M, Masuta C, Takanami Y, Kuwata S (1993) Resistance against cucumber mosaic virus in plants expressing the viral replicon. FEBS Lett 379:26-30
[143] Szittya G, Silhavy D, Molnár A, Havelda Z, Lovasá, Lakatos L, BánfalviZ, BurgyánJ (2003) Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22:633-640
[144] Takiff HE, Chen SM, Court DL (1989) Genetic analysis of the rnc operon of Escherichia coli. J Bacteriol 171:2581-2590
[145] Tavladoraki P, Benvenuto E, Trinca S, Martinis DD, Cattaneo A, Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366:469-472
[146] Tenllado F, Díaz-Ruíz JR (2001) Double-stranded RNA-mediated interference with plant virus infection. J Virol 75:12288-12297
[147] Tenllado F, Llave C, Díaz-Ruíz JR (2004) RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res 102:85-96
[148] Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR (2003) Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol 3:3
[149] Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103-112
[150] Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395:854
[151] Torres RM, Kühn R (1997) Laboratory protocols for conditional gene targeting. Oxford: Oxford University Press, 1997
[152] Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191-3197
[153] Van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR (1990) Flavonoid genes in Petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291-229
[154] Vargas M, Martínez-García B, Díaz-Ruíz JR, Tenllado F (2008) Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids. Virol J 5:42
[155] Vogler H, Akbergenov R, Shivaprasad PV, Dang V, Fasler M, Kwon MO, Zhanybekova S, Hohn T, Heinlein M (2007) Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein. J Virol 81:10379-10388
[156] Voss A, Niersbach M, Hain R, Hirsch, HJ, Liao YC, Kreuzaler F, Fischer R (1995) Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Mol Breed 1:39-50
[157] Watanabe Y, Ogawa T, Takahashi H, Ishida I, Takeuchi Y, Yamamoto M, Okada Y (1995) Resistance against multiple plant viruses in plants mediated by a double stranded-RNA specific ribonuclease. FEBS Lett 372:165-168
[158] Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959-13964
[159] Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for effcient, effective and high-throughput gene silencing in plants. Plant J. 27:581-590
[160] Westphal H, Crouch RJ (1975) Cleavage of adenovirus messenger RNA and of 28S and 18S ribosomal RNA by RNase III. Proc Natl Acad Sci USA 72:3077-3081
[161] Wilson RB, Davis D, Mitchell AP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181: 1868-1874
[162] Wu H, Yang RK, Butcher S, Kang S, Chanfreau G, and Feigon J (2001) A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNaseIII. EMBO J 20:7240-7249
[163] Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119
[164] Yeom KH, Lee Y, Han J, Suh MR, Kim VN (2006) Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing. Nucleic Acids Res 34:4622-4629
[165] Yie Y, Zhao F, Zhao SZ, Liu YZ, Liu YL, Tien P (1992) High resistance to cucumber mosaic virus conferred by satellite RNA and coat protein in transgenic commercial tobacco cultivar G-140. Mol Plant Microbe Interact 5:460-465
[166] Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011-3016
[167] Ying SY, Chang DC, Miller JD, Lin SL (2006) MicroRNA Protocols. Perspectives. Methods Mol Biol. 342:351-358
[168] Yi R, Doehle BP, Qin Y, Macara IG, Cullen B (2005) Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 11:220-226
[169] Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97:5978-5983
[170] Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33
[171] Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 21:5875-5885
[172] Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W (2004) Single processing center models for human Dicer and bacterial RNase III. Cell 118:57-68
[173] Zhu CX, Song YZ, Yin GH, Wen FJ (2008) Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus and Cucumber mosaic virus. J Phytopathol 157:101-107
[174] Ziegler-Graff V, Guilford PJ, Baulcombe DC (1991) Tobacco rattle virus RNA-129K gene product potentates viral movement and also affects symptom induction in tobacco. Virology 182:145-155
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |