【作者】 尹国华；

【导师】 温孚江；

【作者基本信息】 山东农业大学 ， 植物病理学， 2009， 博士

【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%。更多还原

【Abstract】 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.更多还原