【作者】 朱常香；

【导师】 温孚江；

【作者基本信息】 山东农业大学 ， 植物病理学， 2005， 博士

【摘要】 烟草病毒病是全世界烟草栽培区发生普遍,危害严重的一大类病害。目前世界上已报道发现的侵染烟草的病毒达47 种,我国有17 种。在田间,常表现为多种病毒混合侵染,给烟草生产造成重大损失,重病田减产20～50%,夏烟减产70%以上。对病毒病的快速检测和监控技术是病毒病防治的基础性工作,可提高病毒病防治的针对性和时效性;而植物抗病毒基因工程的发展,特别是RNA 介导的病毒抗性策略的提出,为植物病毒病的防治提供了新的途径。围绕着烟草病害的防治,本研究进行了两方面的探索。其一,分离、提纯马铃薯Y病毒(PVY)、黄瓜花叶病毒(CMV)、烟草普通花叶病毒(TMV)、烟草蚀纹病毒(TEV)、马铃薯X 病毒(PVX)、烟草环斑病毒(TRSV),克隆了6 种病毒的外壳蛋白(CP)基因,并进行了序列分析,首次明确了PVY、PVX 和CMV 山东分离物在分类学上的归属地位。制备了不同病毒的抗体和酶标抗体,建立了以点免疫结合技术(DIBA)为基础的快速、多元的病毒检测体系,对山东烟草病毒病进行了初步调查,明确了主要病毒种类。其二,RNA 介导的病毒抗性策略应用于烟草病毒病防治的研究。在已获得不同抗病类型的转非翻译PVY-CP 基因烟草的基础上,研究了RNA 介导的病毒抗性遗传及外源基因的整合方式与RNA 介导的病毒抗性产生的关系,明确了RNA 介导的病毒抗性可稳定地遗传;揭示了转基因的结构对于诱发病毒抗性产生可能起关键的作用,并进而设计了发夹结构DNA 进行了验证。在此基础上,以烟草上危害最为严重的PVY、CMV和TMV 为材料,克隆并构建了包含3 种病毒部分CP 基因的发夹结构DNA 的植物表达载体,转化烟草,首次培育获得了抗3 种病毒转基因烟草。研究结果将为烟草病毒病的防治提供理论依据和技术支持,具有很好的理论和应用价值。具体研究结果如下: 1. 烟草病毒的检测新技术研究—烟草病毒病快速、多元检测技术的建立及应用1.1 病毒的分离从山东感病的烟草和马铃薯上,分离纯化了马铃薯Y 病毒(PVY-SD-TA)、马铃薯X 病毒(PVX-SD1)、烟草花叶病毒(TMV-SD1)、烟草蚀纹病毒(TEV-SD1)、黄瓜花叶病毒(CMV-SD1)和烟草环斑病毒(TRSV-SD1)等6 种烟草上常见的病毒。1.2 病毒外壳蛋白基因克隆及序列分析更多还原

【Abstract】 Tobacco virus diseases occur in all of the tobacco cultivated areas throughout the world, and cause serious yield losses every year. generally, the losses caused by virus infection could reach up to 20%-50%. The viruses infection could lead to more serious losses on summer tobaccos, which could be as high as 70%. Up to 47 viruses have been found naturally infecting tobacco worldwide, and 17 viruses have been reported infecting tobacco in China. The detecting technologies to the viruses are the basis of virus controlling, which can improve the pertinence and efficiency. Also, the development of anti-virus genetic engineering strategy, especially the application of the RNA-mediated virus resistance, may provide a new strategy to control the virus infection. In this study, the potato virus Y (PVY), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), tobacco etch virus (TEV), potato virus X (PVX) and tobacco ring spot virus (TRSV) were isolated, and the rabbits was then immunized using the purified virus particles or the expressed coat proteins as antigens, and the antisera against these viruses were obtained. The quickly and handily analysis technology that was based on the Dot Immune Banding Assay (DIBA) was established, and was used to detect the tobacco viruses in Shandong. On the basis of obtaining CP transgenic plants, the inheritance of the transgene was studied. I also cloned different parts of CP genes of PVY, CMV and TMV, and constructed plant expressing vectors that contain hairpin structure of the chimerical gene. The plant-expressing vectors were then transformed into the tobacco plant of NC89, and he transgenic plants with resistance to PVY, TMV and CMV were obtained.. These research results will provide theory and technology support to tobacco virus controlling. The main results and conclusions presented in this thesis are decribed as follows: 1. Research on new technology to detect tobacco viruses 1.1 Isolation of tobacco viruses Isolates of PVY, TMV, CMV, TEV, PVX and TRSV were obtained from tobacco or potato in Shandong Province, which were named PVY-SD-TA, TMV-SD1, CMV-SD1, TEV-SD1, PVX-SD1 and TRSV-SD1, respectively. 1.2 Cloning and sequence analysis of the coat protein genes of tobacco viruses The coat protein genes of the PVY-SD-TA, PVX-SD1,TMV-SD1, TEV-SD1, CMV-SD1 and TRSV-SD1 were amplified from the virus RNAs by using reverse transcription-polymerase chain reaction (RT-PCR). Sequences analysis results were as follows: The CP gene of PVY-SD-TA CP was 804 nucleotides in length, which encodes the coat protein of 267 amino acids. Sequencing results showed that the homology between PVY-SD-TA CP gene and PVYN and PVYO was 96% and 99%, respectively. According to these results, PVY-SD-TA was identified as an isolate of PVYO strain. The CP gene of PVX-SD1 was 719 nucleotides in length, which encodes the coat protein of 238 amino acids. The nucleotide sequence of the PVX-SD1 CP gene was determined and compared with other isolates reported in GenBank. It shared 80.1%～99.7% and 89.8%～100% homology with the reported isolates at the sequence levels of nucleotide and deduced amino acid, respectively. PVX-SD1 shared 99.7% and 100% homologies at the nucleotide and deduced amino acid levels, respectively; with one of the European strain UK3, indicating that they could belong to the same strain. The CP gene of TMV-SD1 was 480 nucleotides in length, which encodes the coat protein of 159 amino acids, and the CP gene shared 73.7%～98.1% homology with TMV CP genes registered in GenBank. The CP gene of TEV-SD1 was 789 nucleotides in length, which encodes the coat protein of 263 amino acids, and the CP gene shared 94.1%～98.6% homology with TEV CP genes registered in GenBank. The CP gene of CMV-SD1 was 1548 nucleotides in length, which encodes the coat protein of 515 amino acids. The sequences of the CMV-SD1 CP genes were sequenced andcompared with the CP gene sequences of CMV reported in GenBank. The results showed that the sequence of the CMV-SD1 CP gene shared 92.7%~94.9% homology with subgroupⅠstrains of CMV, while only 59.3%~59.5% homology was found when compared with CMV subgroupⅡstrains. According to these results, CMV-SD1 isolate s belong to CMV subgroupI. The CP gene of TRSV-SD1was 1548 nucleotides in length, which encodes the coat protein of 515 amino acids, and the CP gene shared 90.7%-94.6% nucleotides and amino acid homology with TRSV CP genes registered in GenBank. 1.3 Preparation of viuses antibodies and enzymae-labelling antibodies Rabbits were immunized using the purified viruses particles or the expressed coat proteins as antigens, and the antisera were obtained. The IgGs of six viruses were purified from the antisera, and were labeled by phosphatase alkaline. 1.4 Establishment of DIBA assay system After the factors influencing virus detection, such as the concentration of antibodies and enmye-labelled antibodies, diluted concentration and preparation of the leaf juices, the effects of EDTA and reaction times, a highly efficient assay system was developed. Using this protocol, six viruses could be detected simultaneously. 1.5 Investigation of virues infecting tobacco plants in Shandong The investigation results to the virus varieties in Shandong tobacco areas showed that PVY, CMV, TMV, TEV, PVX, and TRSV had been found. Of them, PVY, CMV and TMV were detected in high frequency: the frequencies of PVY, CMV, and TMV was 53.08%, 43.69%, and 38.03%, respectively. 2. Research on new technology to control tobacco viruses 2.1 The genetic analysis of RNA -mediated virus resistance in transgenic tobacco Several T0 progeny transgenic plants with different resistant level and different transgenes copies were selected to further study the transgene and the resistance inheritance from T1-T4 progeny lines. Results showed that the transgene in T0 progeny susceptible transgenic plants which contained 1-2 transgene copies followed one site Mendelian segregation rule in their after progenies, different progenies were all susceptible. T0 progeny resistant transgenic plants, which contained 4-6 transgene copies, followed 15:1 and 63:1 segregation in T1 progeny, but the transgene reconstruction were observed, the inheritance ofRNA-mediated resistance was unstable. The resistant transgenic plants which contained two transgene copies came from T0 progeny resistant transgenic plants also followed one site Mendelian segregation rule in their after progeny, the transgene and resistance can inherit and express stably. The homozygous resistant transgenic plants were obtained. The analysis of transgene integration organization indicated that almost all of the highly resistant plants contained inverted repeat (IR) sequences. 2.2 Effects of hpRNA to RNA-mediated resistance occurrence We designed cDNA constructs using direct repeat (DR) and inverted repeat (IR)which transcript producetion was hpRNA of the coat protein gene segments of potato virus Y (PVY),and introduced these constructs into tobacco plants via Agrobacterium tumefaciens-mediated transformation system. Resistant assays of the transgenic plants showed that highly resistant plants to PVY infection were obtained in both DR-and IR-transgenic plants. However, higher proportion of resistant plants (60%), was obtained from the IR-transgenic plants than that from DR-transgenic plants. 2.3 Production of transgenic tobacco plants with multi-virus resistance According to the CP genes nucleotide sequence of PVYO, TMV and CMV several primers were designed and synthesized. Short fragments of CP genes of PVY (200bp), TMV (200bp) and CMV (250bp) were obtained by PCR. The three CP gene fragments were inserted into cloning plasmid pUC19, and the recombinant plasmid pUPTC was obtained, which contained the first chain of the hairpin DNA (hpDNA) . Using the recombinant plasmid pUPTC as template, the second chain of the hpDNA was obtained by PCR and was inserted into pUPTC, the recombinant plasmid pHPTC was obtained. The chimeric transgenes with hpDNA structure were excised and inserted into plant-expressing vector pROKII, and the recombinant plant-expression vector pRPTV was obtained. The recombinant plant-expression vector pRPTC was introduced into Agrobacterium tumefaciens LBA4404 by direct transferring method, then the target gene was introduced into tobacco NC89. The transformed tissues were selected in the presence pf 100mg/L kanamycin and the transgenic plants were confirmed by kanamycin resistance screening and PCR. One thousand and eighteen transgenic tobacco plants were obtained. The resistance assay showed that the proportion of resistance (immune) plants was up to 18%. Southern blot analysis of更多还原