【作者】 耿显胜；

【导师】 田国忠；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2013， 博士

【摘要】 由植原体引起的泡桐丛枝病（paulownia witches’-broom，PaWB）在我国分布广泛，为害严重，是中国泡桐树种上最严重的病害之一。泡桐丛枝病由种苗种根等无性繁殖材料和韧皮部取食的介体昆虫传播，目前已鉴定的介体昆虫有茶翅蝽（Halyomorpha halys(St l)）、烟草盲蝽（Crytopeltis tenuis (Reuter)）和小绿叶蝉（Empoasca flavescens(Fabricius)）。为了研究泡桐丛枝植原体质粒的功能，进一步明确泡桐丛枝病介体昆虫-植原体之间的分子互作和介体昆虫的传播特性，本论文对以下四个方面进行研究。本研究从实验室保存的含泡桐丛枝植原体质粒pPaWBNy-1的3.0kb的克隆和感病泡桐组培苗中分离到虫传相关基因pPaWBNy-1-ORF5和pPaWBNy-2-ORF4的亲水性肽段编码区，连接到原核表达载体。通过优化表达条件，得到分子量约为15kDa的His-ORF5和分子量约为38kDa的GST-ORF4融合蛋白。纯化融合蛋白，制备了这两个融合蛋白的兔抗体。Western blot分析发现，制备的两个抗体特异性好、灵敏度高，间接ELISA测定两者的效价为1:8100和1:4096。利用制备的抗体，分析了pPaWBNy-1-ORF5和pPaWBNy-2-ORF4在不同材料的表达情况，发现在感病的泡桐组培苗和野外采集的感病泡桐中皆未检测到两基因的表达，而在饲毒的茶翅蝽中检测到分子量约17kDa的pPaWBNy-1-ORF5编码蛋白和18kDa的PaWBNy-2-ORF4编码蛋白。推测pPaWBNy-1-ORF5和pPaWBNy-2-ORF4编码蛋白参与介体昆虫茶翅蝽传播泡桐丛枝植原体。利用原核表达的His-ORF5融合蛋白微注射茶翅蝽，制备了可用于体内分子互作的蛋白样本。以纯化的His-ORF5融合蛋白作为探针，用Far Western blot试验在茶翅蝽胸腹部提取的蛋白中鉴定到分子量为52kDa和36kDa的两个与pPaWBNy-1-ORF5编码蛋白互作的茶翅蝽蛋白，而在血淋巴提取的蛋白中鉴定到分子量为52kDa的互作蛋白。对于这两个蛋白序列的测定，将会有助于确定pPaWBNy-1-ORF5和pPaWBNy-2-ORF4编码蛋白的功能，有助于进一步理解泡桐丛枝植原体与介体昆虫茶翅蝽互作的分子机制。采集了北京中国林科院院内泡桐林中的茶翅蝽，建立了北京茶翅蝽种群的稳定饲养、饲毒和微注射的技术体系。使用网捕法和振落法采集了河南南阳泡桐林和越冬场所的蝽象，一共采集了四种蝽象：茶翅蝽、麻皮蝽（Erhtesina fullo (Thunberg)）、珀蝽（Plautiafimbriata (Fabricus)）和宽胫格蝽（Cappaea tibialis Hsiao et Cheng），它们的数量分别为293、41、8和8头。对越冬场所和泡桐林中的蝽象样本分别提取DNA和蛋白。巢式和半巢式PCR检测带毒情况，在麻皮蝽和宽胫格蝽中没有检测到植原体；而越冬茶翅蝽的带毒率为29.2%，泡桐林中茶翅蝽的带毒率为16.7%。提取的茶翅蝽蛋白进行免疫印迹试验，能够检测到分子量约17kDa的pPaWBNy-1-ORF5编码蛋白表达。使用质粒基因RepA的引物进行半巢式PCR检测珀蝽中的植原体，发现一头珀蝽检测结果为阳性，对PCR产物克隆和测序，序列分析发现其与泡桐丛枝植原体的RepA同源性为99.4%。对阳性珀蝽的蛋白进行免疫印迹试验，鉴定到一个分子量约18kDa的pPaWBNy-2-ORF4编码蛋白。以上结果证明该头珀蝽携带泡桐丛枝植原体。珀蝽是否也是泡桐丛枝植原体的介体昆虫，需要通过传播试验加以证实。更多还原

【Abstract】 Paulownia witches’-broom (PaWB) disease associated with phytoplasma is a devastatingdisease of paulownia in China. Most species of Chinese paulownia are sensitive to PaWBdisease. PaWB disease could be transmitted by vegetative propagation materials（eg: seedlingand root cutting）and phloem-feeding insects. The vector insects of PaWB phytoplasma wereHalyomorpha halys (St l), Crytopeltis tenuis (Reuter) and Empoasca flavescens (Fabricius). Tobetter understand the function of PaWB phytoplasma plasmids and molecular interactions ofPaWB phytoplasma-vector and insect transmission, we conducted the following researches.We use a clone containing a3.0kb segment of PaWB phytoplasma plasmid pPaWBNy-1and genome DNA extracted from infected paulownia plantlets as DNA templates, PCRamplification of pPaWBNy-1-ORF5and pPaWBNy-2-ORF4hydrophilic peptide codingregions. The amplified DNA fragments were inserted into the prokaryotic expression vectorand the recombinant plasmid was transformed into the Escherichia coli Rosseta(DE3) strain.Under the optimal experimental condition, the15kDa of His-tagged ORF5and38kDa ofGST-tagged ORF4fusion proteins were expressed efficiently in E.coli Rosseta(DE3). Thefusion proteins were purified and injected into white rabbit to raise antiserum. The titers of twoantisera were determined to be1:8100and1:4096by indirect ELISA, respectively.Using the two antisera, we analysed the expression of pPaWBNy-1-ORF5andpPaWBNy-2-ORF4genes in different specimens of host plant and vector insect, the tworesultant antibodies didn’t react with the proteins extracted from infected paulownia plantletsand field-collected symptomed paulownia, but detected17kDa of pPaWBNy-1-ORF5codingprotein and18kDa of pPaWBNy-2-ORF4coding protein in H. halys exposing to PaWBphytoplasma infected paulownia. It was inferred that pPaWBNy-1-ORF5andpPaWBNy-2-ORF4might be involved in the insect transmission of H. halys. The in vivointeraction protein specimens were prepared with H. halys by injecting His-tagged ORF5 fusion protein into the hemolymph of H. halys. Then far Western blot experiments were carriedout on H. halys protein blot with phytoplasma proteins as the overlay to identify the protein ofH. halys involved in the interaction with the pPaWBNy-1-ORF5coding protein. Two proteinsof the thoracoab dominal of H. halys were identified, one with a molecular mass of52kDa andthe second with a mass of36kDa. Moreover, a52kDa protein from the hemolymph of H.halys were identified. Sequencing those insect proteins, will help us to determinepPaWBNy-1-ORF5protein function, therefore, to promote to understand the molecularmechanism of PaWB phytoplasma interacting with vector insect H. halys.H. halys nymphs were collected from paulownia plantations in the yard of Chinese Academyof Froestry. We developed a stable rearing, acquisition method as well as abdominalmicroinjection methods of Beijing population of H. halys. Adults of the stink bugs werecollected by means of sweeping net and beating tray in infested paulownia plantations byPaWB phytoplasma and overwintering sites in Nanyang, Henan province. A total of350stinkbugs were collected:293of H. halys,41of Erhtesina fullo (Thunberg),8of Plautia fimbriata(Fabricus) and8of Cappaea tibialis Hsiao et Cheng. DNA and protein were extracted fromstink bugs collected from Nangyang. Semi-nested PCR and nested PCR amplification of theRepA gene specific to PaWB plasmid was performed using the extracted DNA as templates.The results indicated that E. fullo and C. tibialis didn’t carry PaWB phytoplasma, while thepositive detection rate was29.2%of overwinter H. halys, and16.7%of H. halys collected frompaulownia plantations. Western blot analysis using pPaWBNy-1-ORF5antibody revealed thatthat a specific17kDa protein band in H. halys protein, which indicated thatpPaWBNy-1-ORF5gene expressed in H. halys.In semi-nested PCR using RepA gene primers, from a total of8individual stink bugs tested,1of P. fimbriata samples gave a positive singal. The amplified DNA fragment was cloned andsequenced, the sequence alignment revealed that the sequence was99.4%identical to RepAgene paulownia witches’-broom phytoplasma (EF426472). In Western blot analysis, a18kDapolypeptide was detected in total protein from P. fimbriata. This results indicated that pPaWBNy-2-ORF4gene expressed in P. fimbriata. Whether the P. fimbriata is the vector’insect of PaWB phytoplasma, need to be confirmed through the transmissionassay.更多还原