【作者】 薛进；

【导师】 陈剑平； 张恒木； 高必达；

【作者基本信息】 湖南农业大学 ， 生物安全与检疫， 2013， 博士

【摘要】 植物病毒在自然界的传播一般依赖介体,如昆虫、螨、线虫以及真菌等,其中近80%的植物病毒由特定的昆虫传播。南方水稻黑条矮缩病于2001年在我国广东省阳江市首次发现,起初被认为由水稻黑条矮缩病毒(Rice black streaked dwarf virus, RBSDV)的一个分离物引起,至2008年其病原被鉴定为一个水稻病毒新种,暂定名为南方水稻黑条矮缩病毒(Southern rice black streaked dwarf virus, SRBSDV)(或水稻黑条矮缩病毒2,Rice black streaked dwarf virus2, RBSDV-2),属于呼肠孤病毒科(Reoviridae)斐济病毒属(Fijivirus)第2组一个新成员。SRBSDV主要由白背飞虱(Sogatella furcifera)以持久性方式传毒,不经卵、种子、机械传播。在本研究中,首先利用Gateway技术构建了白背飞虱cDNA酵母表达文库,检测分析显示该文库库容量约5.09x107,插入片段大小在0.75-2kb之间,且重组率很高,表明该文库可用于后续的互作筛选实验。同时我们基于已报道的中国广东和海南SRBSDV分离物的基因组全序列设计引物,构建了SRBSDV S1-S4编码的酵母诱饵和靶标重组载体。毒性分析显示,SRBSDV S1-S4所编码的基因对酵母菌株均无显著毒性；自激活检测显示SRBSDV P3蛋白可在酵母中激活报告基因表达,表明P3具有自激活活性。接着我们以SRBSDV结构蛋白P8为诱饵通过Mating法筛选白背飞虱cDNA酵母表达文库,共筛选获得543个阳性克隆,测序分析显示这些阳性克隆代表了5个白背飞虱基因,包括白背飞虱羧酸酯酶(Sogatella furcifera Carboxylesterase, SfCarE)基因、白背飞虱细胞色素氧化酶亚基Ⅰ(Sogatella furcifera cytochrome oxidase subunit I gene, SfCO I)基因和3个功能未知基因。酵母双杂交显示SfCarE和P8在酵母中存在强烈的相互作用；双分子荧光互补(Bimolecular fluorescence comple-mentation, BiFC)实验分析表明SfCarE和SRBSDV P8在植物细胞中也存在强烈的相互作用,而且二者互作形成的黄色荧光主要分布于细胞质内。亚细胞定位实验表明,SRBSDV P8分布于细胞质及细胞核中；而SfCarE主要分布于细胞质中,支持P8与SfCarE互作主要发生于细胞质的结论。此外,为了研究病毒蛋白之间的相互作用,我们以SRBSDV编码的多功能蛋白P6为诱饵,通过Mating法筛选SRBSDV cDNA酵母表达文库,一共筛选到3对互作组合,即P6/P5-1、P6/P6、P6/P9-1。本文重点深入研究了P6/P6、P6/P9-1之间的互作。酵母共转化实验表明在酵母细胞中P6/P6互作强度比P6/P9-1略强。利用双分子荧光互补(BiFC)技术分析显示P6/P6、P6/P9-1在植物细胞内均能相互作用,互作所形成的黄色荧光分布于细胞质,并且荧光主要以点状聚集体的形式存在,阴性对照的细胞中则未发现荧光。缺失突变分析显示4个SRBSDV P6缺失突变体与完整P6蛋白在酵母细胞中均不存在互作,表明SRBSDV P6N端和C端区域对于其自身互作都是必须的。缺失突变分析表明P9-1N端和C端区域也是其与P6互作所必须的,而P6与P9-1互作的关键结构域位于P6N端(aa1-93)。免疫电镜观察分析显示P6抗体的免疫胶体金颗粒可特异性地标记在病株水稻体内具有较高电子密度且无定形的病毒基质(Viroplasm)中,表明P6是病毒基质的成份之一,支持其与病毒基质另一组分P9-1之间的互作,共同参与病毒基质的形成。这些结果为进一步研究病毒蛋白自身互作及其与介体白背飞虱之间的互作打下基础。更多还原

【Abstract】 Plant viruses are naturally transmitted by vectors, including insects, mites, nematodes and fungi, in which approximately80%of plant viruses are insect-borne. Southern rice black streaked dwarf diseases, cuased by Southern rice black streaked dwarf virus (SRBSDV), was first reported in Yangjiang city, Guangdong Province, China. The virus was initially considered an isolate of Rice black streaked dwarf virus (RBSDV). Until2008, it has been identified to be a novel species of rice viruses and tentatively named Southern rice black streaked dwarf virus (SRBSDV) or Rice black streaked dwarf virus2(RBSDV-2), which was a putative member of second group within genus fijivirus, family reoviridae. SRBSDV is transmitted by the white-backed planthopper (WBPH), Sogatella furcifera Horvath, in a persistent manner, but not transmitted by eggs, seeds, andmechanical mode.In order to screen interactions between the genes of WBPH and SRBSDV, the WBPH cDNA expression library was first constructed into the yeast plasmids using Gateway technique in this study. Quality assay of the cDNAlibrary titer total colony-forming units5.09×107CFU, the average insert size was between0.75and2kb and the recombinant percentage of the cDNA library was very high. These results consistently indicated that the cDNA library can be used for screening interactions by the Yeast Two Hybrid system. Based on the published sequences of SRBSDV-GD and-HN isolates, a series of primers were designed and used to construct the recombinant plasmids inserted with the encoding regions of SRBSDV S1-S4in Yeast Two Hybrid (YTH) system. Toxical assay showed that all the proteins encoded on the SRBSDV S1-S4have no significant toxic effects on the growth of yeast strains. Auto-activation tests showed that only P3protein of SRBSDV could autonomously activate the expression of reporter genes in yeast, indicating SRBSDV P3had transcriptional activation effect.Then the P8protein, a core structural protein of SRBSDV, was used a bait for screening the expression cDNA library of the white-backed planthopper (WBPH), Sogatella furcifera Horvath, by yeast mating method. A total of543positive colonies were selected on SD/-Ade/-His/-Leu/-Trp/X-a-Gal medium. Sequence analysis showed insertions of these prey plasmids derived from5genes of WBPH, includingcarboxylest-erase of Sogatella furcifera (SfCarE),Sogatella furcifera cytochrome oxidase subunit I gene (SfCO I), and other3genes with unknown function. There is strong interaction betweenSfCarE and SRBSDV P8in yeast cells. Bimolecular fluorescence complement-ation(BiFC)assay showed the interaction also occurred in plant cells and the reconstituted YFP fluorescence was mainly distributed in the cytoplasm. Subcellular localization assay showed that SfCarE was distributed in the cytoplasm and SRBSDV P8was detected in both the cytoplasm and the nucleus, supporting the interaction between them in cytoplasm.In addition, to understand the interactions of viral proteins, SRBSDV P6, a multifunctional protein, was used as bait and a SRBSDV cDNA library as prey in a yeast two hybrid (YTH) assay by yeast mating method. Total3pairs of interactions, including P6/P5-1, P6/P6, and P6/P9-1, were selected. Here we further studied on the interactions of P6/P6and P6/P9-1. Co-transformed yeast assays showed that there was a stronger interaction of P6/P6than that of P6/P9-1. These interactions were confirmed by bimolecular fluorescence complement (BiFC) assay in plant cells. The reconstituted YFP fluorescence was randomly distributed in the cytoplasm and aggregate into granular structures, which appeared similar to the viroplasm-like structures (VLS). No YFP fluorescence was observed in the control cells. YTH analyses using truncated mutants showed that all the4truncated mutants of P6could not interact with P6, indicating that both N-and C-terminal fragments were necessary for its self-interaction. YTH analyses using truncated mutants also indicated that both N-and C-terminal fragments of P9-1were necessary for its interaction with P6, and the N-terminal region (aa1-93) P6was crucial for its interaction with P9-1. Immunogold labeling showed that the immunogold particles were specifically labeled in amorphous Viroplasm with higher electron-density in infected cell of rice plants, indicating that P6is a component of the Viroplasm and supporting P6was involved in viroplasm formation by interacting with another component, P9-1, of viroplasm in infected plants. These results provided foundation for further study on the self-interactions among the viral proteins and interactions between SRBSDV and its vector, white-backed planthopper (WBPH), Sogatella furcifera Horvath.更多还原