水稻矮缩病毒非结构蛋白Pns10的介体互作因子的筛选与功能验证

发布时间：2018-01-16 17:21

  本文关键词：水稻矮缩病毒非结构蛋白Pns10的介体互作因子的筛选与功能验证　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 水稻矮缩病毒 Pns10 黑尾叶蝉 蛋白互作

【摘要】：水稻矮缩病毒(Rice dwarf virus,RDV)属于持久增殖型病毒,主要由介体黑尾叶蝉传播。在叶蝉内,病毒利用自身编码的非结构蛋白Pns10形成包裹病毒粒体的小管通道,采取"小管运输病毒"的策略进行安全扩散,以抵御叶蝉的各种免疫攻击。这种安全运输病毒策略需要介体蛋白共同参与才能顺利完成,先前研究表明Pns10通过酵母双杂交筛选到介体互作蛋白肌动蛋白(actin)、肌球蛋白(myosin)等,黑尾叶蝉胞质型actin与Pns10的特异性互作决定黑尾叶蝉专化性传播RDV,这些为Pns10互作介体蛋白的筛选提供了基础。鉴于此,本研究继续利用核蛋白互作的Clontech酵母双杂交系统筛选数量相等的成虫和若虫的黑尾叶蝉cDNA库,获取阳性克隆。在NCBI Blastx进行序列比对分析后,预测筛选的基因功能多为与昆虫生长发育、细胞成分等相关,挑选6个可能互作的蛋白:原肌球调节蛋白(tropomodulin,Tmod),线粒体孔蛋白(mitochondrial porin,Mito P),运脂蛋白前体(lipophorin precusor,LP),高密度脂蛋白结合蛋白(Vigilin),凋亡诱导因子(apoptosis inducing factor,AIF),卵黄原蛋白(vitellogenin,Vg),将这6种蛋白的酵母质粒与pGBKT7-Pns10再次回转酵母感受态细胞AH109,确定Pns10与候选蛋白互作。双分子免疫荧光互补技术(BiFC),证明Tmod、Vg和LP与Pns10互作,而Mito P、AIF和vigilin与Pns10不互作。在本氏烟共定位表达系统中,Tmod 和 Vg,vigilin,LP 均能与 RDV Pns10 共定位,而 Mito P、AIF不与Pns10共定位。为进一步明确6个候选蛋白在RDV扩散中发挥的作用,利用RT-qPCR实验检测6个候选蛋白基因在带毒培养细胞和黑尾叶蝉中的相对表达量。结果表明除Mito P与对照组无明显差别外,其余5个基因的相对表达量均高于对照组。由于Tmod作为actin慢生长端的唯一盖帽蛋白,是actin的相关蛋白,而包裹病毒Pns10小管运动是沿着actin纤维丝进行病毒的运输。利用GST-Pull down实验证明Tmod能与RDVPns10在体外发生特异性互作;Tmod荧光抗体免疫标记带毒培养细胞和黑尾叶蝉消化道,Tmod定位在微绒毛上,与Pns10共定位,Pns10小管能够穿出actin形成的微绒毛。RT-qPCR分析Tmod、Pns10、RDVP8在带毒叶蝉中的表达趋势相似。利用dsTmod抑制Tmod在介体中的表达,发现Tmod、Pns10和P8表达量明显下降。并且干扰Tmod表达的第14天,虫体的带毒率也下降了一半。因此,Tmod对Pns10行使功能有正调控作用,Pns10小管依赖于actin的小管动力(ABTM)充分利用Tmod在介体中的调控作用,以至于actin能够无限延伸,反向便捷了Pns10小管插入邻近细胞,甚至为突破介体组织膜屏障提供动力,最终实现病毒扩散。Vg为卵黄蛋白前体,是卵发育的营养物质,在脂肪体上合成释放到血淋巴中,最终被卵巢吸收。利用GST-Pulldown实验进一步确定Pns10与Vg互作。免疫荧光抗体标记培养细胞,发现Vg可与Pns10共定位。此外,Pns10与Vg还可在卵巢滤泡细胞、菌胞和卵巢管柄处共定位,但当Pns10与Vg大量共定位于卵巢管柄时,卵内并未标记到Pns10蛋白,因此推测卵巢管柄是RDV侵入卵的初侵染点,Vg与Pns10互作帮助了病毒在卵内的扩散。
[Abstract]:Rice dwarf virus (Rice dwarf, virus, RDV) belong to the persistent virus proliferation, mainly by the mediator cincticeps spread. In the use of their own within the leafhopper, virus encoding the non structural protein Pns10 formation of tubular channel virus particles package, take the "tubular transport virus" strategy for safe diffusion in various immune attack against the virus. The security strategy of leafhopper transport needs mediator protein to participate in the completion of Pns10, the yeast two hybrid screening to mediated protein interaction with actin (actin), previous research has shown that myosin (myosin), specific cincticeps cytoplasmic actin and Pns10 interaction decided cincticeps designed of the RDV transmission, which provides the basis for screening Pns10 interaction mediated protein. In view of this, this study continues the Clontech interaction of nuclear proteins by yeast two hybrid screening system is equal to the number of adults and nymphs The leafhopper cDNA library, to obtain positive clones. The sequences of NCBI in Blastx after the screening of gene function prediction for the growth and development of insects, and cellular components, select 6 protein may interaction: tropomodulin (tropomodulin, Tmod), mitochondrial porin (mitochondrial porin, Mito, P) transport of lipoprotein precursor (lipophorin precusor, LP), high density lipoprotein binding protein (Vigilin), apoptosis inducing factor (apoptosis inducing, factor, AIF), vitellogenin (vitellogenin, Vg), the yeast plasmid and pGBKT7-Pns10 of these 6 proteins again turning yeast competent AH109 cells, Pns10 and determination of the candidate protein interaction. Double immunofluorescence molecular complementary technology (BiFC), Tmod, Vg and LP and Pns10 interaction, and Mito P, AIF and vigilin and Pns10 interaction. In n.benthamiana co expression system, Tmod and Vg, vigilin, LP can and RDV Pns10 and Mito P, CO localization, AIF co localized with Pns10. In order to further clarify the 6 candidate proteins play the role in the diffusion of RDV, using RT-qPCR test to detect 6 candidate protein genes in infected cells and relative expression of leafhoppers in culture. The results showed that except Mito P and control group obvious difference, the relative expression of other 5 genes were higher than the control group. The Tmod as the only protein blocks actin slow growing end, is related to protein actin, and wrapped the virus Pns10 movement is carried out along the tubular virus actin filament transport. Using GST-Pull down experiments show that Tmod can specifically with RDVPns10 in vitro interaction; Tmod fluorescent antibody virus immune markers in cultured cells and leafhoppers in digestive tract, Tmod was localized in the microvilli, CO localization with Pns10, Pns10 can actin the formation of microvilli were tubular.RT-qPCR analysis Tmod, Pns 10, similar to RDVP8 in the expression. The expression trend of poison leafhopper, inhibition of Tmod mediated by dsTmod in Tmod, Pns10 and P8 expression decreased significantly. The expression of Tmod and interference for fourteenth days, the virus carrying rate of worms also fell by half. Therefore, the Tmod of Pns10 has positive regulation function effect of tubular actin dependent Pns10 tubule (ABTM) Regulation to make full use of Tmod in the medium, so that actin can be extended indefinitely, reverse Pns10 tubular insertion of adjacent cells is convenient, even to provide power for the breakthrough of mediator membrane barrier, and ultimately realize the spread of the virus.Vg yolk protein precursor, nutrition the material egg development, synthesis and release of blood in Bazhong with fat body, ovary was eventually absorbed. Experiments using GST-Pulldown Pns10 to further determine the interaction with Vg. Immunofluorescence antibody labeled cells, Vg was co localized with Pns10. In addition, Pns10 And Vg can also in ovarian follicular cells, bacteria and ovarian tube handle of the co localization of Pns10 and Vg, but when a large number of colocalized in ovarian tube handle, the egg does not mark to the Pns10 protein, suggesting that ovarian tube handle is RDV into eggs of primary infection, Vg interacts with Pns10 to help spread the virus in the egg.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.11

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