【作者】 赵霄晨；

【导师】 徐炎；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2012， 硕士

【摘要】 在前期研究中，构建了葡萄白粉病原菌诱导的华东葡萄‘白河-35-1’叶片cDNA文库，获得华东葡萄病程相关蛋白PR10.1（Pathogenesis related protein10）基因EST序列，采用RACE克隆技术获得该基因全长序列。本研究通过重叠嵌套延伸PCR方法对VpPR10.1中三个保守氨基酸进行点突变，将获得的野生型和突变型VpPR10.1基因进行原核表达，纯化目的蛋白。体外对纯化VpPR10.1蛋白的核酸酶活性、抑菌活性、诱导细胞程序性死亡和FITC标记蛋白定位进行分析，为华东葡萄病程相关蛋白PR10.1抗病机理提供理论依据，取得的主要研究结果如下：1、运用重叠嵌套延伸PCR方法对VpPR10.1蛋白三个保守氨基酸残基（lys55、glu149和tyr151）进行点突变，分别替换为asn55、gly149和his151。构建重组原核表达载体pGEX-4T-1，转化原核表达菌株BL21（DE3），通过优化目的蛋白表达条件，在30℃，0.1mM IPTG诱导表达3h，获得大小为43KDa的野生型和突变型可溶性重组蛋白。诱导后的菌体用细菌裂解液裂解，收集可溶性蛋白，用GST亲和树脂进行纯化目的融合蛋白；用凝血酶去除重组蛋白的GST标签，获得17KDa的野生型和突变型VpPR10.1蛋白。2、纯化的目的蛋白体外进行核酸酶活性分析。将80μg的VpPR10.1野生型及其突变型蛋白分别与酵母总RNA、华东葡萄总RNA和DNA进行反应，用1%的琼脂糖凝胶电泳检测。结果表明，野生型VpPR10.1蛋白具有明显的核酸酶活性。与野生型相比，三个突变体能不同程度的降低VpPR10.1蛋白的核酸酶活性，而突变型VpPR10.1-K55N/E149G明显降低了VpPR10.1蛋白核酸酶活性。3、烟草赤星病抑菌试验分析表明，不同蛋白量的VpPR10.1融合蛋白与烟草赤星病孢子共培养5天后，赤星病病原菌生长出现差异。随着蛋白量的增大，赤星病病原菌生长明显受到抑制。VpPR10.1重组蛋白和突变型蛋白抑菌活性与其核酸酶活性是一致的。80μg野生型VpPR10.1蛋白对烟草赤星病病菌有显著抑制作用，VpPR10.1-Y151H在一定程度上降低了对烟草赤星病病原菌抑制作用，而突变型VpPR10.1-K55N和VpPR10.1-E149G蛋白能明显地降低其抑制作用。将VpPR10.1基因及其突变体构建到植物诱导表达载体pER8，用农杆菌介导的真空渗透法侵染欧洲葡萄佳丽酿叶片，之后接种葡萄白粉病，11d后观察叶片菌丝生长情况，过量表达VpPR10.1蛋白能抑制葡萄白粉病原菌生长。结果表明：在离体和体内条件下，VpPR10.1蛋白均表现有抑制植物病原菌的活性。4、华东葡萄病程相关蛋白（VpPR10.1）诱导细胞程序性死亡分析。将VpPR10.1重组蛋白进行时间梯度和浓度梯度处理烟草BY-2悬浮细胞，诱导后的悬浮细胞用EvansBlue进行染色。结果表明，当蛋白浓度为100μg/mL时，能明显观察到悬浮细胞死亡；在诱导0~24h之内，悬浮细胞死亡数目随时间的延长而增多。提取VpPR10.1重组蛋白与烟草BY-2悬浮细胞共培养24h的DNA，进行DNA降解分析，发现诱导后的悬浮细胞DNA有明显的降解，表明外源VpPR10.1蛋白能够诱导植物细胞的程序性死亡。5、异硫氰酸荧光素(FITC)标记1mg VpPR10.1重组蛋白。将10μL标记的重组蛋白（FITC-VpPR10.1）分别与100ng/mL葡萄基因组DNA和葡萄总RNA进行反应，1%琼脂糖凝胶电泳进行检测分析表明标记的重组蛋白（FITC-VpPR10.1）仍然具有核酸酶活性。然后，用12μg/mL的FITC-VpPR10.1诱导烟草BY-2悬浮细胞24h，用荧光显微镜检测VpPR10.1蛋白在细胞内的定位。结果显示，诱导后的细胞内都观察到明显的荧光信号，说明外源VpPR10.1蛋白能够有效的进入诱导细胞内。更多还原

【Abstract】 In the previous study, a PR10EST sequence has been obtained from cDNA library preparedfrom Chinese wild Vitis pseudoreticulata clone Baihe-35-1leaves，which is high resistant toUncinula necator from years’ investigation. The full length cDNA sequence of PR10hasbeen cloned using RACE. This further study was to analysis of VpPR10.1gene function.Along with the wild type, three substitution mutants were constructed by site directedmutagenesis using overlap extension PCR. The coding sequence of VpPR10.1was cloned intothe pGEX-4T-1vector. The purified protein was used to analyze nuclease activity,antibacterial activity, programmed cell death and the localization of FITC-labeled protein.The main results obtained are as follows:1. Along with the wild type, three substitution mutants (K55N, E149G, and H151H)were constructed by site directed mutagenesis using overlap extension PCR. The codingsequence of VpPR10.1was cloned into the pGEX-4T-1vector, and then the pGEX-VpPR10construct was transformed into E.coli BL21. Expression of the VpPR10fusion protein insolubility was induced with0.1mM IPTG at30°C for3h. The bacterial cells were pelletedafter incubation and suspended in lysis solution. Fusion protein was purified withGlutathione-Sepharose resin by affinity chromatography. The removal of GST tag wasperformed using the Thrombin Cleavage Capture Kit.2. Analysis of nuclease activities of the purified proteins in vitro. Used the wild type andthe mutant protein of VpPR10.1reacted with the total RNA of yeast, total RNA and DNA ofbaihe-35-1respectively,then detected by1%agarose gel electrophoresis. The results indicatedthat wild type protein VpPR10.1can clearly degraded DNA and RNA, and mutant proteindecreased the nuclease activity. The mutant VpPR10.1-Y151H slightly decreased the nucleaseactivity, while the mutant VpPR10.1-K55N/E149G obviously decreased the nuclease activity.3. A. alternate were grown on PDB medium in the presence of purified wildrecombinant VpPR10.1proteins at different concentrations and evaluated after incubated for5days. Then, mycelium showed the different states. With the increase of protein, theantibacterial activity was significantly enhanced.80μg wild type VpPR10.1protein showedgrowth inhibitation against tobacco fungus Alternaria alternate. The results showed that, theVpPR10.1protein antibacterial activity was consistent with the nuclease activity. The VpPR10.1protein showed significant antibacterial activity. Three mutant proteins decreasedthe antibacterial activity.The mutant VpPR10.1-Y151H slightly decreased the antibacterialactivity, while the mutant VpPR10.1-K55N/E149G obviously decreased the antibacterialactivity.The coding region of VpPR10.1was sub-cloned into expression vector pER8and then,were transiently transformed into V. vinifera cv. Carignane using Agrobacterium-mediatedvacuum method. Grape leaves inoculated with powdery mildew, the hyphal growth observedat the11d. The results showed excessive express VpPR10.1proteins can inhibit hypha growthof powdery mildew. VpPR10.1proteins showed activity of inhibit growth of pathogens invitro and in vivo.4. To know the sensitivity of VpPR10.1protein in inducing cell death, dose-andtime-dependent experiments were performed. Cell death sensitivity was determined bystaining the treated cells with Evans blue.100μg/mL VpPR10.1protein co-incubated withtobacco BY-2for24h can caused cell death obviously. Moreover, increase in cell death wasalso dependent on post-treatment time, where cell death started to increase after0h andcontinued till24h. Since DNA fragmentation is a hallmark of PCD, it was monitored ongenomic DNA isolated from suspended cells treated with VpPR10.1protein for24h. Resultsshowed DNA fragmentation by the exogenous VpPR10.1protein treatment.5. Fluorescein isothiocyanate (FITC) labeled the VpPR10.1reorganization protein. TheFITC-VpPR10.1reacted with the total RNA and DNA of baihe-35-1respectively, thendetected by1%agarose gel electrophoresis. The FITC-VpPR10.1protein still has a nucleaseactivity. When FITC-VpPR10.1protein was incubated with BY-2cells for24h, an intensefluorescent signal was observed inside in almost all cells. This result indicates thatFITC-VpPR10.1is effectively translocated inside into the intact BY-2cells.更多还原