甘蓝黑腐病菌Xanthomonas campestris pv. campestris topoIB基因的克隆及功能分析

论文摘要

野油菜黄单胞菌野油菜致病变种（Xanthomonas campestris pv.campestris,Xcc）可以引起十字花科植物的黑腐病,在全世界范围内造成严重的农业减产。同时该细菌也是研究植物与细菌相互作用的模式菌株,具有较高的理论研究和利用价值。DNA拓扑异构酶是催化DNA拓扑学异构体相互转变酶的总称,是细胞中一类重要的酶,能够调节DNA的拓扑结构,因而在细胞的DNA复制、重组和转录过程中起关键作用。但有关植物病原细菌中拓扑异构酶功能研究,目前尚未见文献报道。本实验以Xcc 8004中Xc0034基因,预测编码的TopoIB蛋白作为研究对象,通过生物信息学对TopoIB蛋白的理化性质进行初步分析,结果表明TopoIB蛋白含有核定位信号和信号肽,无跨膜结构域。RT-PCR实验表明,topoIB基因可以表达,但表达量较低。构建亚细胞定位载体p1305-GFP-TopoIB,在农杆菌介导烟草叶片中瞬时表达,发现p1305-GFP-TopoIB融合蛋白定位于烟草叶片的细胞核上。通过SOE-PCR法构建topoIB基因的突变体△TopoIB,检测结果显示△TopoIB菌株无论是在植物体内还是培养基中与野生型Xc8004的生长状况无明显差异,且胞外多糖产量基本相同,其他常见致病因子如胞外蛋白酶、胞外纤维素酶和胞外淀粉酶产量也与野生型Xc 8004基本一致。将野生型Xcc 8004和△TopoIB突变体接种于拟南芥和甘蓝（京华一号）,结果显示△TopoIB菌株致病性并没有受到影响。构建topoIB基因的原核表达载体并进行高效表达,经IPTG诱导,在大肠杆菌BL21菌株中成功表达了与His标签融合的TopoIB蛋白,分子量大小约为43 kDa,利用镍柱在200mM咪唑洗脱下可以得到纯化蛋白,浓度为788ng/μL,表明topoIB基因的体外诱导表达成功。体外酶活检测结果显示,394 ng的TopoIB重组蛋白反应1 min就可以将超螺旋DNA解旋,反应15min可以完全解旋,表明TopoIB蛋白具有拓扑异构酶活性。综上所述,本实验明确了Xc 8004中的topoIB基因可以低水平表达,且topoIB基因编码产物作用于植物细胞核内,topoIB基因的缺失对其菌株自身的胞外多糖、胞外酶和致病性无明显影响,体外原核表达分析显示,通过异源表达纯化获得的重组蛋白具有较高的解旋活性,为今后病原细菌中topoIB基因的研究提供重要理论依据。

论文目录

文章来源

类型: 硕士论文

作者: 张迎

导师: 张清霞

关键词: 缺失突变,亚细胞定位,原核表达

来源: 扬州大学

年度: 2019

分类: 基础科学,农业科技

专业: 生物学,农业基础科学,植物保护

单位: 扬州大学

基金: 国家自然科学基金委员会(项目编号:31772210),山东省农业微生物重点实验室开放基金(项目编号:SDKL2017015)

分类号: S432.42

DOI: 10.27441/d.cnki.gyzdu.2019.000227

总页数: 67

文件大小: 6250K

下载量: 31

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标签：缺失突变论文;  亚细胞定位论文;  原核表达论文;