棘孢木霉和球毛壳菌免疫诱导蛋白及其功能分析-immunopotentiator proteins of trichoderma spinosum and chaetomium globosum and their functional analysis.docx

在识别寄主和侵袭寄主上的能力没有变化。该基因对棘孢木霉 T4 菌丝的疏水 性有影响，而对其孢子的疏水性没有影响，对球毛壳菌 W7 的菌丝和孢子的疏 水性均没有影响。基因的缺失对它们定殖植物根部的能力均无明显改变。将棘孢木霉的野生型，基因缺失体和过表达体分别用于大豆诱导试验。结 果表明野生型和过表达体能够诱导大豆大部分病原相关蛋白基因的上调表达， 引起大豆叶片的防御相关反应，并能增强其对病原真菌的抗病能力，而基因缺 失体与对照相比差异不显著。棘孢木霉不仅能增强大豆的局部抗性，还能引发 其系统抗病性。将球毛壳菌的野生型，基因缺失体和过表达体分别对大豆进行 诱导试验。得到的结果与棘孢木霉基本相似，但病原相关蛋白的诱导能力有所 差异，并且在进行诱导大豆抗灰斑病抗病试验时发现，EplW7 基因的缺失并不 能使其诱导抗性能力完全丧失，说明球毛壳菌内可能还存在其它诱导物质。为研究EplT4和EplW7调控的分子机制，分别构建了棘孢木霉T4和球毛壳菌 W7的cDNA文库，并分别将两个基因的启动子构建到报告载体pHIS2，与连有 cDNA 文库的 pGADT7-Rec2 表达载体 共转化到酿酒酵母 （ Saccharomyces cerevisiae）Y187中，利用酵母单杂交系统分别筛选棘孢木霉和球毛壳菌cDNA 文库中Epl基因的上游调控因子。共筛选到5个可能作用于EplT4的反式作用因子， 分别是磷酸酶、信号识别受体、乙酰转移酶、假定蛋白1和假定蛋白2等。筛选 到4个可能作用于EplW7的反式因子，分别是WD重复结构域蛋白、棕榈酰转移 酶和2个假定蛋白。关键词：棘孢木霉 T4；球毛壳菌 W7；免疫诱导蛋白；酵母表达；诱导抗性； 调控因子AbstractBiocontrol agents inhibit the pathogens by a variety of mechanisms, one of which is to induce the defense responses of the host plants, and elicit their local or systemic acquired resistance (SAR). Current researches mainly focus on the interactions between biocontrol agents and pathogens or pathogens and plants. However, there is very few study focuses on the relation between biocontrol agents and plant. In order to explore biocontrol related genes, cDNA libraries of the mycelium from both Trichoderma asperellum T4 and Chaetomium globosum W7 were constructed in our laboratory. By sequencing and bioinformatics analysis of these two libraries, the gene which encodes the immunity-inducing protein in these two organisms were found and designated as EplT4 and EplW7 respectively. The relatively low(65.22%) protein sequence identity of these two genes’ products suggests possible structural and functional differences between them that need to be investigated separately.In order to get plenty of proteins for functional studies of EplT4 and EplW7, both genes were cloned and expressed in Pichia pastoris GS115. In order to facilitate subsequent protein purification, the N terminal signal peptides of both proteins were replaced by a signal peptide of the Sacch