【作者】 林玲；

【导师】 王宗华； 汪世华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 稻瘟病菌（Magnaporthe grisea）是重要的植物病原真菌，也是研究丝状真菌的重要模式生物。稻瘟病菌丝氨酸蛋白酶可能作为稻瘟病菌分泌到细胞外与寄主受体蛋白起作用的激发子之一。稻瘟病菌的MGG₀7965．5基因编码丝氨酸蛋白酶。提取稻瘟病菌70-15菌株的总RNA，逆转录成cDNA后扩增MGG₀7965．5基因，构建了pPICH-ser表达载体，并转入毕赤酵母GS115中。利用pPICH载体上带有的6×His标签，表达和纯化丝氨酸蛋白酶。为了研究丝氨酸蛋白酶在植物防卫反应中的作用，构建pBD-GAL4-ser诱饵质粒，将其转入酵母菌株AH109中，判定其是否有自激活发生，并鉴定了重组诱饵蛋白的毒性。结果表明，重组诱饵质粒无自激活发生，并且重组诱饵蛋白无毒性。以转化诱饵质粒的AH109菌株与水稻的cDNA酵母文库进行杂交，筛选与丝氨酸蛋白酶有互作关系的酵母克隆。经过两轮的缺陷型培养基SD／Trp^-／Leu^-／Ade^-的筛选和SD／Trp^-／Leu^-／Ade^-／X-α-gal筛选，得到了32个克隆。提取这些酵母克隆菌株的质粒后转入大肠杆菌DH5α中，重新提取大肠杆菌的质粒，并将其一一进行回复性杂交实验。然后用β-半乳糖苷酶检测阳性克隆内LacZ报告基因的表达同时，以pAD-GAL4载体上的通用引物扩增阳性质粒，并将其送去测序。最终获得了12个阳性克隆菌株并对其进行生物信息学分析。结果表明，12个阳性克隆菌株中有4个捕获到的序列为pAD-GAL4载体序列。将剩下的8个菌株进行分类，与蛋白质代谢有关的有3个，与脂类代谢相关的有1个，与离子通道相关的有1个。另外2个未知功能。研究结果将有助于进一步研究稻瘟病菌丝氨酸蛋白酶与水稻相应受体的互作机制和生物学功能。更多还原

【Abstract】 The rice blast fungus, Magnaporthe grisea, is an important disease fungus, is also taken as a model for the study of filamentous fingi. It’s thought that serine proteases maybe one of the elicitors which will be secreted outside the cell and will interact with receptor proteins. MGG₀7965.5 gene encodes the serine proteases.In this research we found that the intron which is shown in the gene dabatase of MGG₀7965.5 gene may be an extron as the sequences of cDNA and genome show the same result. We constructed the pPICH-ser expression vector successlly, and transformed it into Pichia pastoris GS115. We expressed and purified the Serine Protease by 6×His tag which is partaked in pPICH vector.In order to study the role of serine protease in plant defense, we constructed the recombinant bait plasmid pBD-GAL4-ser. Then we transformed it into Saccharomyces cerevisae AH109 and tried to test the bait vector for automatic transcriptional activation as well as toxicity effects. The result showed that the bait vector is inactive and avirulent to AH109. AH109 strain transformed with recombinant bait plasmid mated to rice cDNA to find objected gene having interaction with bait plasmid. Thirty-two clones were selected from two series of SD/Trp^-/Leu^-/Ade^- plates and SD/Trp^-/Leu^-/Ade^-/X-α-gal plates. The vector containing objected genes were transformed into E.coli DH5α. After that, we extracted the plasmid of E.coli and retested them by yeast two-hybrid system. We got 12 contransformation clones on SD/Trp^-/Leu^-/Ade^-/X-α-gal plate. At the same time, we amplified the prey DNA fragment by PCR using consensus primers in the pAD-GAL4 vector. These clones were sequenced. At last, we obtained 12 positive clones and analysed the sequences by blast in NCBI. The results showed that 4 pAD-GAL4 vector sequences were prayed among the 12 positive clones. The residual stains were classified as follows: three of them were relative with protein metabolism, one was relative with lipid metabolism, another one was relative with ion channel, while the others were unknown. The results will facilitate further studies on interaction between the serine protease and the possible receptors and their biological functions.更多还原