【作者】 吴小燕；

【导师】 井金学； 王美南；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2009， 硕士

【摘要】 稻瘟病菌（Magnaporthe grisea）引起水稻的重要病害--稻瘟病,了解其致病分子机制对稻瘟病的防治意义重大。1、稻瘟病菌过氧化物酶体的发生与增殖的研究过氧化物酶体是真核细胞中一类单层膜包裹的细胞器,参与并调节脂肪代谢过程。过氧化物酶体基质蛋白在细胞质中合成,靠自身序列所具有的过氧化物酶体定位信号（Peroxisome targeting signal,PTS）（PTS1与PTS2）而被识别和转运进入基质内部。本文利用嵌合引物,通过构建融合载体,利用GFP与RFP对PTS1与PTS2进行了共定位,并且通过荧光定位研究了过氧化物酶体的发生与增殖。研究结果如下:（1）以MPG1、ATG4及GPD基因启动子为绿色荧光蛋白启动子,构建了双元表达载体pHGpdGFPA、pHAtg4GFPA与pHMpg1GFPA。在共聚焦显微镜下观测转化子的荧光表达,筛选了强启动子MPG1启动子作为GFP和RFP表达启动子。（2）以MPG1基因启动子调控表达,分别构建GFP、GFP-PTS1与GFP-PTS2的双元表达载体;以及RFP和RFP-PTS1的双元表达载体。（3）通过农杆菌介导的转化,将GFP、GFP-PTS1、GFP-PTS2、RFP和RFP-PTS1转入稻瘟病菌野生型菌株Guy11中。结果显示,GFP-PTS1、GFP-PTS2及RFP-PTS1转化子细胞中荧光呈点状,多位于细胞周围,而不含PTS的GFP和RFP转化子中荧光均匀分散于细胞质。利用绿色荧光蛋白和红色荧光蛋白对过氧化物酶体进行了共定位。在相同条件下,GFP-PTS2转化子的荧光背景高于GFP-PTS1;孢子中荧光点的密度高于菌丝和附着胞中;孢子萌发与附着胞形成过程,荧光点密度会产生相应的规律性变化。（4）将GFP-PTS2转入已经得到的稻瘟病菌PEX6突变体中,发现PTS2信号途径被阻断。（5）通过GFP表达转化子,研究了稻瘟病菌过氧化物酶体的发生与增殖等动态变化过程。2、稻瘟病菌MGBIR1基因的克隆了解M. grisea侵入后病菌的定殖、菌丝的分化和扩展等过程的分子机制是目前M. grisea致病机理研究的重点。凋亡抑制蛋白（Inhibition of apoptosis proteins, IAPs）在许多模式系统中抑制细胞凋亡。BIR（Baculoviral inhibition of apoptosis protein repeat domain）为凋亡抑制蛋白（IAPs）的重复结构域。本文克隆了BIR1在M. grisea中的序列同源基因MGBIR1,并成功构建了同源基因置换载体。研究结果如下:（1）利用裂殖酵母菌（Schizosaccharomyces pombe ）BIR1p （NP₅87866.3）蛋白序列在M. grisea基因组数据库中进行同源序列检索,检索到1个假设基因MGG₀4912,结合基因的保守区段分析,定名为MGBIR1。（2）利用MGBIR1基因编码的蛋白序列,通过Blast P程序在GeneBank中进行同源基因查找,并进行了序列比对。（3）构建了MGBIR1基因置换载体p1300-MGBIR1::HPH。（4）进行了五次ATMT敲除转化,已获得130个潮霉素抗性转化子,通过PCR扩增的方法对已获得的转化子进行基因敲除PCR验证。明确M. grisea中是否存在BIR1的同源基因,有助于深入理解M. grisea菌丝的分化与扩展过程。更多还原

【Abstract】 Magnaporthe grisea is a well-known ascomycete that causes rice blast. A better understanding of molecular basis of rice blast is beneficial for rice blast control.1. Localizated Analysis of Peroxisome in Magnaporthe griseaPeroxisome is one class of eukaryotic cell organelle, with single-layer membrane,that regulated the metabolism process of lipids. The peroxisome mechanism in yeast and mammals have been researched thoroughly.The targeting and import of proteins depended on their two peroxisomal targeting signals （PTS1 and PTS2） in sequences as we have known. In this study, the vectors of the fluorescent fusions with PTS were constructed and introduced into Magnaporthe oryzea Guy11 cells. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal amounts during the pre-penetration processes were investigated as well. The results are as follows:1. MPG1, ATG4 and GPD were selected as promoters of GFP , with which we have constructed the binary vectors p1300BMGFPA, p1300BMGFPA and p1300BMGFPA respectively. Strong expression promoter of GFP and RFP, MPG1 promoter has been screened by fluorescence microscopy.2. MPG1 as promoter of GFP and RFP, binary vectors GFP, GFP-PTS1 and GFP-PTS2 were constructed in the pCAMBIA1300. MPG1 as promoter of GFP and RFP, the binary vectors RFP and RFP-PTS1 were constructed in pCAMBIA1300 as well.3. Co-location transformants were obtained with GFP and RFP by fungal ATMT transformation twice. The results showed that, PTS1 and PTS2 could be located into peroxisome by GFP and RFP. Moreover, the fluorescence background of GFP-PTS2 transformants is higher than that of GFP-PTS1; the density of fluorescent spots in spores is higher than that in the mycelium and appressorium; there is a corresponding regularity change of fluorescence-point density in the germination of spores and the formation of appressorium.4. Through importing GFP-PTS2 into the Magnaporthe grisea PEX6 mutant, the PTS2 signal pathway’s being blocked as same as PTS1 was found.5. Using the transformants of with GFP, the inducement of peroxisomes and the dynamic of peroxisome amounts during the pre-penetration processes were investigated.2. Cloning of MGBIR1 in Magnaporthe griseaThe pre-penetration processes of M. grisea, especially appressorium differentiation and formation, have been studied throughly; the post-penetration processes which is essential for infection cycle of M. girsea, including hypha differentiation and colonization, still left much to be studied. The inhibitor of apoptosis （IAP） family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat domains. BIR（Baculoviral inhibition of apoptosis protein repeat domain）domain were found in inhibitor of apoptosis proteins and other proteins, acting as a direct inhibitor of caspase enzymes. In this study, MGBIR1 gene from M. grisea, a sequence homologue of BIR1 was cloned. The role of MGBIR1 gene was analyzed by gene replacement. The results are as follows:（1） MGG₀4912,a hypothetical gene was found by blast with the amino acid sequence of BIR1 in M. grisea genome database, and was assigned MGBIR1.（2） The homology sequence of MGBIR1 protein was studied in GeneBank by Blast P, and the alignment analysis was done.（3） The replacement vector p1300-MGBIR1::HPH of MGBIR1 gene was constructed.（4） After 5 transformations, One hundred and thirty hygromycin-resistance transformants were obtained. The transformants were identified by PCR, and MGBIR1 gene knocked out mutants have not been obtained.The clarification of homologues to BIR1 and its function in M. grisea will benefit the understanding of the molecular metabolism in post-penetration of M. grisea.更多还原