【作者】 张磊；

【导师】 林福呈； 卢建平；

【作者基本信息】 浙江大学 ， 微生物学， 2007， 硕士

【摘要】 稻瘟病是水稻上重要的病害之一，其病原菌为子囊菌Magnaporthe grisea。了解M．grisea的致病机理不仅有利于稻瘟病的防治，而且作为研究植物病原真菌与寄主互作的理想模式系统，对于了解其它真菌的致病机理也有重要意义。细胞自噬广泛存在于真核细胞内，在饥饿条件下，它可以调节细胞内长寿蛋白和细胞器的降解，降解产物再被细胞重新利用。因此，真核生物在分化和发育的过程中，细胞自噬过程被认为与细胞的形态建成相关，在细胞器和蛋白质周转的过程中发挥重要作用。本文克隆了稻瘟病菌中的自噬基因MgATG3、MgATG4和MgATG7，通过基因取代的方法分析了MgATG4基因在稻瘟病菌致病性及其他相关过程的作用。具体研究结果如下：1．利用酵母中ATG3、ATG4和ATG7基因序列，在稻瘟病菌中找到同源基因，定名为MgATG3、MgATG4和MgATG7。2．通过构建MgATG3、MgATG4和MgATG7基因置换载体和敲除转化，得到MgATG4 Knock-out突变子。通过PCR和Southern杂交分析鉴定出2个Knock-out突变子，明确MgATG4在Guy11菌株中为单拷贝。3．⊿MgATG4突变子的菌落形态与野生型不同，生长速度无明显改变。在N饥饿，C饥饿，高渗(1M NaCl)胁迫条件下的菌落形态，生长速度与野生型比较无明显差异。4．⊿MgATG4产孢能力下降，萌发速度与野生型相比明显减缓。⊿MgATG4突变子对大麦的致病力比野生型大大下降。5．MgATG4基因PBNEATG4荧光蛋白表达载体的设计和构建。更多还原

【Abstract】 Magnaporthe grisea is an important ascomycete that causes rice blast. Studying of the disease-caused basis of M. grisea is not only beneficial for rice blast control, but also serve as a model for revealing other fungal pathogen-plant interactions.Autophagy is a ubiquitous process that degrades and recycles long-lived proteins and organelles under starvation in all eukaryotic cells. So autophagy has been presumed to be involved in cellular architectural changes that occur during differentiation and development, presumably via its role in organelle and protein turnover.In this study, MgATG3, MgATG4 and MgATG7 genes from M. grisea were cloned. The role of MgATG4 in pathogenicity was analyzed by targeted gene replacement. The results are showed as follows:1. Using the sequence of ATG3、ATG4 and ATG7 genes in yeast , we found the homologous sequence from M. grisea genome databases, named as MgATG3、MgATG4 and MgATG7.2. MgATG3、MgATG4 and MgATG7 genes replacement vector were constructed, and hygromycin-resistance transformants were obtained by MgATG4 gene fungal transformation. Knockout mutants were identified by PCR and Southern blot. Single copy was detected by genomic Southern blot in the genome of Guy11 for MgATG4 gene.3. Compared to Guy11, the colony modality ofΔMgATG4 was much different. The growth rate of the mutants on CM, CM-C, CM-N and CM with 1M NaCl had no obvious change.4. Conidia germination and appressoria formation of mutantΔMgATG4 was suppressed, compared to wild type. The pathogenicity ofΔMgATG4 mutant was partially reduced on barley.5. MgATG4 eGFP fusion expressing vectors, named as PBNEATG4, is constructing.更多还原