【作者】 王政逸；

【导师】 李德葆； Nicholas J.Talbot；

【作者基本信息】 浙江大学 ， 植物病理学， 2002， 博士

【摘要】 Magnaporthe grisea可引起水稻上重要的病害-稻瘟病。了解M．grisea的致病分子机理不仅对稻瘟病的防治，而且它作为一个理想的研究体系对了解其它植物病原真菌与寄主的互作也具有重要意义。显然通过致病相关基因的鉴定是达到这一目标的关键所在。M．grisea能分化产生特殊的侵染结构-附着胞，并以机械压力穿透寄主角质层。附着胞中积累高浓度甘油是细胞膨压和机械压力产生的原因所在。令人感兴趣的问题是：附着胞中高浓度的甘油是如何合成的呢?前人通过细胞生物学和酶学的研究表明，脂肪可能是产生甘油的重要前体物质之一，通过脂肪酸代谢合成甘油的途径必须涉及乙醛酸循环。异柠檬酸裂解酶(ICL)是乙醛酸循环中的关键酶，催化异柠檬酸裂解为乙醛酸和琥珀酸，然后通过糖异生途径产生甘油。本文克隆了编码异柠檬酸裂解酶的M．griseaICL1基因，并通过目标基因取代的方法分析了ICL1基因在M．grisea致病过程中的作用。研究结果如下： 1． 根据异柠檬酸裂解酶基因的同源序列设计二个引物，以M．grisea Guy11基因组DNA为模板进行PCR扩增后获得340bp的核苷酸序列，并以此序列为探针从M．grisea Guy11基因组文库和cDNA文库中筛选、克隆了完整的M．grisea Guy11 ICL1基因，获得了包括启动子区域和完整阅读框(ORF)的ICL1全基因序列。ICL1基因共有5个外显子，由4个内含子序列分开，ICL1基因编码547个氨基酸，与Neurospora Crassa(acu3)和Aspergillus nidulans(acuD)的蛋白序列具有很高的同源性，分别达到85.5％和76.3％。ICL1基因的分子量大约为60KD，与acu3和acuD编码的酶蛋白分子量相当。所不同的是M．grisea ICL1基因有4个内含子，而acu3和acuD只有2个内含子，其中ICL1基因的前面2个内含子与acu3和acuD的位置是类似的。基因组Southern杂交的结果显示M．grisea ICL1基因是单拷贝基因。 2． 通过构建ICL1基因取代载体和真菌转化，共获得55个转化子。利用PCR和Southern杂交的方法鉴定出3个ICL1基因的knock out转化子。M．grisea△ICL1突变体在以橄榄油(脂类)或醋酸盐为C源时，不能正常生长与产孢，而以葡萄糖为C源时能正常生长和产孢，说明△ICL1突变体脂肪酸β-氧化产 生的乙酚－COA不能进入乙醛酸循环，该代谢途径己经被阻断。 3．A ICL］突变体在 CM培养基上的菌落形态为灰白色，而 GUyll为灰褐色，产 ”抱量也略有减少，但菌落生长速度没有改变。在塑料盖玻片上的抱子萌发试 验结果表明，A ICLI突变体的抱子萌发推迟，芽管比 Guull和表型转化子明 显增长，附着胞形成率显著下降。 4．通过染色的方法对A ICLI突变体附着抱的形成过程中脂肪微滴的移动过程 作了初步的观察，结果显示凸1a1突变体脂肪微滴的移动较为缓慢。采用细 胞蹋陷的方法，对萌发 24h和 48h时的A ICLI突变体附着胞膨压测定结果表 明，尽管附着胞的膨压有所下降，但这种下降十分有限。说明脂肪代谢对附 着胞中的甘油合成和膨压产生起一定作用，但附着胞中甘油的合成途径可能 十分复杂，并不是单一的前体物质或代谢途径起主导作用，抱子内的其他储 存物质如碳水化合物也可能参与甘油合成。 5．在洋葱内表皮上的穿透试验表明，凸 ICLI突变体（I－10）对洋葱内表皮的穿 透率为45．3％，比Guyll的73．5％和表型转化子的69．8％明显下降。 6．当用 A ICLI突变体接种水稻时，产生稻瘟病症状的时间比 GUyll和表型转 化子推迟，推迟的原因是否与抱子萌发和附着胞形成较晚有关，尚有待于进 一步证实。A ICLI突变体对水稻的致病力也有所下降。 7．通过ICLI基因的启动子与绿色荧光蛋白基因（GFP）融合表达的研究也表 明，ICLI基因在分生抱子中的表达比菌丝体中强，而在 W＋sodium acetate培 养基上生长时菌丝体和分生抱子的ICLI基因均强烈诱导表达。在分生抱子形 成附着胞的过程中，分生抱子、芽管和附着胞均有不同程度的表达，尤以附 着胞中的ICLI基因表达水平较高，直至48h时，附着胞中的ICLI基因表达 仍然维持极高的水平。这一结果表明，ICLI基因的表达与附着胞的发育过程 紧密相关。 综合上述，我们认为ICLI基因对从 grjsea的完全致病性是需要的。但附 着胞中甘油的合成途径可能十分复杂，并不是由单一的前体物质或代谢途径决 定的，而可能涉及多个途径的共同作用。更多还原

【Abstract】 Magnaporthe grisea is best known as the causal agent of theeconomically important blast disease of rice worldwide. A betterunderstanding of the molecular basis of this disease is not onlybeneficial for rice blast control, but also can serve as a model forunderstanding other fungal-plant interactions. Obviously, theidentification of genes required for pathogenicity is a key step towardachieving this goal. The fungus elaborates a specialized infection cellcalled an appressorium to penetrate the rice cuticle mechanically. Togenerate mechanical force, appressoria produce enormous hydrostaticturgor by accumulating molar concentration of glycerol. How glycerol issynthesized in such large amounts in M. grisea appressorium is anintriguing question. Previous studies on cytobiology and enzymaticactivities showed glycerol generation from lipids in a conidium istheoretically possible. Glyoxylate cycle is involved in the pathway ofglycerol synthesis from fatty acid. Isocitrate lyase(ICL) is a key enzymein glyoxylate cycle, catalyzing the conversion of isocitrate toglyoxylate and succinate, which generate glycerol by gluconeogenesispathway. In this study, M. grisea ICL1 gene encoding isocitrate lyase wascloned and the role of ICL1 gene in pathogenicity was analyzed by targetedgene replacement. The results are showed as follows:1. Two primers designed by conserved oligonucleoitides of isocitratelyase genes from other organisms were used for PCR amplification withM. grisea Guyll genomic DNA as template. The expected 340bp product wasobtained. By using the PCR product as a probe, two positive clones wereisolated from M. grisea Guyll genomic DNA library and conidial cDNAlibrary respectively. Both the ICL1 genomic DNA included promoter regionand open reading frame and cDNA clones were sequenced. The results showedM. grisea ICL1 gene contain 5 exons and 4 introns, and encode 547 predicted amino acids. The ICL1 protein sequence was very high homologous to the proteins of Neurospora crassa(acu3) and Aspergillus nidulans(acufl), 85.5% and 76.3% respectively. The molecular weight of ICL1 protein was about 60KD and similar to that of acu3 and acuD. But acu3 or acuD only had 2 introns witch were similar positions with the first 2 introns of ICL1 gene. Moreover, single copy of ICL1 gene in M. grisea was showed by Southern blot.2. Fifty-five transformants were obtained by fungal transformation with ICL1 gene replacement vector. Three ICL1 gene knock out mutants were identified by both PCR and Southern blot. These A ICL1 mutants could not grow and conidate on the medium with olive oil or sodium acetate as the sole carbon source. This result showed the acetyl-CoA from fatty acid JJ-oxidation could not feed glyoxylate cycle for further metabolism by these A ICL1 mutants, i.e. the fat metabolism had been blocked.3. The colonies of A ICL1 mutants on CM plates showed grey-white and different to the grey-dark colour of Guy 11, and the conidiation of these AICL1 mutants were also reduced. But the growth rate between AICL1 mutants and Guy 11 was no different. Moreover, the results of germination test on plastic coverslips showed the conidial germination and appressorium formation of A ICL1 mutants were delayed compared to Guyll, and the germ tubes of A ICL1 mutants were usually much longer than those of Guy11. Meanwhile the appressorium formation rate of AICL1 mutants was also reduced.4. Conidia of A ICL1 mutants were allowed to germinate on plastic coverslips in water drops, and the water was removed at different intervals during germination and stained with Nile Red for the presence of lipid droplets. The results showed the lipid mobilization of A ICL1 mutants seemed slower than that of Guyll. Furthermore, theappressorium turgor at intervals of 24h and 48h period was measured by a cytorrhysis assay. We found the turgor pressure of A ICL1 mutants was reduced, but this reduction was not significantly high. These preliminary data showed l更多还原