玉米弯孢叶斑病菌呋喃型毒素生物合成途径研究

As an novel non-host specific toxin causing maize Curvularia leaf spot（Curvularia lunata）, methyl 5-(hydroxymethyl)furan-2-carboxylate is molecularly and biochemically investigated in its biosynthesis pathway. This study would make a great contribution to understanding the mechanism of the furotoid toxin production and favors screening gene targets from the pathogen potential for anti-toxin maize molecular assistant breeding. Using approaches of comparative genomics, molecular and biochemical analysis to clone the major members of core gene clusters related to toxin biosynthesis is performed mainly based on the homologous sequence identified in other genetic closely related pathogenic fungal species, then the function analysis with each of those cloned genes is carried out under varied conditions of illumination and nutrition. The interaction between members of the core cluster genes for synergistic expression, toxin subcellular accumulation process corresponding to the time course of gene dynamic expression are detected globally in order to construct a genetic linkage map for furanoid toxin biosynthesis. Furthermore the catalytic center of toxin synthase encoded by the gene clusters will be biochemically characterized. Taken together,a comprehensive biochemical and molecular pathway of furanoid toxin from Curvularia lunata in maize is proposed. The result as a first work in this field would build a solid basis for entirely understanding the toxin production mechanism.

甲基-（5-羟甲基）呋喃-2-羧酸盐是玉米弯孢叶斑病菌产生的非寄主专化性毒素，是引起玉米叶斑病的重要致病因子。本项目在分子和生化水平上研究该毒素生物合成途径，对于揭示病菌产生毒素机理、筛选抗毒素作用靶标具有重要意义。通过比较基因组学、基因功能分析和生物化学等方法，筛选鉴定呋喃型毒素生物合成核心基因簇成员和毒素合成中间产物；在不同光照和营养条件下研究毒素生物合成核心基因簇成员互作性质、协同表达时序性及其与中间产物合成和积累的对应关系，构建呋喃型毒素生物合成核心基因簇成员相互关系连锁图；研究毒素生物合成核心基因簇编码酶蛋白活性中心结构特征和催化效应，综合提出玉米弯孢叶斑病菌呋喃型毒素生物合成分子和生化途径，填补国际空白，为全面揭示该病菌产生毒素分子机理奠定理论基础。

甲基-（5-羟甲基）呋喃-2-羧酸（M5HF2C）是玉米弯孢叶斑病菌（Curvularia lunata）产生的非寄主专化性毒素，是引起玉米叶斑病的重要致病因子。本项目在分子和生化水平上研究该毒素生物合成途径和调控机制，对于揭示病菌产生毒素机理及病原菌致病机制具有重要意义。. 转录组分析表明，PKS途径与弯孢菌毒素M5HF2C合成密切相关。通过敲除PKS18基因，发现该基因是M5HF2C毒素合成的关键相关基因。与野生株(16.3ng/mL)相比在∆PKS18菌株(3.05ng/mL)中，毒素含量下降极显著。. 构建了MAPK信号通路相关基因（Clk1、Clm1、Clh1）的缺失突变株，△Clk1不产生呋喃毒素，△Clh1和△Clm1虽然检测到呋喃毒素，但与野生型菌株CX-3相比，含量明显降低（P<0.01）。通过敲除次生代谢调控相关基因clvelB发现，M5HF2C毒素的合成量大幅度下降，而回补株M5HF2C毒素合成量显著恢复。上述结果证明，MAPK基因和clvelB是呋喃毒素合成的调控因子。. 为了鉴定毒素的合成前体和中间产物，通过酵母双杂交筛选到了与毒素合成调控因子Clt-1互作的木聚糖酶（ClXyn24）、乙酰木聚糖酯酶（ClAxe43）。进一步通过构建ClXyn24和ClAxe43缺失突变株及这两个基因的双缺失突变株株，发现突变株中乙酰辅酶A和丙二酰辅酶A的累积明显减少。不仅如此，Q-PCR验证发现PKS18及其所在基因簇下游的13个基因，在ΔClXyn24、ΔClAxe43和ΔClXyn24&ClAxe43表达量均显著下调。证明PKS18通路的表达与木聚糖代谢有关。因此认为Clt-1通过其BTB结构域与ClXyn24、ClAxe43发生互作，作用于木聚糖代谢的上游途径，产生了毒素合成的重要底物乙酰辅酶A。为了深度挖掘M5HF2C毒素合成前体物质，我们检测了PKS18敲除突变株代谢组，发现与毒素结构非常类似的化合物糠醛（a-呋喃甲醛）明显积累，即PKS18负调控糠醛形成。综合分析认为，木糖/木聚糖代谢与糠醛的形成及毒素合成发挥了重要作用。