水杨酸代谢在绿僵菌致病及抗逆中的作用

Salicylic acid can be produced by plants and microorganisms. As an immune and stress response signaling molecule, salicylic acid plays an important role in resistance to biological and non-biological stress in plants. Exogenous salicylic acid also has multiple functions in human. However, the biological roles of salicylic acid in pathogenic microorganisms are still not clear. Analysis of the genome sequence reveals that the salicylate synthase and salicylate hydroxylase are present in the entomopathogenic fungus Metarhizium acridum, indicating that insecticidal fungus has a similar salicylate metabolism pathway as bacteria. In this proposal, we will clone the genes encoding key enzymes in salicylate metabolism: one salicylate synthase gene (MaSalS) and two salicylate hydroxylase genes (MaSalH1, MaSalH2), in M. acridum.The three enzymes will be charactereized by expression in yeast. To determine the biological function of salicylate in entomopathogenic fungi, the production of salicylic acid will be altered by up-regulation or down-regulation of the expression of salicylate synthase and saliylate hydroxylase genes using over-expression and knock-out techniques,respectively. The growth characteristics, stress tolerance, conidium production and pathogenicity will be evaluated in the engineered M. acridum strains with varied salicylic acid production. Based on these results, we will summarize the roles of salicylic acid in stress tolerance and pathogenesis in entomopathogenic fungi. To clarify the genes and pathways regulated by salicylate in entomopathogenic fungus and its host insect, gene expression profile will be analyzed among strains with altered salicylate production and host insect after infection with these M. acridum engineered strains using Digital Gene Expression technique. This results will help to clarify the mechanism of salicylate in some vital characteristcs, such as pathogenicity and stress tolerance, in pathogenic microorganisms.

植物和微生物都能够合成水杨酸。水杨酸作为一种免疫信号分子及逆境响应信号分子，在植物抵抗生物和非生物胁迫中发挥着重要作用。外源水杨酸在人体中具有多种重要作用。病原微生物中水杨酸的作用尚不清楚。本课题组通过分析昆虫模式病原真菌"绿僵菌"的全基因组序列发现，绿僵菌中存在水杨酸合成酶及水杨酸羟化酶基因，具有与细菌类似的水杨酸代谢途径。本项目拟克隆绿僵菌水杨酸代谢关键基因：1个水杨酸合成酶、2个水杨酸羟化酶基因，通过酵母表达，分析酶学特性；采用基因敲除及超表达等技术，上调或下调表达这三个基因，改变菌株的水杨酸代谢，分析水杨酸水平改变后菌株生长、抗逆及致病性等性状的变化，确定水杨酸代谢在昆虫病原真菌抗逆及致病等过程中的作用；通过分析水杨酸代谢改变后真菌及侵染后寄主昆虫的全基因表达谱变化，弄清水杨酸代谢调控病原真菌及其寄主昆虫的主要途径及基因，解析水杨酸代谢对病原菌致病和抗逆等重要性状的调控机制。

植物和微生物都能够合成水杨酸。水杨酸作为一种免疫信号分子及逆境响应信号分子，在植物抵抗生物和非生物胁迫中发挥着重要作用。外源水杨酸在人体中具有多种重要作用。病原微生物中水杨酸的作用尚不清楚。通过分析昆虫模式病原真菌“绿僵菌”的全基因组序列发现，绿僵菌中存在水杨酸合成酶MaSalS及水杨酸羟化酶(MaSalH1,MaSalH2)基因，具有与细菌类似的水杨酸代谢途径。本研究中，通过基因敲除技术分别敲除水杨酸合成酶和水杨酸羟化酶，研究了水杨酸代谢对绿僵菌生长、抗逆和致病性的影响。结果表明，高浓度外源水杨酸对真菌生长具有抑制作用，降低蝗虫存活率。水杨酸合成酶在孢子时期表达最高，在虫菌体时期表达较弱；水杨酸羟化酶在孢子和虫菌体时期表达量高。抗逆性分析显示，水杨酸合成酶和水杨酸羟化酶不影响真菌抗氧化，高渗和细胞壁破坏剂等逆境能力。毒力分析显示，水杨酸合成酶敲除后，菌株毒力下降，而水杨酸羟化酶(MaSalH1,MaSalH2)敲除后，菌株毒力上升，说明水杨酸代谢途径影响菌株致病性。检测菌株侵染寄主昆虫后，ΔMaSalS菌株处理后起吞噬作用血细胞数量多于野生型；而ΔMaSalH1和ΔMaSalH2菌株处理后起吞噬作用血细胞数量比野生型显著减少。发现采用GC-MS检测水杨酸合成酶敲除菌株中水杨酸含量，结果显示，相比野生型，水杨酸合成酶敲除后菌体水杨酸量显著减少。血细胞是无脊椎动物免疫系统中第一道防线。因此，理解昆虫血细胞在细胞免疫中的作用有助于开发高效的杀虫真菌。但目前，尚无有效的方法鉴定蝗虫血细胞和吞噬细胞的种类。本项目中，我们建立了用Wright–Giemsa染色方法观察昆虫感染病原菌后的血细胞种类和数量变化。