重寄生真菌盾壳霉胞外蛋白酶的分离纯化与生防功能研究

生防菌盾壳霉是核盘菌的重寄生菌。它通过寄生核盘菌菌核，减少初侵染来源，从而取得防病效果。我们前期研究结果表明：盾壳霉在重寄生过程中大量产生蛋白酶，而且蛋白酶的产生受碳氮源和环境pH值调控。目前，对盾壳霉胞外蛋白酶的种类、产生调控机制、及其生防功能等问题都不明确。本项目提出对盾壳霉胞外蛋白酶进行分离纯化，研究其酶学特性；克隆蛋白酶基因，研究在重寄生过程中的表达模式；通过基因敲除/互补试验和酵母表达系统研究蛋白酶在盾壳霉重寄生过程中的功能；最后构建高效寄生特性的盾壳霉工程菌株。项目的完成将揭示盾壳霉胞外蛋白酶在重寄生过程中的功能，有助于认识盾壳霉的重寄生机制，同时也为提高盾壳霉重寄生效果开辟新途径。

The biocontrol agent Coniothyrium minitans is a mycoparasite of Sclerotinia sclerotiorum. It can parasitize sclerotia of S. sclerotiorum, reducing apothecial production and hence disease incidence. Our previous studies showed that C. minitans secreted a lot of proteases in the infection process, and the production of proteases by C. minitans was regulated by carbon sources, nitrogen source and ambient pH. So far, types of C. minitans extracellular proteases, mechanisms involved in proteases production by C. minitans, and the biocontrol function of proteases remain unknown. In this project, we proposed to purify C. minitans extracellular proteases, to study the proteases properties, to clone the proteases genes and confirm the function, to analyze the expression patterns of proteases genes in the process of C. minitans mycoparasitic, to express the proteases in Saccharomyces cerevisiae and determine the biocontrol function of proteases, to create efficient parasitic strain of C. minitans. The objective of this project is to reveal the biocontrol function of proteases in the process of C. minitans mycoparasitic. The results will broaden our understanding of the mechanism of C. minitans mycoparasitic, and also will be useful for genetically improving biocontrol efficacy of C. minitans against S. sclerotiorum.