武夷菌素生物合成基因簇的克隆及功能分析

Wuyiencin are a new agricultural antibiotics and microbial fungicide.Its main composition is a nucleoside antibiotic with nevel chemical stucture firstly discovered by institute of plant protecton, the chinese academy of agricultural sciences. Wuyiencin used as bio-pesticde for controlling many crop diseases with the characteristics of wide-spectrum,low-toxicity,non-pollution and high-effect.Especially to vegetable pathogenetic fungi, wuyiencin's control effect on cucumber powdery mildem(sphaerothea cucurbitial),tomato leaf mold(cladosporium fulvm) is more than 85%, comparable to that of chemical pesticide Triadimefon.At present Wuyiencin has manufactured in factory and commercially applied in biological control of many plant diseases in China. . To further reduce the cost of manufacture of Wuyiencin and overcome the shortcomings of mutation breeding to select the high-yield producing strains, the aim of the project is to study and establish the site-directed breeding based on understanding biosynthesis gene clusters and machanisms of biological metabolic regulation of Wuyiencin in the producing strain. This project includes the following research works, the firstly, constructure of genome Fosmid bank of Wuyiencin producing strain, designing and selection of the specific primers,and then clone the complete biosynthesis gene culsters of Wuyiencin. The second, By the method of Wuyiencin biosynthesis gene deletion and over-expression in producing strain, at same time, observeration the changes of producing strain on the biological and culture characteristis , the inhibiting spectrum to pathogenc fungi and the compositin of Wuyiencin in fermentation liquid checked by HLPC. make sure the key genes among the gene cluster which decides the Wuyiencin biosynthesis of producing strain, futher more deduce the gateway of Wuyiencin biosynthesis and make sure the mechanism of biosynthesis regulation. . The expected outcomes will lead to establishing a new metheod of site-directed breeding to improve the yield of Wuyiencin and to get Wuyiencin's derivatives from the producing strain. The studies has important significance in enhancing the level of industrial production of Wuyiencin and the ability of independent innovation of biopesticide.

武夷菌素具有我国自主知识产权的新型生物农药，具有低毒、广谱、高效的特点，对多种蔬菜真菌病害具有良好的防治效果，并已产业化生产和大面积应用。为了进一步降低武夷菌素的生产成本，突破采用传统诱变方法选育武夷菌素高产菌株面临的效率低、目标产物不明确的限制，建立以明确武夷菌素生物合成基因簇及其代谢调控机理为前提的定向育种方法，本项目拟通过构建武夷菌素产生菌的基因组Fosmid文库，设计特异性引物进行筛选，克隆完整的武夷菌素生物合成基因簇。通过目的基因缺失和过表达，结合产生菌的生物学特性、抑菌谱和发酵液中武夷菌素有效成分的高压液相色谱测定，确定基因簇中与武夷菌素合成相关的关键基因，推断武夷菌素的生物合成途径。该成果将为进一步阐明武夷菌素的合成机制，利用定向育种技术和代谢工程提高武夷菌素产量或获得更高活性的武夷菌素衍生物提供理论、技术支持，对提高武夷菌素产业化水平和增强我国生物农药创新能力具有重要意义。

武夷菌素具有我国自主知识产权的新型生物农药，具有低毒、广谱、高效的特点，对多种蔬菜真菌病害具有良好的防治效果，并已产业化生产和大面积应用。为了进一步降低武夷菌素的生产成本，突破采用传统诱变方法选育武夷菌素高产菌株面临的效率低、目标产物不明确的限制，建立以明确武夷菌素生物合成基因簇及其代谢调控机理为前提的定向育种方法。本项目成功构建武夷菌素产生菌的基因组Fosmid文库，设计特异性引物进行筛选，克隆14个完整的ORF，确定武夷菌素生物合成基因簇。该成果将为进一步阐明武夷菌素的合成机制，利用定向育种技术和代谢工程提高武夷菌素产量或获得更高活性的武夷菌素衍生物提供理论、技术支持；对提高武夷菌素产业化水平和增强我国生物农药创新能力具有重要意义。