美极梅奇酵母菌生防功能决定因子普切明酸生物合成关键基因的筛选及基因功能验证

Metschnikowia pulcherrima, as biocontrol agents, have been widely used to control plant pathogens on fruits. M. pulcherrima also showed strong activities against some human pathogens including Proteus vugaris and Candida krusei. Morevoer, this antagonist has the capability of degrading the mycotoxin (Patulin) and promoting plant growth. The study by traditional experiments indicated that the biocontrol activities of M. pulcherrima controlling pathogens are based on the production of pulcherriminic acid, which chelates the ferron irons from the surroundings. It is known that the ferron iron is a necessary nutrient for the survival of most microbial pathogens. However, at present it is still unknown that how M. pulcherrima produces pulcherriminic acid. No genes involved in the biosynthesis of pulcherrimic acid by the yeast was reported. Non-colored pulcherriminic acid chelating ferron irons produced red compound named pulcherrimin. Based on this characteristic, we will use transposon-inserting methods to screen the pulcherriminic acid biosynthesis related genes, and use the gene-knocking technologies to study the gene functions. Our study will benefit to establishing the high throughput screening method for antagonistic yeast and for constructing the engineered antagonistic M. pulcherrima.

梅奇酵母 Metschnikowia pulcherrima 作为一种生防菌剂已广泛应用于果实病害防治；该酵母对一些人体病原菌也有抑制效果，还具有降解展青霉素和促进植物生长的功能。传统生物学实验表明M. pulcherrima对病原菌的抑制是通过其分泌的普切明酸（pulcherriminic acid）与环境中的铁离子（Fe2+）结合而竞争吸收环境中铁离子来实现的。但该酵母如何合成pulcherriminic acid 这一机制却并不明确，且其生物合成相关基因也未见报道。基于无色普切明酸一旦与铁离子结合便形成肉眼可见的红色物质pulcherrimin这一特性，本项目拟利用转座子插入技术筛选普切明酸生物合成的关键基因，并用基因敲除和过量表达的方法验证其基因功能。意义：为建立M. pulcherrima 的高通量筛选方法和构建高产普切明酸工程菌株奠定基础。

梅奇酵母Metschnikowia pulcherrima作为一种生防菌剂已广泛应用于果实病害防治；该酵母菌对一些人体病原菌也有抑制作用。前期研究表明M. pulcherrima对病原菌的抑制是通过其分泌的普切明酸与环境中的铁离子结合而竞争吸收铁离子来实现的；铁离子是大多数病原菌生长所必要的元素；但普切明酸生物合成相关基因未见报道；基于无色的普切明酸一旦和铁离子结合便形成肉眼可见的红色物质pulcherrimin且红色物质的颜色深度和普切明酸产量有正相关关系这一特性，项目拟利用转座子插入技术筛选普切明酸生物合成的关键基因，初步确定其基因功能，为建立M. pucherrima 高通量筛选方法和构建高产普切明酸工程菌株奠定基础。本项目完成了（1）通过对梅奇酵母菌M. pulcherrima 菌株DPP2在含有不同浓度底物L-亮氨酸的MM中进行培养，利用颜色反应，初步确定了不同浓度底物对DPP2产普切明酸的影响；（2）利用Illumina平台结合第3代测序方法对M. pucherrima DPP2基因组DNA进行测序，结果表明梅奇酵母菌DPP2 基因组大小约22M bp，GC 含量为 45.8%，基因6,086个，基因平均大小为1,403bp，基因占基因组大小约为38.5%；（3）通过对DPP2在不同培养基上不同条件下产孢数量的统计，优化了其产孢条件，明确了DPP2较佳产孢条件；（4）利用含有卡那霉素抗性和潮霉素抗性基因的质粒pCAMBIA1300-HPH转化农杆菌，在经农杆菌转化酵母菌DPP2，结合野生梅奇酵母菌能够产生普切明酸和铁离子反应形成肉眼可见红色物质这一特点，建立了梅奇酵母菌普切明酸生物合成关键基因筛选体系，并获得了参与梅奇酵母菌生物合成关键基因1个，命名为Pycm，该基因长度为2046 bp的基因，没有内含子，编码681个氨基酸；对该基因功能初步分析，Pycm可能属于WD40家族蛋白；蛋白序列进化分析结果表明Pycm和子囊菌中的同类蛋白血缘关系较近。本研究获得了梅奇酵母菌DPP2全基因组序列，并完成了序列分析，优化了梅奇酵母菌产孢条件，基于普明酸螯合培养基上铁离子形成红色物质这一特点，利用含有卡那霉素和潮霉素抗性基因质粒pCAMBIA1300-HPH及农杆菌介导，建立了简洁有效筛选普切明酸生物合成关键基因的方法。