象耳豆根结线虫生防微生物功能基因挖掘及作用机理研究

In order to avoid the traditional technology of separation and purification,metagenomic libraries were constructed using total genomic DNA extracted from rhizosphere microorganisms in Hainan and were used together with a fosmid vector,pCC1FOS.Active clones and novel bioactive protease genes which could kill the Meloidogyne enterolobii were screened and characterized.Skim milk was served as substrate, method of function-driven screening was used to screen protease genes. At the same time, Meloidogyne enterolobii was served as target to screen the nematicidal protease genes among the selected protease genes for the future. After construction and screening of the subclones, plasmid DNA was isolated from the active subclone and sequenced.The whole sequences of the nematicidal protease genes were obtained. Sequence analysis was carried out using the DS Gene 1.5 program. Then prokaryotic expression system of the target genes were constructed in E.coli. Methods of column chromatography, high performance liquid chromatography, biological mass spectrometry and bioassay were performed to separate, purify and identify active protein. Subsequently, protease activity was determined by measuring the release of acid-soluble material from azocasein. Effects of temperature and pH on enzyme activity and stability would be tested. And the structures of protease were be disussed. The other physical and chemical properties of these proteases would be understood. Also, electron microscopy was carried out in order to find out the changes of nematode cuticles before and after the treatment of active clones or active proteases.

避开传统分离、纯化培养的技术手段，构建和筛选根际土壤微生物宏基因组文库，获取对象耳豆根结线虫有生物活性的、新型的蛋白酶基因并研究其作用机制。以脱脂奶为底物，功能驱动筛选蛋白酶基因，并以象耳豆根结线虫为靶标，进一步筛选对其有活性的蛋白酶基因；构建和筛选亚克隆，通过序列分析，获得活性蛋白酶基因核苷酸序列并预测氨基酸结构；在大肠杆菌中克隆并原核表达目的基因；利用柱层析、高效液相色谱、室内活性跟踪和生物质谱，分离、纯化并鉴定活性蛋白；解析蛋白结构和功能，并测定酶活、其它理化性质和杀线活性，通过电镜观察，研究其对象耳豆根结线虫的作用机制。

M. enterolobii给果蔬造成毁灭性的危害，寄主范围广，抗性强。普通化学药剂不易防治。本研究以M. enterolobii为研究对象，依托热带作物根际微生物资源，利用宏基因组技术挖掘新的杀线蛋白酶并构建其高效表达体系，为杀线剂的研发和线虫防控提供技术支持。具体如下：.1.番石榴园Fosmid文库构建和分析：构建了宏基因组Fosmid文库，包含31008个克隆，平均插入片段36.5kb，库容超1 Gb。随机插入率98.1%，物种多样性丰富，无同源序列87.79%；.2.杀线蛋白筛选：筛选出111个含蛋白酶基因的Fosmid克隆。进一步杀线生测，筛选出21FA6、5BA8、5FA8、20AA2、60HA9、14BA10、83FA2、83HA8、178FA8等9个克隆。其中，21FA6效果最好，8h后线虫的死亡率100%。预测可能是一种胞外碱性蛋白酶；.3.亚克隆构建、筛选和评价：构建了亚克隆文库espro21FA6。筛选出活性亚克隆espro21FA6-7a2。其4 h、8 h和12 h，线虫致死率分别为78%，92%和98%。12h后，能够看到被破坏的线虫角质。盆栽效果原液高达83.7%，稀释10倍也有68.7%；.4. espro21FA-7a2处理前后Meloidogyne enterolobii转录组分析：丝氨酸/苏氨酸激酶和酪氨酸激酶等蛋白酶增多。尤其参与亮氨酸、谷氨酸、半胱氨酸以及膜蛋白降解的基因大量表达。可能是espro21FA-7a2中活性物质的加入，激活了角质降解基因，使其体表角质降解，死亡；.5.亚克隆测序和序列分析：获得2个活性亚克隆。其中，espro 60HA9与Geobacter sp. M21肽酶peptidase M48氨基酸序列相似性为43%；espro21FA-7a2与Maricaulis maris丝氨酸蛋白酶S9家族47.52%的同源性，与Kangiella koreensis的丝氨酸蛋白酶S9有44.31%同源性。是新型的丝氨酸蛋白酶，有保守的催化三元组：Asp384,His461和Ser372；.6. 蛋白酶基因的克隆、表达和评价：该基因有2条独立启动子。构建了espro21fa6原核表达载体和体系。蛋白质62KDa，为胞外分泌蛋白。盆栽防效82.8%。.发表国内核心7篇，会议论文1篇，申请国家发明和实用新型专利各2项。