温室黄瓜根结线虫发生地土壤微生物宏基因组文库的构建及其一个杀线虫蛋白酶基因的筛选

Construction of microbial metagenomic library and screening of a nematicidal protease gene in greenhouse cucumber soil infested with Root-knot nematodes

【目的】本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。【方法】采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库,同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛。【结果】该文库库容31008个克隆,平均插入片段36.5kb,包含1.13Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选,通过功能初筛,筛选到1个含杀线虫蛋白酶基因的Fosmid克隆(pro12)。进一步构建和筛选出亚克隆(espro124a5),通过对基因结构进行了初步分析发现:espro124a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no.YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,是一种新型的丝氨酸蛋白酶,有其保守的催化三元组:Asp469、His541和Ser348。【结论】密度梯度离心法提取到的DNA纯度高、片段长,完全能满足构建宏基因组Fosmid文库的要求;同时,构建的宏基因组Fosmid文库库容大,有利于我们从中筛选其他的微生物基因资源。

[Objective] In order to search novel nematicidal protease genes,a metagenomic fosmid library was constructed and screened by uncultured method.[Methods]Density gradient centrifugation was used to extract and purify total greenhouse soil microbial DNA.After end-repair,ligation,packing and transformation,metagenomic fosmid library was constructed.At the same time,in order to screen the library,function-driven screening was used as a potential strategy,skim milk was served as substrate and root-knot nematodes as targets.[Results]The library contained 31,008 clones with the average insert fragment of 36.5 kb,including 1.13Gbp microbial genetic information,so it was suitable for large-scale microbial functional gene screening.By the function-driven screening,fosmid clone pro12 which contained the nematicidal protease gene was screened.Then,subclones were constructed and screened.A subclone named espro124a5 was screened.After analysis of gene structure,espro124a5 is a secreted extracellular protease and a database search for homologies revealed it possessed 45% identities with peptidase S15 from Maricaulis maris MCS10 （ accession no.YP_ 756822 at NCBI）.It is a novel serine protease.Besides these,it has the serine protease-conserved catalytic triad residues,Asp469,His541 and the catalytic nucleophile Ser348.[Conclusion]DNA obtained from the method of Nycodenz density gradient centrifugation had high purity,long fragment,and can meet the requirements of constructing metagenomic fosmid library.At the same time,the metagenomic fosmid library contains a lot of microbial genetic information,which is suitable for the screening of the other microbial genetic resources.