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【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】 The purpose of this study is to construct and screen a metagenomic library by non-culturing methods in order to find novel nematicidal protease genes. 【Method】 The total DNA of soil microorganism in greenhouse was extracted and purified by density gradient centrifugation. The metagenomic Fosmid library was constructed by blunting, ligation, packaging and transfection. At the same time, skim milk was taken as the substrate and root knot nematode as the target , The library of functional screening. 【Result】 The library contained 31008 clones with the average insert size of 36.5kb and contained 1.13Gbp of microbial genome information, suitable for large-scale screening of microbial functional genes. Through functional screening, a Fosmid clone containing nematicidal protease gene was screened out (pro12). The subclone (espro124a5) was further constructed and screened. Based on the preliminary analysis of the gene structure, espro124a5 is a secreted extracellular protease, which is similar to the serine protease S15 from Maricaulis maris MCS10 (accession no. YP_756822at NCBI) 45% homology, is a novel serine protease with its conserved catalytic triad: Asp469, His541 and Ser348. 【Conclusion】 The DNA extracted by density gradient centrifugation has the advantages of high purity and long fragment, which can fully meet the requirement of constructing the metagenomic Fosmid library. At the same time, the constructed metagenomic Fosmid library is large, which will help us to screen other microbial gene resources.