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 1.13 Gbp 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.