Construction And Functional Screening Of Tianmu Mountain Soil Metagenomic Library For Clones With Chitinase Activity

Metagenome is the collective microbial genomes assaemblage found in an environment. Soil is amajor reservoir of microbial genetic diversity. However, more than99%of the microorgnismspresent in soil are not readily culturable in standard laboratory conditions and therefore notaccessible for biotechnology or basic research. Consequently, the traditional cultivation-dependentmethods for screening active substances are coming to the bottleneck. Metagenomic approachesfacilitate the accessibility of the genetic reservoir of soil microbial communities and their potentialapplication. In this paper, we explored the bacterial metagenomic DNA extraction and purificationmethods of soil from Tianmu Mountain in Zhejiang province, China, and constructed a soilmetagenomic library. Followed by screening chitin-degrading clone from the constructedmetagenomic library, a gene encoding chitinase was cloned. The results in detail were described asfollowing.In the beginning, we optimized the soil bacterial DNA extraction and purification strategy byanalyzing different methods reported hitherto, and the extracted DNA meet the requirement forcosmid library construction. An optimized soil DNA extraction and purification method wasestablished on the basis of comparing the soil dispersion slurry differential centrifugation method,soil dispersion slurry sucrose density gradient centrifugation method, agarose gel embedding method,lysis method, dialysis method, gel extraction kit, etc. In summary, soil was homogenated with0.2%sodium pyrophosphate for dispersion, then soil dispersion slurry was conducted differentialcentrifugation，in which the lower speed and higher speed were600×g and10,000×g, respectively.The soil bacteria extracted were lysed by heat-aided CTAB alkaline lysis solution. Bacterial DNAextract were purified by method that combined low-melting point agarose gel electrophoresis withagarase.Using the methods described above, a soil bacterial cosmid-based metagenomic library of TianmuMountain was constructed. The extracted soil bacterial metagenomic DNA was partial digested bySau3AⅠ, and the suitable digested bacterial DNA was ligated with the Bam HⅠ/ScaⅠ digestedcosmid pLAFR-5. The ligation product was used for Lambda bacteriolphage protein packaging andE.coli DH5α transfection. Titering reaction results deduced that the library contained approximate10,000positive clones, which qualified the metagenomic library. Consequently, clones of chitin degradation activity were screened by assay in the medium containingcolloidal chitin. One clone which exhibited significant chitin-degrade zone was obtained.Interestingly, this clone also exhibited significant antagonistic activity against the phytopathogenBotrytis cinerea. The inserted DNA in this clone was sequenced, and a putative chitinase gene waslocated. The chitinase gene contained1,623bp which encoding541amino acid. Protein sequenceanalysis revealed that it belongs to GH18_chitinase-like superfamily, which has potential inantagonistic application against phytopathegenic fungi and nematodes.In conclusion, a soil bacterial metagenomic library of Tianmu Mountain was constructed in thisresearch. A chitinase showed effective antagonistic activity against pathogenic fungus was obtainedbased on metagenomic library. The results in this reseach would contribute to exploring bacterialresources of Tianmu Mountain. Addtionally, a new way to explore bacterial functional genes aspesticide resources was established.