Construction Of Microbial Metagenomic Fosmid Library In Soil And Studies Of The Nematicidal Protease Genes

In the natural environment, microbial resources is extremely rich. However, only less than 1% of microorganisms can be cultured, over 99% of microorganisms cannot be cultured by the traditional culturable methods. For a long time, the traditional cultureable methods could be used to study the environmental samples, greenhouse soil samples were not exception, so it hindered the study of microbial diversity, it also restricted the excavation and utilization of benefical microbial resources. In order to understand the greenhouse soil microbial community composition fully, find out the interaction between microorganisms and greenhouse plant cultivars, plant pathogenic root-knot nematodes and among of themselves; at the same time, and to screen useful genetic resources from the microorganisms which related directly to the occurrence and the prevention of greenhouse crop diseases and pests. In these studies, uncultured methods could be used to survey the microbial community composition and to explore the microbial functional genes, especially the development and utilization of the nem-aticidal protease genes. The results were showed as follow:Firstly, in order to develope and utilize rich microbial resources in the particular greenhouse ecological environment, and to understand the interaction between micro-bes and plants, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constru-cted to analyze the composition of archaeal and fungal community structure in green-house soil samples. Total greenhouse soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS heating treatment and treatment of CTAB. Archaeal and fungal universal primers were used to amplify their specific ge-nes. After retrieving, ligating, transforming, screening of white clones, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constructed. The sequences of archa-ea and fungi were defined into operational taxonomic units(OTUs) according to 97% similarity threshold for OTU assignment using the software program DOTUR. Phylo-genetic analysis showed that Crenarchaeota and unidentified-Archaea were the prima-ry two sub-groups and only a few of Euryarchaeota existed in the archaeal clone libra-ry, total 45 OTUs. All the Crenarchaeota belonged to Thermoprotei; except Basidiom- ycota, the other four sub-group fungi were discovered in the fungal library, total 24 OTUs. The diversity of archaea was very abundant and a few Euryarchaeota (methan-ebacteria) existed in the archaeal clone library. It is may be directly related to the long-term high temperature, high humidity, high content of organic matter environ-memt. The limitation of oxygen was the other reason to cause this phenomenon; Asc-omycotina(over 80%) were the dominant subgroups in fungal library. It was because most of the plant fungus diseases belonged to soil-borne diseases which gone through the winter by the ways of scierotium or perithecium and became the source of primary infection.Secondly, in order to study the bacterial diversity in cucumber rhizosphere soil samples,16S rRNA gene clone library and metagenomic end rondom sequencing were combined to analyze the bacterial diversity preliminarily.35 OTUs were obtained fro-m the library according to sequences similarity of 97%, which affiliated toγ-Proteob-acteria,α-Proteobacteria,δ-Proteobacteria,Firmicutes and so on.γ-Proteobacteria we-re the dominant bacterial groups followed by Firmicutes, and Bacillus were the domi-nant bacterial species.In terms of dominant bacteria, the results revealed by metagen-omic end rondom sequencing were similar to that of 16S rRNA gene library. However, method of metagenomic end rondom sequencing contained most of the vulnerable populations, so it could reflect the true level of bacterial diversity. Comparing to the exposed soil bacterial 16S rRNA gene library, the bacterial diversity was very low. This phenomenon may be directly related to Continuous cultivation for many years and plant a single vegetable species.Thirdly, in order to find out the novel nematicidal protease genes, a metagenomic fosmid library was constructed and screened by uncultured method. Method of dens-ity gradient centrifugation was used to extract and purify total greenhouse soil micro-bial DNA. After end-repair, ligation, packing and transformation, metagenomic fos-mid library was constructed. This library contained 31,008 clones with the average in-sert fragment of 36.5 kb, including 1.13Gbp microbial genetic information, so it was suitable for large-scale microbial functional gene screening. At the same time,in order to screen the library, function-driven screening was used as a potential strategy, skim milk was served as the substrate and root-knot nematodes were served as targets,11 Fosmid clones which contained putative protease genes could be screened, among them,8 Fosmid clones contained the putative nematicidal protease genes, included p11 and p7.Finally, further research was done on P11. The subclone library was constructed and the subclone espl14a5 which could degraded the protein was screened. After ana-lysis of gene structure, espl14a5 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.Indor biological nematicidal and potted tests showed that cloning broth could kill the nematodes without inducer and the promoter which existed in the clone plasmids. So this protease gene had its own promoter. At the same time,this protease is a secreted extracellular serine protease.