Construction Of Metagenomic Library And Establishment Of A Molecular Method For Characterization Of Soil Microbial Community

An efficient method was developed to isolate the microbial nucleic acid from soils. In this way the DNA yields from two soil samples were 14.32u g/g.dry soil and 10. 06 ug/g.dry soil, and recovery rates were 91. 66% and 89. 02% respectively. The method was also resultful for extraction of DNA of G+ bacteria. The crude DNA pellets were recovered by dialyzing and then could be used for molecular operation such as amplification of 16S rRNA gene and digestion of restriction enzymes.The plasmid pUC19 was prepared and digested with BamHI.Then the vector was dephosphated with CIAP to minimize the probabilities of self-ligation of cohesive ends. At the same time, the purified soil DNA was partially digested by enzyme Sau3AI, and the 2-6kb and 6-9kb fragments were recovered by dialyzing respectively. Subsequently they are further purified and ligated with the treated vectors. The ligation product was transformed to E. coli DH5 a , respectively by the means of competence and eletroporation. The cells were cultured on Amp plates and later plasmids of the survived clones were examined to determine whether foreign fragments have inserted in them. In the competence way, the transformation rate of 2-6kb fragment was about 50% while that of 6~9kb was about 30%. For the 2-6kb fragment, the rate of transformation by eletroporation was 10-70 times higher than by competence. However, for the 6~9kb fragments the two means gave no obviously different results. To examine the quality of the library of 2-6kb fragments, 100 randomly selected white clones were screened for their plasmid size. The result showed that an average insert size of the library is 2. 7kb, with 44% of the inserts are larger than 3kb, 83% of the inserts are between 2~4kb. We estimated in this way the library contains 6000-10000 combinatant per gram dry soil.The total DNA was extracted from nine methyparthion-degrading strains and then performed ERIC-PCR. The agarose electrophoresis showed every bacterium present particular profiles that can be stably repeated. According to these fingerprints, the similarity was analyzed among the strains, which were then clustered. The result was primly agreed with the analysis based on comparison of their 16S rDNA sequence. It was hence proved that ERIC-PCR was a powerful means to discriminate different strains and determinate a given isolate. After the PCR conditions were well established, the adequately diluted soil DNA was performed ERIC-PCR. The electrophoresis profiles present complex banding patterns.