Identification Of The Composition Of Guanzhong Soil Microbial Community And The Development Of Quantitative Identification Technique

As one of the important component of ecosystem, soil is a precious naturalresource.While soil microorganisms, which is the active constituent underground, playedan important role in keeping the balance of nature, such as the transporting of nutrients,the decomposition of organic matter and the purification of environmental pollutions.Besides, the variety of the composition and activity of soil microbial communitystructure is regarded as a significant indicator in measuring soil quality, soil fertility andthe productivity of crops.Here, we firstly describe a PCR-based rRNA methods library, which used aGuanzhong soil microbial metagenome as its amplification template to successfully cloneand sequence the gene of16S rRNA in prokaryotes and ITS in eucaryote, and by this wepreliminarily analyzed the structure of guanzhong soil microbial community. Secondly,the16S sequences were targeted to design phylum-specific PCR primers based on thesimple allele-discriminating technique by which mismatched bases were introducedartificially when primers were designed to increase the primers’specificity. Thus, primersof five phyla were obtained and these primers can be used in rapid quantitativeidentification. After screening and optimization of PCR conditions, these primer werefinally applied to real-time fluorescence quantitative PCR of different mixing ratios ofplasmid templates and new soil microbial metagenome, thereby we can achieve the rapidquantitative identification of these phyla.Phylum-specific PCR primers based on the simple allele-discriminating specificprimer design technique breaks the limitation of traditional ones and is more effective inenhancing primers’ specificity, reaching a basis of rapid quantitative identification.Since rapid quantitative identification is able to track the microcosmic changes in soilmicrobial community under different environmental conditions fastly and cheaply, and isalso helpful for the study of soil microbial community structure, it can surely promoteprotection of soil microbial diversity and the reasonal development and utilization of soilmicrobial resources.