Efficient Classiifcation And Identiifcation Of Industrial Microbial ResourcesBased On Molecular Markers

Microorganism is one of the most important biological resources on the earth, also thebiological basis for the industrial biotechnology. Microbial resources applied in biotechnologyhave been contributing greatly to economic and social benefits. Identification of microbialresources is the key point to the development of biotechnology. Conventional microbialidentification has been based on the phenotype and biochemical characteristics. With theadvances in polymerase chain reaction (PCR) and DNA sequencing technologies, moleculartechniques have progressively been developed as standard approaches in studies dealing withmicrobial classification. The microbial taxonomy has shift from phenotypic to DNA-basedmolecular technology. In recent years, several molecular identification methods have beenestablished by making use of the variable domains of the rDNA sequences. With the aim toevalueate the feasibility and value of rDNA sequences applied in industrial microbialidentification, this study focused on the establishment of rapid method for microbial genomicDNA extraction and the applicability of molecular markers (bacterial16S rDNA, internaltranscribed spacer of yeasts and filamentous fungi) used in microbial taxonomy. The mainresults are as follows:(1) A rapid, efficient approach for molecular markers prepartion in bulk wasestablished based on a novel rapid microbial genomic DNA extraction method. In thisstudy, a novel rapid and efficient DNA extraction method based on alkaline lysis which candeal with a large number of microbial isolates (96n) in the same batch was established. Thegenomic DNA required only20minutes to prepare, and eliminated the need for complexDNA purification protocols such as solvent extraction. The extracted genomic DNA can bedirectly used as template for molecular markers amplification using PCR assay. The amplifiedrDNA sequences were easy to identify by analysis. The extracted DNA also can be used toamplify other protein-coding genes for molecular identification. This method can be used forrapid systematic microbial identification. Up to now, we have already successfully identified5296strains (including bacteria, actinomycetes, filamentous fungi and yeast) based on thismethod.(2) Evaluated the feasibility of16S rDNA sequence for systemic classification andidentification of bacterial isolates,82.5%of test strains can be assigned to species leveldirectly. The16S rDNA sequences of3726bacterial strains deposited in CICIM-CU (Cultureand information center of industrial microorganism of China universities) were amplifiedusing PCR reaction. The sequences were compared to reference data available at the GenBankdatabase by using BLAST (Basic local alignment search tool). The results shown all of teststrains were identified to genus level successful by16S rDNA sequence-based anaylsis. Mostof isolates (82.5%) were identified to species level which assigned to210species belongingto45genera. Some closely-related Bacillus and Geobacillus species have highly similar16SrDNA sequences, making16S rDNA sequence analysis-based identification problematic.(3) Assessment of the sensitivity of ITS sequence for classification of filamentousfungal isolates,82.2%of test strains were assigned to species level successfully. The ITS regions of832fungal isolates were amplified by PCR and sequenced. The results of ITSsequences analysis shown most of fungal isolates (82.2%) were identified to species levelsuccessfully which assigned to84species belonging to33genera.143fungal isolates (17.2%)were identified to genus level merely which difficult to distinguished from each other becausepoor interspecies homology of ITS sequence, especially in genus Aspergillus and Penicillium.Only5strains (0.6%) cannot be identified based on ITS sequences analysis. Although ITSsequence may not be sufficiently sensitive for identifying some closely related taxa to specieslevel, it can be used as first-round molecular marker to systemic identification of bulk fungalisolates efficiently.(4) Assessment of the sensitivity of ITS sequence for classification of yeast isolates,94.3%of isolates were assigned to species level by ITS sequence variability. The738yeast isolates deposited in CICIM-CU were identified by ITS regions sequence-basedalignment, the result shown696test strains (94.3%) were identified to species levelsuccessfully which assigned to42species belonging to18genera.40strains (5.4%) wereassigned to genus level merely.2strains (0.3%) cannot be identified based on ITS sequencesanalysis.The results shown the ITS sequence exhibits enough differences between yeasts, andcan be used in the discrimination of intra-and inter-species relationships.In summary, based on the novel efficient method of microbial identification, this researchconfirmed the sequences of16S rDNA and ITS have sufficiently sensitive for identifyingbacterial, yeast and fungal isolates, respectively. Most of microbial isolates in this study canbe assiganed to species or genus level successfully by using these two molecular markers. Thepresent results clearly demonstrate that the16S rDNA and ITS region are sufficient asfirst-round molecualer markers used in microbial isolates identification. Therefore, microbialidentification can and should starts from the rDNA sequences.