Establishment of a PCR [polymeric chain reaction]-based detection method for the photosynthetic rhizobia as a preliminary study in the determination of their diversity and distribution in the Philippines
2009
Cantera, J.J., Laviña, W.A., Encarnacion, G.D., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Biological Sciences
Detection of the nod A gene and diversity studies using the Internal Transcribed Spacer (ITS) region was done for selected photosynthetic rhizobia. Different techniques were used to extract the DNA of the bacterial isolates, these were Cetyltrimethyl ammonium bromide (CTAB) method, boiling method and freeze-thaw method. Boiling method and freeze-thaw method produce genomic DNA approximately less than 25 ng/micro ul as compared to 50 ng/micro ul of DNA from the CTAB method. Least smeared DNA comes from the CTAB treated with RNAse. Total Genomic DNA isolated from all photosynthesis rhizobia were used as template for the PCR amplification of the nod A gene. Amplification of the gene resulted to 666 bp product, a slightly longer fragment for a non-photosynthetic rhizobium pLB1 (750 bp), and no amplification product for the non-nodulating control organism, Escherichia coli. DNA directly extracted from the stem nodules when used as template did not produce any amplicons and DNA directly extracted from the root nodules when used as template for the PCR reaction resulted to the production of an amplicon longer than the expected product. Differences in the bonding patterns were observed when genomic DNA extracted using the different techniques were used as template for PCR amplification of the ITS region. Based on the results, no two isolates share a common bonding pattern. Dendrograms created based on the similarities in bonding pattern reveal similar groupings of the isolates as compared to previous studies which made use of phenotypic characteristics (Ladha and So, 1994) and sequence analysis of nod A and nif A genes (Cantera et al., 2002).
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