Molecular Techniques to Study Polymorphism between Closely Related Microorganisms in Relation to Specific Protein Phosphatase
Phylogenetically, the Sebacinaceae that include P. indica recognized a new order, the Sebacinales. The order primarily contains the genera Sebacina, Tremelloscypha, Efibulobasidium, Craterocolla, and Piriformospora. In this study, attempts were made to purify acid phosphatase from P. indica. The fungus produced only one form of intracellular acid phosphatase irrespective of the phosphate concentration. The enzyme was possibly a constitutive enzyme showing molecular mass of 66 kD as separated by SDS-PAGE. The enzyme showed the pH and temperature optima of 5.3 and 40°C, respectively. K m for p-NPP (monoester) was 0.35 mM. Antibodies raised against cytosolic acid phosphatase using gel band in Native PAGE after selective precipitation with ammonium sulfate followed by gel filtration and ion exchange chromatography gave sufficient quantities of antibodies based on immunoblot analysis.
Its reaction with native protein as well as denatured protein was significant. The antibody immunoprecipitated a single band of approximately 66 kD protein in SDS gel. The antiserum localized the enzyme on the vacuoles, cell wall, and cytoplasm of the mycelium, indicating the possible sites of phosphate metabolism.
The acid phosphatase in P. indica and S. vermifera were similar in their molecular mass. The immunoblot showed the strong reactivity of antiserum with protein of S. vermifera. P. indica antiserum blotted the bands in S. vermifera at similar location of P. indica on SDS-PAGE. The antiserum also localized the enzyme in S. vermifera by immunofluorescence technique, showing strong relationship of this fungus with P. indica. The immunogold labeling of antiserum from P. indica precisely localized the enzyme in cytoplasm and vacuoles of S. vermifera supporting the immunological link between these two fungi.
Using α-naphthyl phosphate as substrate, different isoforms of ACPase were obtained by Fast Garnet GBC staining. P. indica and S. vermifera CF and W/MF showed three distinct isoforms of ACPase each, one with higher molecular mass and two other with lower molecular masses. The patterns of isoforms were similar in both the fungi.
Piriformospora indica and Sebacina vermifera sensu showed similar morphology, functions, and isozymes. However, they show distinct genetic variation based on the random amplified polymorphic DNA (RAPD) analysis. Un-weighted Pair Group method with Arithmetic Mean (UPGMA) cluster analysis clustered the isolates into two distinct groups. Statistical analysis of the data was preformed using the NTSYS-pc (Numerical Taxonomy System, Applied Biostatistics) program. An average genetic similarity between both the fungi was 0.58 (i.e., 58%), can be considered to place in species of same roof. Results illustrated the potential value of RAPDs technique for detecting polymorphism among fungal.
KeywordsSodium Dodecyl Sulphate Sodium Acetate Buffer Water Bath Shaker Native Page Average Genetic Similarity
Rajani Malla is thankful to Dr. Ashok K Chauhan, Founder President, Amity-RBEF, New Delhi for his encouragement and support. The author is also thankful to Pham Giang from International Center for Genetic Engineering and Biotechnology, Ram Prasad from Amity Institute, Dr. Upendra and Sweta from All India Institute of Medical Sciences.
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