MOLECULAR ANALYSIS OF MEDICINAL Schizophyllum commune FUNGI

ABSTRACT

This article presents information about the molecular phylogeny of the Schizophyllum commune fungus strain, which was compared with the strains of the fungus from different countries, based on the read nucleotide sequence of the 1,5.8 S ribosomal RNA gene fragment, the electrogram of the amplification products.

АННОТОЦИЯ

В статье представлены сведения о молекулярной филогении штамма гриба Schizophyllum commune, который сравнивался со штаммами гриба из разных стран, на основе считывания нуклеотидной последовательности фрагмента гена 1,5,8S рибосомальной РНК, электрограммы продуктов амплификации.

Keywords: DNA, sequencing, fragment, electrophoresis, ITS-1 and ITS-4 primers, Amplification, phylogeny, genome.

Ключевые слова: ДНК, секвенирование, фрагмент, электрофорез, праймеры ITS-1 и ITS-4, амплификация, филогения, геном.

Introduction

Molecular identification of medicinal fungus Schizophyllum commune was carried out by a group of foreign scientists. Preliminary DNA sequencing and molecular analysis of the fungus S. commune was done by (Anderson, Campbell and Prossers, 2003) [1], identification and variation analysis of rDNA isolates of the fungus by [2] and the initial phylogenetic tree of fungus was carried out by [3].

Construction of more than 50 primer copies as a result of repeated transcription in the genome of S. commune based on the unit consisting of 18S-5.8S-28S rRNA was carried out by (Moore and Frazer, 2002)[4]. Searching for homologies with nucleotide and ITS sequences of fungal 18S RNA A search for small ribosomal RNA gene sequences in GenBank was reported in a research by [5].

Yadav and Tyagi (2005) [6] identified the 18S rDNA sites responsible for the schizophyllan biopolymer produced from the culture fluid of medicinal S. commune using ITS-1 and ITS-4 primers and placing them in NCBI sequences.

J. Sherkulova and E. Eshonkulov [7], [8] are working on obtaining pure cultures of this fungus and studying its strains in Uzbekistan.

As a result of our research, for the first time in the territory of Uzbekistan, the identification of 1, 5.8 S ribosomal RNA gene fragment from the isolates of the medicinal fungus S. commune grown in liquid nutrient medium, searching for nucleotide sequence similarities in the gene bank, molecular phylogeny within the species was carried out.

Materials and method

Genome DNA isolation of S. commune fungus was carried out in the "Molecular Microbiology" Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan and the "Genetics and Genomics" laboratories of the Department of Microbiology and Biotechnology of Karshi State University. 600 μl of the culture liquid of the fungus grown in liquid nutrient medium for 14 days was measured and placed in eppendorf  test tubes. It was precipitated by centrifugation at 14,000 rpm. Then the liquid was washed 3 times with distilled water, centrifuged and drained. 600 μl of GTE (glucose-25 mM, tris-NSL- 20 mM and 10 mM EDTA) was dissolved in the cleaned and washed sediment. Lysozyme was added to the solution at a concentration of 2 μg/ml and kept in an ice container for 15 minutes.

It was incubated at 370° C for 30 minutes after addition of SDS solution with a final concentration of 2%. NaAs solution with a final concentration of 0.3 M was added to the mixture and kept on ice for 30 min. After centrifugation, one volume (600 μl) of phenol (tris-saturated, pH-8.0) solution was added to them, mixed well for 2 minutes, and centrifuged at 13,200 rpm for 10 minutes.

In the next step, the upper supernatant was weighed, transferred to new plastic test tubes, and NaAc solution (pH-5.3) with a final concentration of 0.3 M was added. Then 2.5 volumes of cold 96% ethanol were added and refrigerated for 2 hours to precipitate the DNA. Then, it was centrifuged at 13,200 rpm for 5 minutes and dried in an eppendorf thermoblock at 50°C for 20 minutes. In the final step, the DNA in the test tube was dissolved in 60 μl of TE buffer [9].

The separated DNA fragment was electrophoresed. Amplification and electrophoresis of the fungal 1.5.8 gene region was performed on a Biorad PCB, Main Board, CFX96/CFX96 Deep Well/CFX384 [10].

Intraspecific molecular phylogeny of gene fragments of different national strains of the fungus S. commune was performed in the Mega X program (Shu, C.-H., Hsu, H.-J., 2011) [11].

Results and discussion

According to the research results, molecular identification of the strains isolated from the districts of Kashkadarya region was carried out with specific ITS-1 and ITS-4 primers. According to the result of identification, an electrogram of the amplification products of the 1.5.8 gene region was obtained (Fig. 1).

Figure 1. Electrogram of products of 1,5.8 gene region amplification of Schizophyllum commune fungus with ITS-1 and ITS-4 primers

The identification and nucleotide sequence of the 1,5.8 S ribosomal RNA gene fragment from the fungus S.commune is as follows

The .S. commune fungus, 1.5.8 S ribosomal RNA gene fragment sequence results compared to the S.commune Sequense ID: MT647523.1 sample available in the gene database using BLAST at the National Center for Biotechnology Information (nih.gov) gave 99% similarity (Fig. 2).

Figure 2. Schizophyllum commune fungus, 1.5.8 S ribosomal RNA gene fragment compared to Sequense ID: MT647523.1 available in the gene database

According to the obtained results, a molecular phylogenetic tree of the S. commune fungus, 1,5.8 S ribosomal RNA gene fragment, based on comparison with other strains of the world, was created using the Mega X program. In it, the molecular phylogeny of the molecularly identified species in 

Uzbekistan was compared with the strains of S. commune found in 14 countries of the world (Fig. 3).

Figure 3. An intraspecific molecular phylogeny of the 1,5.8 S ribosomal RNA gene fragment of the fungus Schizophyllum commune based on comparison with other strains of the world

According to the research results, the nucleotide sequence of the 1.5.8 S ribosomal RNA gene fragment of strains of the medicinal fungus S. commune in Uzbekistan was studied. The electrophorogram of the gene region amplification products was carried out. The molecular phylogeny of the strain identified in Uzbekistan was compared with the strains of S. commune found in 14 countries, and the molecular phylogeny within the species was analyzed.

References:

1. Anderson, I.C., Campbell, D.C. and Prosser, I.J., 2003. Potential bias of fungal 18S rDNA and internal transcriber spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Environmental Biology, Volume 5, pp. 36-47.
2. Maheshwari, R., 2011. Ribosomal DNA. In: J. Bennett and P. A. Lemke, eds. Fungi experimental methods in Biology. 2nd ed. Bangalore: CRC Press, p. 277.
3. Deacon, J., 2006. Fungal genetics, molecular genetics and genomics. In: Fungal Biology. UK: Blackwell, pp. 158-183.
4. Moore, D. and Frazer, L. N., 2002a. Diversity in the kingdom fungi: Chytridomycota, Zygomycota, Ascomycota, and Basidiomycota. In: Essential Fungal Genetics. Manchester: Springer, pp. 8-13.
5. Gontia-Mishra, I., Deshmukh, D., Tripathi, N., Bardiya-Bhurat, K., Tantwai, K. and Tiwari, S., 2013. Isolation, morphological and molecular characterization of phytae-hydrolysing fungi by 18S rDNA sequence analysis. Brazillian Journal of Microbiology, 44(1), pp.317-323.
6. Yadav, P. and Tyagi, R., 2005. Microbial production and processing of the polysaccharide Schizophyllan. In: Experimental Biotechnology. New Delhi: Discovery Publishing House, pp. 35-39
7. Erkin E., Dilmurod M., Navbakhor K. MEDICINAL SCHIZOPHYLLUM СOMMUNA FR. THE FIRST REPORT OF THE FUNGUS WHICH IS DISTRIBUTED IN THE TERRITORY OF UZBEKISTAN //Universum: химия и биология. – 2022. – №. 11-3 (101). – С. 13-16.
8. Erkin E., Jamila S., Dilmurod M. SCHIZOPHYLLUM СOMMUNASI FR. ON THE TERRITORY OF UZBEKISTAN ISOLATION OF PURE CULTURE OF MEDICINAL FUNGUS //Universum: химия и биология. – 2022. – №. 6-4 (96). – С. 4-7.
9. Moore, D. and Frazer, L. N., 2002c. Origins. In: Essential fungal genetics. Manchester: Springer, pp. 2-8
10. Prabha, T.R., Revathi, K., Vinod, M.S., Shanthakumar, S.P. and Bernard, P., 2013. A simple method for total genomic DNA extraction from water moulds. Current Science, 104(3), pp.345-347.
11. Shu, C.-H. and Hsu, H.-J., 2011. Production of schizophyllan glucan by Schizophyllum commune ATCC 38548 from detoxificated hydrosylate of rice hull. Journal of Taiwan Institute of Chemical Engineers, Volume 42, pp. 387-393.