INTRODUCTION:

Fungal genetic studies require a rapid method of isolating DNA from a large number of samples for restriction enzyme analysis. Previous methods used are limited by relatively low yield of 50 µg DNA/0.1g lyophilized mycelium. The rapid isolation of large quantities of easily digested total genomics DNA from several species including Neurospora crassa, Talaromyces flavus, etc., Yield was increased to 200 µg DNA/0.1 g lyophilized mycelium, and isolated DNA from 2 to 3 times as many as samples can be achieved using this rapid method (Current record is 64 isolated in one day versus 24 using previous methods) (Zolan and Pukkila, 1986).

DNA isolation from some fungal organisms is different because they have cell walls or capsule that is relatively unsusceptible to lysis. Beginning with yeast, Saccharomyces cerevisiae genomic DNA isolation method, a 30 min DNA isolation protocol was developed for filamentous fungi by combining cell wall digestion with cell distruption by glass beads. High-quality DNA was isolated with yield from the hyphae of Crinipellis perniciosa, which causes witches broom disease in cocao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from yeast, S. ceravisiae.

Genomic DNA was suitable for PCR of specific action primers of C. perniciosa, allowing it to be different from fugal contaminants, including its natural competitor, T. stromaticum (Melo et al., 2006).

MATERIALS AND METHODS:

Molecular Characterization of fungi:

Isolation of chromosomal DNA:

The DNA extraction was performed by the methods of Melo et al. (2006). Approximately 200 mg of washed mycelia of Trichoderma harzianum culture was transferred in to a micro centrifuge tube and suspended in 200 µl buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM Na₂EDTA). When DNA was extracted from T. harzianum that had been grown in liquid culture, with shaking, the balls that had formed were washed three times with cold-sterile distilled water and the DNA extraction buffer had 10 – fold EDTA, 200 µl phenol chloroform – alcohol isoamylic (25:24:1) mixture and 0.3 g sterile glass beads (Sigma, G1277). The suspension was vortexed at top setting for 5 min. To each tube, 200 µl TrisEDTA, (pH 8.0), was added mixed, and the suspension was centrifuged for 5 min. at 13,500 rpm. The supernatant was transferred in to a new micro centrifuge tube, and the nucleic acids were precipitated by adding 1 ml absolute ethanol. Suspensions were mixed and centrifuged (13,500 rpm) for 2 min. and the pellet was resuspended in 400 µl Tris-EDTA, (pH 8.3) µl RNase (10 mg/ml) and incubated for 5 min. Then, it was centrifuged again with 10 µl ammonium acetate (4 M) for 3 min at 13,500 rpm and the supernatant was discarded. The DNA pellet was dried in airflow for 15 min and finally resuspended in 40 µl sterile distilled water. The genomic DNA was verified by 1.2% agarose gel electrophoresis, a single band of high molecular weight DNA was allowed to develop.

PCR amplification of 18S rDNA:

The extracted DNA was used for PCR, which was performed in 25 µl reaction volumes containing 20 ng genomic DNA, 100 µM dNTPs, 1 mM MgCl₂, 2.5 µl 10 x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, pH 8.3), 0.2 µm of each primer pair and 1U Taq DNA polymerase (Invitrogen^®); distilled water was added to complete the final volume of the reaction cycling were initial denaturation step at 95°C for 3 min., followed by 30 cycle, each consisting of 95°C for 50s, annealing temperature 58°C for 50_s and 72°C for 1 min. with a final extension at 72°C for 7 min. T. harzianum specific action primers (Dforward: 5’ ACC CGC TGA ACT GC 3’; DReverse: 5’ GGT CCG TGT TTC AAG ACG 3’) were added to the extracts of the few different fungi and single reaction were performed in an Eppendorf Master cycleris^® Thermocycler. The quantity of the PCR reaction was monitored in 1% Tris-acceteate-EDTA-agarose gel, and bands were visualized by staining with ethidium bromide. Image were made and stored with the Kodak - EDAS^® system. Full length D₂ region of 18S rDNA gene fragment was amplified by PCR from the above isolated genomic DNA. A single discrete PCR amplification band of 596 bp was observed when resolved on agrose gel. To identify the fungus for phylogenitic analysis either ITS (Internal Transcribed spacer) region of 18S rDNA region (LSU: Large subunit) with the D₂ set of primer were used.

18SrRNA sequencing of fungal isolate:

Consensus sequence of 18S rRNA gene was generated from forward and reverse sequence data using aligner software and sequential using the facility available at Xcelris Labs Ltd., Ahmedabad, India.

Nucleotide sequence accession:

The 18S rDNA sequence for the T. harzianum have been deposited in Gene Bank http://www.ncbi.nim.nih.gov./genebank.

Phylogenetic analysis:

The D₂ region of 18S rRNA gene sequence was used to carry out BLAST with the NR database of NCBI genbank database (URL http://www.ncbi.n/m.nih.g). Based on maximum identity scores first ten sequence were selected and Global pair wise sequence similarity between the sequence were performed using Needleman and Wunsuh algorithm available with the emboss sequence analysis suite. Multiple sequence analysis were performed using alignment program CLUSTAL W. The phylogenetic tree was constructed using MEGA 4.

The evolutionary history was inferred using the neighbour – joining method of Saitou and Nei (1987). The bootstrap consensus tree inferred from 500 replicated was taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicated tree in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches, (next to the branches). The evolutionary distance were computed using the Kimura 2- parameter method (Kimura, 1980) and are in the units of the number of base substation gaps substations per site Codon position included were 1^st+ 2^nd+3^rd+ Noncoding. All positions gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 663 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 4 (Tamura et al., 2007). Tree visualization was done with the tree view program.

18S rRNA secondary structure prediction of fungal isolate:

The secondary structure of Trichoderma harzianum were predicted using the Bioinformatics tools available in online www.genebee.msu.su/service/rna2-reduced.html.

Restriction site analysis of 18S rRNA of fungal isolate:

The restriction sites in 18SrRNA of Trichoderma harzianum were analyzed by using NEB cutter programme version 2.0 in online www.neb.com/ NEBcutter 2/index.php.

RESULTS AND DISCUSSION:

Since, the currently available DNA extraction protocols are rather costly and time consuming (Wilson, 1990; Syn and Swarup, 2000; Sambrook and Russel, 2001). Burke et al. (2000) adapted a rapid DNA isolation method from yeast, combining chemical reagent digestion with mechanical (glass beads) shearing for lysis the hyphae of Crinipellis perniciosa and three other hyphal fungi, followed by DNA isolation.

Fig 1.Identification of chromosomal DNA from

Trichoderma harzianum

Fig:2

GenBank: GU646678.1

Hypocrea lixii strain PSMT1 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence

LOCUS GU646678 592 bp DNA linear PLN 03-APR-2010

DEFINITION Hypocrea lixii strain PSMT1 18S ribosomal RNA gene, partial

sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene,

and internal transcribed spacer 2, complete sequence; and 28S

ribosomal RNA gene, partial sequence.

ACCESSION GU646678

VERSION GU646678.1 GI:292385262

KEYWORDS .

SOURCE Hypocrea lixii (anamorph: Trichoderma harzianum)

ORGANISM Hypocrea lixii

Eukaryota; Fungi; Dikarya; Ascomycota; Saccharomyceta;

Pezizomycotina; Leotiomyceta; Sordariomyceta; Sordariomycetes;

Hypocreomycetidae; Hypocreales; Hypocreaceae; Hypocrea.

REFERENCE 1 (bases 1 to 592)

AUTHORS Senthil Kumar,G., Panneerselvam,A., Mubarak Ali,D. and Thajuddin,N.

TITLE 18S Sequence Analysis of Trichoderma harzianum

JOURNAL Unpublished

REFERENCE 2 (bases 1 to 592)

AUTHORS Senthil Kumar,G., Panneerselvam,A., Mubarak Ali,D. and Thajuddin,N.

TITLE Direct Submission

JOURNAL Submitted (02-FEB-2010) Microbiology, Bharathidasan University,

Palkalaiperur, Tiruchirappalli, Tamilnadu 620 024, India

FEATURES Location/Qualifiers

source 1..592

/organism="Hypocrea lixii"

/mol_type="genomic DNA"

/strain="PSMT1"

/isolation_source="paddy field"

/db_xref="taxon:5544"

/country="India: Tamilnadu, Thanjauvr"

misc_RNA <1..>592

/note="contains 18S ribosomal RNA, internal transcribed

spacer 1, 5.8S ribosomal RNA, internal transcribed spacer

2, and 28S ribosomal RNA"

ORIGIN

1 tcggattccg taggtgaacc tgcggaggga tcattaccga gtttacaact cccaaaccca

61 atgtgaacgt taccaaactg ttgccttggc gggatctttg ccccgggtgc gtcgcagccc

121 cggaccaagg cgcccgccgg aggaccaacc aaaactctta ttgtataccc cctcgcgggt

181 ttttttacta tctgagccat ctcggcgccc ctcgtgggcg tttcgaaaat gaatcaaaac

241 tttcaacaac ggatctcttg gttctggcat cgatgaagaa cgcagcgaaa tgcgataagt

301 aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg cgcccgccag

361 tattctggcg ggcatgcctg tccgagcgtc atttcaaccc tcgaacccct ccggggggtc

421 ggcgttgggg atcggccctt tacggggccg gccccgaaat acagtggcgg tctcgccgca

481 gcctctcctg cgcagtagtt tgcacactcg catcgggagc gcggcgcgtc cacagccgtt

541 aaacacccca aacttctgaa atgtgacctc ggatcaggta ggaatacccc aa

//Bottom of Form

Amplification with acting primer occurred only with DNA of C. perniciosa and not with the DNA from A. blazei, T. steromaticum L. edodes, and S. cerevisiae, and thus confirming species – specificity of the acting gene.

In the preset study, amplification with acting primers (D forward: 5’ ACC CGC TGA ACT TAA GC 3’ and D reverse, 5’ GGT CCG TGT TTC AAG ACG 3’) occurred only with DNA of Aspergillus ochraceous of gene thus confirming species specificity of the acting gene. Full length D2 region of 18S rDNA gene fragment was amplified by PCR from the above isolated genomic DNA. A single discrete PCR amplicon band was observed when resolved on agarose gel (Fig.1).

Sandhu et al. (1995) reported that the 28S ribosomal gene was selected to provide adequate species – specific to distinguish closely related organisms. Additionally, ribosomal genes in fungi were present as many as 100 or more copies per genome (Maleszka et al., 1993), and this would provide good detection sensitivity. This reasoning was validated when primer U1 and U2 successfully amplified 28S rDNA from the equivalent of 0.2 genomes of Saccharomyces cerevisiae. In the present investigation the consensus sequence of 592 bp of D2 region of 18S rDNA gene were analysed, and it was generated from forward and reverse sequence data using aligner software.

The 18S rDNA sequence of T. harzianum was performed to get accession number (GU.646678). In the present investigation the 18S rDNA of T. harzianum has been deposited in Genebank http://www.ncbi.nl.lh.gov/ genebank (Fig.2). A phylogenetic tree was also constructed by Neighbour – joining method. The present investigation concludes that the culture of T. harzianum closely related to Hypocera tixii (at 99% level) based on nucleotide homology and phylogenetic analysis(Fig.3). The secondary structure of T. harzianum 18S rDNA (GU646678) showed 25 stems, 24 bulge loops and 14 hairpin loops in their structure(Fig.4). A large number of restriction enzyme sites were observed in the fungal isolates. However the cleavage sites and the nature of restriction enzyme of T. harzianum is 40 sites. The GC content of this species was determined to be 56 per cent and AT content found to be 44 per cent using NEB cutter programme(Fig.5).

ACKNOWEDGEMENT:

The authors are grateful to Secretary and Correspondent A.V.V.M. Sri Pushpam College, Poondi – 615 503 for providing laboratory facilities.

REFERENCE:

1. Burke, D., Dean, D., and Tim, S., 2000. Methods in Yeast Genetics 2000: A cold spring Harbor Loboratory Course Manual. New York: Cold Spring.

2. Felsentsein, J., 1985. Confidence limits on phlogenies. An approach using the bootstrap. Evolution, 39: 783-791.

3. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mole. Evol., 16: 111-120.

4. Maleszka, R., and Clark-Walker, G.D., 1993. Yeasts have a four-fold variation in ribosomal DNA copy number. Yeast, 9: 53-58.

5. Melo, S.C.O., Pungartnik, C., Cascardo, J.C.M., and Brendel, M., 2006. Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniosa. Gent. Mol. Res., 5(4): 851-855.

6. Saitou, N., and Nei, M., 1987. The neighbor-joining method. A new method for reconstructing phylogenetic trees. Mol. Biol. Evoln., 4: 406-425.

7. Sambrook, J., and Russel, D.W., 2001. Rapid isolation of yeast DNA. In: Molecular Cloning: A Laboratory Manual [Sambrook, J and Russel, D.W. (eds)]. New York: Cold Spring Harbor Laboratory, pp.631-632.

8. Sandhu, G.S., Kline, B.C., Stockman, L., and Roberts, G.D., 1995. Molecular probes for diagnosis of fungi infections. J. Clin. Microbiol., 5(11): 2913-2919.

9. Syn, C.K., and Swarup, S., 2000. A scalable protocol for the isolation of large-sized genomic DNA within an hour from several bacteria. Anal. Biochem., 278: 86-90.

10. Tamura, K., Dudley, J., Nei, M., and Kumar, S., 2007. MEGA 4: Molecular Genetics Analysis (MEGA) Software Version. 4.0 Mole. Biol. Evoln., 24: 1596-1599.

11. Wilson, K., 1990. Preparation of genomic DNA from bacteria. In: Current protocols in molecular biology (Ausbel F.M. and Brent, R. eds.) Greene Publ. Assoc. and Wiley Interscience, New York. 241-245.

12. *Zolan, M.E., and Pukila, P.J., 1986. Mol. Cell Biol., 6: 195-200.

Received on 04.04.2011 Modified on 14.05.2011

Asian J. Research Chem. 4(8): August, 2011; Page 1225-1230