DISCUSSION

Amplification of a fragment of fungal DNA with homology to a chitinase gene was achieved using PCR techniques with degenerate primers designed from conserved regions of a number of known fungal chitinases. The gene fragment, ChiA shows homology to plant, yeast and fungal chitinases. Unfortunately, only one isolate gave an amplicon out of the eleven isolates that showed chitinase activity. It is a fundamental weakness of PCR that a single base mismatch at the 3’ end of the primer prevents extension of the strand and the chain reaction is prevented (Cline et al. 1996). To overcome this problem, either new primers should be designed or a DNA polymerase with 3’-5’ proofreading exonuclease activity (eg. Pfu DNA polymerase) could be used to remove 3’ mismatches so that strand extension can occur (Cline et al. 1996).

5.4.1.1 Genome walking method

The method used to determine the full-length sequence, the genome walking or PCR- vectorette method, did not allow the full-length gene sequence to be determined. The nested PCR used to amplify single-locus products resulted in smearing that may have been caused by the primers annealing to more than one site. A single nested primer, NPL 1 and an adaptor primer AP2 amplified a 250 bp fragment. This fragment proved to be homologous to the known sequence. The failure to amplify and sequence the flanking regions using the genome walking method may be because there were no restriction enzyme recognition sites close to the sequence. When the sequences were searched for restriction enzyme recognition sites, the enzymes used, EcoRV and SspI, were absent (Appendix 1, Fig A2). These restriction enzymes were chosen so that

digestion of the genomic DNA did not produce many small fragments. It is possible that the lengths of the fragments, produced after digestion, were too large for amplification.

A restriction map of the T. asperellum nucleotide sequence also revealed that there were two AluI sites at positions 156 and 448 downstream from the 5’ end. If indeed EcoRV and SspI produced fragments that were too large for amplification, this can be confirmed by “long range” PCR or Southern blotting. Other researchers have amplified segments as large as 10 kbp using the PCR vectorette method (Jeffreys et al. 1988).

More likely, the anchor template failed to ligate to the ends of the restricted fragment library so that amplification would not occur. The smears produced during the primary PCR may correspond to non-specific DNA amplification likely due to non-specific priming, lack of optimization of the PCR conditions, poor quality genomic DNA, or non specific primers. The nested primers reduced the background. Although in this case the PCR-vectorette technique did not produce additional fragments of the gene, there are ways to improve the technique. Restriction enzyme recognition sites incorporated in the anchor primer and specific primer ought to increase cloning efficiency (Roux and Dhanarajan 1990). Also, the full-length gene can be isolated using inverse PCR and random amplification of cDNA ends (RACE) (Schaeffer et al. 1994). The complete sequence could also be obtained by constructing a cDNA library and a genomic library to obtain the sequences of the transcript and the full-length gene respectively.

5.4.2 Isolation of a chitinase gene using specific primers

To overcome the failure to isolate a full-length gene, Trichoderma spp. specific primers (Viterbo et al. 2002) were used to amplify the fungal genomic DNA of all eleven isolates. The sequence obtained from isolates 04-000 and 04-001 showed 98%

homology to an endochitinase gene from T. harzianum indicating that the sequence was part of a chitinase gene. There is high homology (98%) of this sequence to the published

sequence by Viterbo et al (2002), however they are not identical. This provides evidence on possible genetic diversity between this isolate and that used by the authors.

This sequence was named ChiB. A sequence analysis will be presented in Chapter 6.

5.4.3 Glucanase gene

Initially a 1257 bp nucleotide sequence corresponding to 400 amino acids was amplified from isolate 04-013 with glucanase degenerate primers. The deduced amino acid sequence showed identity to other known glucanases.

5.4.3.1 Genome walking method

This method was successfully used to obtain a complete glucanase gene that showed homology to a glucanase from A. oryzae. A total of 2844 bp were obtained from the putative glucanase gene, named Glu1, from the isolate 04-013.

DNA fragments were amplified in a single PCR reaction from an oligonucleotide vectorette library with a single genomic-specific primer in combination with a vectorette specific primer. Except for the sequence corresponding to the 3’ end of the gene obtained after primary amplification, no distinct products were produced possibly because the restrictions sites were located too far away from the glucanase gene to be amplified with the genome walking method used in this study. Others have also amplified flanking regions of known sequences in a single PCR reaction from an oligocassette library (Kilstrup and Kristiansen 2000).

To obtain the rest of the putative glucanase gene, libraries were amplified in a secondary PCR. While a few non-specific background products were seen on the gel, the dominant products were extracted and sequenced. As a result, a downstream fragment was obtained that added another 1176 bp towards the end of the gene. Using this nucleotide sequence, a further 320 bp sequence was attained, corresponding to the

3’ end of the gene as shown by BLASTX (Altschul et al. 1997) alignment with known glucanases.