Isolation and characterisation of B-1, 3-glucanase gene from Piper spp.
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Date
2005
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
The study entitled isolation and characterization of β-1, 3- glucanase gene in Piper spp was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara from December 2003 to September 2005. β-1, 3-glucanase is reported to be involved in the defense mechanism of Piper against the Phytophthora foot rot disease. Attempts were undertaken to isolate the gene from tolerant species P. colubrinum and susceptible species P. nigrum.
The information of plant β-1, 3- glucanase gene sequence available in the public domain NCBI was collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among different plant species. Based on the data, eight degenerate primers were derived and out of this, three sets of forward and reverse primers were synthesized for gene isolation. Genomic DNA isolated from Piper spp namely P. nigrum and P. colubrinum was subjected to PCR with the designed degenerate primers at various combinations at different annealing temperatures. The amplification with the primer combination GluF1R2 yielded multiple bands in both the selected Piper species, whereas the primer combination GluF2R1 yielded multiple bands in P. colubrinum only. The primer combination GluF2R2 and GluF3R3 yielded single intact band in both P. nigrum and P. colubrinum. The amplicons obtained were eluted and cloned in pGEM-T vector and transformed into competent cells. High level of recombination was observed on blue and white screening. Recombination of the insert was confirmed by PCR and restriction analysis of the plasmid isolated from white colonies. The cloned fragments were sequenced.
The fragments Pnglu from P. nigrum and Pcglu from P. colubrinum sequenced and subjected to BLAST search revealed, significant level of homology to β-1, 3-glucanase genes reported from other plants deposited in the public domain. Two other fragments cloned and sequenced from P. nigrum did not show homology to β-1, 3-glucanase genes deposited in the public domain. The sequences of the fragment Pnglu and Pcglu were subjected to other sequence analysis utilizing bioinformatics tools including BCM Search Launcher, ORF finder, GENSCAN, Biology workbench, Conserved domain database, Interproscan and CATH. Multiple sequence alignment of nucleotide of Pnglu and Pcglu with the selected nucleotides of -1, 3- glucanase gene sequence from different plant species indicated several conserved regions. The cloned fragment Pnglu and Pcglu had largest open reading frame of size 450 and 372 bp respectively. Internal exons of size 423 bp and 348 bp respectively were detected in the fragment Pnglu and Pcglu respectively. Restriction analysis revealed that both Pnglu and Pcglu have restriction sites for the frequent cutter AluI and the rare cutter NaeI. The fragments had high GC content of 56.1 per cent and 56.9 per cent for Pnglu and Pcglu respectively.
The major amino acid composition deduced from Pnglu were alanine, leucine, arginine and proline, while in Pcglu major amino acids were alanine, glycine, arginine, leucine and valine. The secondary structures predicted for the polypeptides deduced from Pnglu and Pcglu had a high proportion of helices. Conserved domains detected from deduced amino acids of Pnglu and Pcglu suggested that both belong to glycosyl hydrolase family 17, which is a single domain family. Both fragments lack transmembrane regions. Domain structure comparison indicated that domains of Pnglu and Pcglu had similarity to glycosidase, which is the sequence super family of -1, 3-glucanases. The sequence information obtained from Pnglu and Pcglu confirmed that they are genomic counter part of -1, 3- glucanase gene from Piper spp and can be further utilized for the isolation of full length gene from Piper spp.
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