Cloning of ech33 and ech36 From Trichoderma spp. and Expression in Saccharomyces cerevisiae (INVSc1)
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Date
2011
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UAS, Dharwad
Abstract
The present study was conducted to isolate and clone ech36 and ech33 coding
for endochitinase from Trichoderma harzianum and Trichoderma virens respectively
and to study the expression of these genes in Saccharomyces cerevisiae (INVSc1
strain). Using specific primers, gene encoding ech36 (1.2 kb) and ech33 (1.2kb) were
cloned into pTZ57R/T vector. The clones were confirmed through PCR and
restriction analysis. BLAST analysis of ech36 showed 98 per cent homology at
nucleotide level and 99 per cent homology at amino acid level. BLAST analysis of
ech33 showed 99 per cent homology at nucleotide level and 95 per cent at amino acid
level with sequence available in the NCBI databases. The cloned ech36 and ech33
were further cloned into yeast expression vector pYES2/CT and expressed in
Saccharomyces cerevisiae INVSc1. They were named as pYESB36 and PYESB33
respectively. The expression of the cloned genes were confirmed by SDS-PAGE,
substrate conversion assay and bioassay against Sclerotium rolfsii and it was
compared with two previously cloned genes chit18-13 and ech42 to find out the most
efficient endochitinase genes for further use in plant transformation. Chitinolytic
activity was highest in pYESR18-13 and it was 3.15 times higher than control (yeast
with plain vector pYES2/CT). Minimum chitinolytic activity was shown by pYESB36
and it was 1.44 times higher than the control (yeast with plain vector pYES2/CT).
PDA plates with 1000 ıg of crude protein from recombinant yeast clone with ech42
(pYEGB42) showed maximum of 66.67 per cent inhibition of S. rolfsii where as yeast
clone with ech36 (pYESB36) showed minimum inhibition of S. rolfsii (43.99%).
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Plant Biotechnology