CLONING AND FUNCTIONAL CHARACTERIZATION OF ENDOCHITINASE GENE FROM TRICHODERMA SPP.
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Date
2007-08-30
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University of Agricultural Sciences GKVK, Banglore
Abstract
The present study was conducted to isolate Thchoderma spp. from different soil
samples, to screen them for chitinase activity using glycol chitin plate assay, to clone
full-length genes encoding endochitinase from Trichoderma species and expression of
cloned endochitinase gene in Saccharomyces cerevisiae. During the study 44
Trichoderma isolates were obtained from 48 soil sample collected. Among the thirty
three isolates tested for chitinase activity (glycol chitin plate assay), isolates T. virens
lABT 1010, T. konlngiil^Ell^l^ and T. poiysporumlk^l were efficient producers
of chitinase enzyme. Further, using specific primers, genes encoding endochitinase
chiMS (1.6kb) from T viride and T. harzianum (1.5kb) were cloned into pTZ57R/T
vector. The clones were confirmed through PGR amplification and restriction analysis.
The clones were sequenced and analyzed for homology at nucleotide and protein level
to find out conserved domain of protein. Genes encoding endochitinase from both the
species have 95 % and 99.5% homology with reported sequence both at nucleotide
and protein level. Phylogenetic analysis of cloned genes indicated that both the genes
fall in same cluster and closely related to T harzianum endochitinase gene.
Endochitinase gene cloned from T. v/re/75 (cloned earlier) was expressed in S. cerevisiae
using pYES2/cr vector. Substrate conversion assay using cell lysate indicated 9.8 times
more activity in recombinant clones compared to control. Further, cell lysates of
recombinant clones significantly inhibited the growth of Scierotium roifsiL In addition,
reduced expression of the same gene was observed when 173bp upstream sequence
was removed.
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