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Cloning and Characterization of endoglucanase genes from Trichoderma spp.

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Title Cloning and Characterization of endoglucanase genes from Trichoderma spp.
 
Creator Ziyauddin J.Shaikh
 
Contributor Sumangala Bhat
 
Subject Plant Bio-Technology
 
Description The present study was conducted to isolate Trichoderma spp. from different soil
samples, to screen them for glucanase activity and to clone full-length genes encoding
endoglucanase from Trichoderma species. Out of ten different soil samples collected, three
Trichoderma species were isolated. These isolates along with five others available in the
department were screened for beta-1, 3-glucanase activities by growing on minimal synthetic
medium supplemented with laminarin (beta-1, 3-glucan) as a sole carbon source in one set
and dried mycelium of Sclerotium rolfsii in another. Highest glucanase activity was observed
when dried mycelium of Sclerotium rolfsii was used. Out of eight different isolates, an isolate
identified as T. polysporum (IABT-4) shown highest activity.
Further attempts were made to clone endoglucanase genes from Trichoderma species.
Using specific primers genes encoding beta-1, 6-glucanase (1.3 kb) from T. virens and beta-1,
4- glucanase (825 bp) from T. reesei were cloned into pTZ57R/T vector. The clones were
confirmed through plasmid minipreparation, PCR amplification, sequencing and homology
search. The clones were sequenced and analyzed for homology at nucleotide, protein and the
conserved domain of protein. Gene encoding beta-1, 4- glucanase shown 100% homology
with reported sequence both at nucleotide and protein level, where as beta-1, 6-glucanase
shown 98% and 99% homology at nucleotide and protein level respectively. Further to
facilitate transformation into crop plants -1, 4-glucanase and -1, 6-glucanase has been
cloned into a plant transformation vector pHS100 using restriction enzymes BamH1 and
Xba1. The recombinant clone pSGRH-1 having -1, 4-glucanase was mobilized into
Agrobacterium tumefaciens LBA4404 by triparental mating. The A. tumefaciens with
recombinant clone pSGRH-1 was used to transform tobacco.
 
Date 2016-10-17T12:03:33Z
2016-10-17T12:03:33Z
2005
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/80700
 
Format application/pdf
 
Publisher UAS, Dharwad