Advertisement
CHAPTER
6
SUMMARY
In the present study, plant transformiition cassettes with antifungal protcin
genes, chitinase and lipid transfer protein 2nd cyanamide hydratase marker gene have
been constructed. Chitinase gene, Bur2chi was amplified from the cDNA library
clone of barley using gene-specific primers. The amplified chitinase showed 1. I kb
size with an ORF. 318 amino acids. The predicted amino acid sequence of BarZchi
has 95.3% homology with chitinase of rye and other plant chitinases. Lipid transfer
protein gene, Lrp 3Fl was amplified from the cDNA library clone of wheat using
gene-specific primers. The amplified Lrp 3F1 showed 345 bp size with an
ORF,
115
amino acids. The predicted amino acid sequence of Ltp 3F1 has 80% homology with
barley and other LTPs of monocots.
BarZchi gene was cloned into PET 28a+ and overexpressed in
E,
coli. The
recombinant chitinase was purified from the inclusion bodies of bacterial pellets.
SDS-PAGE and Western blot analyses with an antiserum against barley chitinase
confirmed the production of 35 kDa recombinant chitinase in
E.
coli. Ltp 3F1 gene
was cloned into pMAL-p2x and overexpressed in
E.
coli as soluble protein.
SDS-
PAGE analysis confirmed the production of 51 kDa LTP fusion protein.
Purified chitinase showed a broad-spectrum antifungal activity even at a
concentration of 100 pg (0.42
U)
against fungi such
as
Botryris cinerea (blight of
tobacco), Pestaloria rheae (leaf spot of tea), Bipolaris oryzae (brown spot of rice),
Alternuria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and
Advertisement
