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Cloning and Characterization of Cysteine Protease Inhibitor Genes (CPIs) From Maize (Zea mays L.) and Rice (oryza sativa L.)

KrishiKosh

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Title Cloning and Characterization of Cysteine Protease Inhibitor Genes (CPIs) From Maize (Zea mays L.) and Rice (oryza sativa L.)
 
Creator J.H.Mudiyappanavar
 
Contributor M.S.Kuruvinashetty
 
Subject Plant Bio-Technology
 
Description Plant cysteine protease inhibitors (CPIs) can be effective in developing transgenic
crop plants resistant to insect pests. These genes have no problems related to codon bias,
RNA processing and protein stability. In the present study, an attempt was made to clone CPI
genes from maize (Zea mays) and rice (Oryza sativa). Using specific primers, genes encoding
maize cysteine protease inhibitor (MCPI) and rice cysteine protease inhibitor (RCPI) were
amplified and cloned in pTZ57R/T vector, sequenced and analyzed in silico. Cloned MCPI
gene had 97 and 99 per cent homology with reported MCPI gene at nucleotide and protein
level, respectively. Similarly, cloned RCPI gene had 99 and 96 per cent homology with
reported RCPI gene at nucleotide and protein level, respectively. Multiple alignment of
derived amino acid sequences of MCPI and RCPI clone herewith those available in the public
databases showed the presence of CPI signature motif, QxVxG.
To facilitate transformation of crop plants, MCPI and RCPI genes were subcloned
into a plant transformation vector pHS100 at XbaI and BamHI restriction sites. The
recombinant pHS-MCPI11 and pHS-RCPI1 were mobilized into Agrobacterium tumefaciens
LBA4404 by tri-parental mating. A. tumefaciens with recombinant clones pHS-MCPI11 was
used for transforming tobacco. A number of kanamycin resistant plants were generated. Of
the 13 checked for PCR with MCPI specific primers, 12 were positive. The expression of
MCPI in tobacco was confirmed in two plants by RT-PCR. Copy number, the presence of
respective proteins in transgenic plants and their effect of tolerance to insect pests are to be
studied.
 
Date 2016-10-17T13:37:32Z
2016-10-17T13:37:32Z
2005
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/80705
 
Format application/pdf
 
Publisher UAS, Dharwad