GENERATION OF TRANSGENIC GROUNDNUT (Arachis hypogea L.) PLANT USING EPSPS GENE AS A MARKER
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Date
2009-07-10
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University of Agricultural Sciences, Bangalore
Abstract
Foot-and-mouth disease (FMD) is the most feared, viral disease of
cloven-footed animals causing heavy losses to the livestock industry. The
conventional vaccines for FMD have their several limitations which
includes safety, temperature sensitivity and duration of immunity.
Attempts have been made to overcome these limitations using
recombinant DNA technology. Amongst the newer vaccines, edible
vaccines are cost-effective, easy-to-administer and socio-culturally
acceptable. Further, the use of marker gene to identifying transgenics
helps in value addition to the product. The detection methods for the
available biochemical markers like GUS protein or antibiotic selection are
cumbersome. Hence in the present study, the P1-2A gene of FMDV ‘O’
serotype along with the selectable EPSPS was used. The P1-2A gene is
coding for structural proteins of FMDV. EPSPS is a key enzyme in the
shikimate biosynthetic pathway necessary for the production of aromatic
amino acids. The herbicide glyphosate is a competitive inhibitor of
EPSPS. Thus, the gene product titrates the herbicide. The EPSPS gene
was cloned into pCambia vector by replacing GUS gene. The cloned gene
was confirmed by colony PCR, restriction digestion and sequencing. The
CaMV P I-2A gene was cloned into pCambia EPSPS vector and confirmed
by colony lysis and restriction digestion. The construct was transferred to
Agrobacterium and used for transformation of groundnut seeds. A fully
matured leaves of groundnut plants were treated with glyphosate at a
concentrations of 1, 2, 3 gm per litre of water. The plants that showed
tolerance to glyphosate at 3 gm per litre of water were further screened
for presence of FMD VP1-2A gene by PCR. Of the 45 transformants, 8
were found positive both for marker gene and FMDV immunogen. The
positive plants are to be further evaluated as edible vaccine.
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