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Characterization and expression of codon optimized soybean <i style="mso-bidi-font-style:normal">phytase</i> gene in <i style="mso-bidi-font-style:normal">E. coli</i>

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Title Characterization and expression of codon optimized soybean phytase gene in E. coli
 
Creator Singh, Pritee
Punjabi, Mansi
Jolly, Monica
Rai, R D
Sachdev, Archana
 
Subject Glycine max
Phytase gene
SOE-PCR
Prokaryotic expression
E. coli
 
Description 537-547
Phytic acid, the major storage form of
phosphorus in plant seeds is degraded by the phytases to yield inositol and
free phosphate, contributing thereby to the improved bioavailability of phytate
phosphorus and essential minerals in plant foods and simultaneous reduction in
phosphorus pollution of the terrestrial and aquatic ecosystems. As a possible
strategy for altering seed phytate levels, the approach involving reduction of
phytate content by ectopically expressing endogenous phytase gene during seed
development of soybean (Glycine max
L. cv. Pusa-20) was attempted in the
present study.
Semi-quantitative RT-PCR revealed the maximum expression of phytase gene transcripts in germinating
cotyledons (~10 days after germinations), compared to other vegetative tissues.
A full-length phytase cDNA was amplified from the germinating seedlings by
splicing by overlap extension (SOE)-PCR and its sequence analysis revealed an
open-reading-frame of 1644 bp, including an N terminal signal peptide of 28
amino acids. Predicted amino acid sequence (547-aa) of molecular mass 62 kDa on
alignment with related purple acid phosphatases in other plants shared five
conserved domains and seven invariant amino acids involved in coordination of
the metals in the binuclear center of purple acid phosphatases. Owing to a
large number of E. coli low-usage
codons in soybean phytase gene, the
modified gene was cloned into a prokaryotic expression vector pET-28a (+) and
its expression in E. coli was
confirmed by SDS-PAGE and Western blot analysis. Bioassay of the crude
expression product in E. coli revealed a functional phytase gene, showing a great potential for developing low phytate
transgenic soybean through its seed-specific overexpression in the early stages
of seed development.
 
Date 2013-12-27T11:43:30Z
2013-12-27T11:43:30Z
2013-12
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/25169
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.50(6) [December 2013]