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Characterization and expression of codon optimized soybean phytase gene in E. Coli

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Title Characterization and expression of codon optimized soybean phytase gene in E. Coli
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Creator Pritee Singh
Manasi Punjabi
Monica Jolly
RD Rai
Archana Sachdev
 
Subject Glycine max
Phytase gene
SOE-PCR
Prokaryotic expression
E.coli
 
Description Not Available
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 (approximately 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.
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Date 2019-08-19T06:54:33Z
2019-08-19T06:54:33Z
2013-12-01
 
Type Article
 
Identifier Not Available
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/22192
 
Language English
 
Relation Not Available;
 
Publisher CSIR-NISCAIR