Functional analysis of oleate desaturase gene expression from Brassica juncea
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Date
2008
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Abstract
Higher plants express one or more microsomal oleic acid desaturase (FAD2;
EC1.3.1.35) isoforms which catalyze the insertion of a double bond between carbons
12 and 13 of oleic acid at both the sn-1 and sn-2 positions of phosphatidylcholine. In
the present study, full length genomic and cDNA sequences of fad2 gene have been
isolated by using PCR approach from Brassica juncea. Analysis of genomic structure
of fad2 gene revealed that isolated genomic sequence (2526bp) contains 1238 bp 5’
untranslated region which is having a large single intron of 1081 bp, 143 bp 3’
untranslated region, and a CDS of 1155 bp. Analysis of cDNA sequence (1445 bp)
corresponding to genomic sequence (2526bp) revealed that isolated cDNA sequence
exactly corresponded with the genomic sequence containing 5’UTR of 147 bp, CDS of
1155 bp and 3’UTR of 143 bp. Southern blot analysis using partial fad 2 genomic
sequence as probe confirmed that there are atleast 2 copies of fad2 gene present in the
Brassica juncea genome. ClustalW alignment of deduced amino acid sequences of the
BjFAD2 protein together with the other plant fatty acid desaturases showed that all
these membrane-bound desaturases contained the three conserved histidine clusters
[HXXXH,HXXHH and HXXHH] that have been shown essential for desaturase
activity-acting as potential ligands for non-heme iron atoms. Phylogenetic analysis of
deduced amino acid sequences of BjFAD2 with other plant microsomal (FAD2) and
plastidial (FAD6) oleate desaturases showed that a BjFAD2 was positioned in a
subgroup with FAD2 enzymes that exhibit a house keeping pattern of expression.
Study of developmental expression of fad2 gene from developing seeds of Brassica
juncea through RT-PCR revealed that expression of fad2 gene was induced in early
stages of seed development (15 DAF), peaked in mid maturation stage (30 DAF) and
declined as seeds matured (45 DAF). Study of temperature dependent expression of
fad2 gene through RT-PCR revealed that expression of fad2 gene increased under
lower temperature treatments with incubation time as compared to the higher
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temperature treatments where transcript level of fad2 gene was drastically reduced.
Effect of temperature on fad2 gene expression through Real-Time PCR showed that
expression of fad2 gene was one fold increased under lower temperature (100C) and
three fold decreased in higher temperature treatment (320C) as compared to normal
temperature treatments (210C).Fatty acid analysis in temperature treatments by gas
chromatography showed that there were significant differences in linoleic and
linolenic acid content between the treatments with incubation time. Study of
differential expression of fad2 gene in different lines of Brassica having high erucic
acid content (Pusa Bold) and low erucic acid genotypes (LES-39 and LES 1- 27)
through Real - Time PCR showed that expression of fad2 gene was two fold increased
in LES-39 and 4 fold high in LES 1-27 as compared to Pusa Bold. Fatty acid analysis
in high erucic acid (Pusa Bold) and low erucic acid (LES-39 and LES 1- 27) lines by
gas chromatography revealed that there were significant differences in oleic acid,
linoleic acid and erucic content between all three genotypes. Oleic acid in high erucic
acid line like Pusa Bold was found to be significantly low (15.94%) as compared to
low erucic acid genotypes (LES-39 and LES 1-27) having 38.47% and 36.75% of
oleic acid and 0.94% and 0.87% of erucic acid respecticely which was significantly
low as compared to Pusa Bold having 25.48% of erucic acid content.
Description
t-8043