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Genomics and Applied Biology, 2010, Vol.1 No.2
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Table 1
AhKR
,
AhHD
and
AhENR
genes in peanut
Protein
GenBank
accession
No.
The
length of
aa
5’ upstream
region (bp)
3’ downstream
region (bp)
Molecular
mass (kD)
pI
Localization
The length
of CTP
KR
FJ768728
323
197
399
33.855
9.22
Chloroplast
73
HD
FJ768727
216
39
134
23.422 2
8.91
Chloroplast
43
ENR
FJ768731
389
200
330
41.505 4
8.64
Chloroplast
70
Note: CTP represents chloroplast transit peptide
1.1.1 Cloning and phylogenetic analysis of
AhKR
gene
In Figure 1, the peanut β
-
ketoacyl-ACP reductase
protein sequence was compared for similarity with
those of plants and bacteria using ClustalW. A triad of
Ser217, Tyr230 and Lys234 residues involved in
catalysis and substrate binding was revealed by
sequence alignments, characteristic of the SDR family.
The tyrosine and lysine residues are involved in actual
catalysis, whereas serine participates in substrate
binding and alignment (Price et al., 2001; Li et al.,
2009). To examine the relationships among different
sources of
KR
genes, the neighbour-joining method
was used to construct the phylogenetic tree (Figure 2).
As shown in the phylogenetic tree, all of the
KR
genes
fell into two subfamilies: the bacteria subfamily and
the cyanobacteria/green algae/mosses/higher plants
subfamily. The
AhKR
gene from peanut clustered with
those from higher plants, and the genes from
cyanobacteria may be the origin of genes from higher
plants, mosses and eukaryotic algae.
1.1.2 Cloning and phylogenetic analysis of
AhHD
gene
As shown in Figure 3A, multiple sequence alignment
indicated that AhHD protein contained a histidine at
amino acid 116 and a glutamine at amino acid 130,
which were in positions that were conserved in the
FabZ of
Escherichia coli
(Cronan et al., 1988). The
location of these residues corresponded to the
catalytically active site of the enzymes in
Plasmodium
faliciparum
and
Escherichia coli
. (Cronan et al., 1988;
Heath and Rock, 1996; Sharma et al., 2003).
Specifically, there were distinct differences in the
active site residues, an Asp in FabA and a Glu in FabZ
(Figure 3B). In addition, similar to PaFabZ (Kimber et
al., 2004), five key motifs, LPHRFPFLLVD, GHFP,
PGVL, EAMAQ, and AGD, were also conserved in
AhHD
. Phylogenetic analysis suggested that
fabZ
and
fabA
genes fell into two separate subfamilies (Figure 4).
The
AhHD
gene from peanut clustered together with
genes from higher plants, and were separated from
those of green algae. The
FabZ
genes from
cyanobacteria may be the origin of genes from higher
plants and eukaryotic algae.
1.1.3 Cloning and phylogenetic analysis of
AhENR
gene
FabV, FabI and FabL were distant members of the
SDR superfamily, although FabV aligned only weakly
with FabI or FabL even when many gaps were
allowed (Figure 5A). FabI and FabL contained a
Tyr-X
6
-Lys active site motif. In FabV other variation
was seen, a Tyr-X
8
-Lys motif (Figure 5A). The
Figure 2 Neighbor-joining tree based on the deduced amino
acid sequences of
KR
homologs
Note: Sequences were shown by their accession numbers (locus
tags) and strain names; Bootstrap values from neighbor-joining
analyses were listed to the left of each node, with values more
than 50 were shown