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Legume Genomics and Genetics (online), 2010, Vol. 1, No.3, 11-17
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12
1994; Slabaugh et al., 1995; Chen and Post-Beitten-
miller, 1996). In addition, cDNAs of KAS
Ⅱ
have
also been isolated from many species including soy-
bean, rape, perilla and Arabidopsis (Hwang et al., 2000;
Carlsson et al., 2002; Aghoram et al., 2006). It has
been demonstrated that the altered expression levels of
KAS
Ⅱ
and KAS
Ⅲ
lead to change of oil content and
qualities in
A. thaliana
(Abbadi et al., 2000; Dehesh et
al., 2001; Pidkowich et al., 2007). KAS
Ⅰ
has been
purified from
Spinacia oleracea
leaves, and a gene
encoding KAS
Ⅰ
has also been characterized in bar-
ley (Shimakat et al., 1983; Kauppinen, 1992). How-
ever, the function and the regulation of KAS family
were still not well understood in higher plants.
Peanut is one of the four most important oil crops widely
grown in the world. It would be of great importance to
study the fatty acid biosynthesis pathway for impro-
ving oil quality and increasing oil content of peanut.
In this study, we isolated and characterized a cDNA
containing the complete coding region of KAS
Ⅰ
gene,
and analyzed its expression in different organs and at
different developmental stages of seeds.
1 Results
1.1 Molecular cloning of
AhKAS
Ⅰ
gene from peanut
One full-length
AhKAS
Ⅰ
cDNA clone was identified
from a peanut seedling full-length cDNA library (un-
published data) based on the amino acid similarity.
The
AhKAS
Ⅰ
gene is 1 912 bp in length containing a
1 413 bp ORF, starting with an initiating codon at 238
bp and ending with a stop codon at 1 650 bp (ace-
ssion number FJ768729). The predicted protein pro-
duct of
AhKAS
Ⅰ
comprises 470 amino acids with the
calculated molecular mass of 49.958 9 kD and a pI of
8.46. Prediction of subcellular location suggested that
AhKAS
Ⅰ
protein probably located in chloroplast. The
first 48 amino acids at the N-terminal end of the de-
duced protein have a high proportion of hydroxylated
and small, hydrophobic amino acids, typical of chloro-
plast transit peptide. We have tentatively identified the
transit peptide cleavage site at amino acid 49 based on
the ChloroP 1.1 Server. A Blast search revealed that
the primary structure of
AhKAS
Ⅰ
shared 90.2%, 84.8%,
84.5%, 81.3% identity with
KAS
Ⅰ
genes from
Gly-
cine max
,
Ricinus communis
,
Helianthus annuus
and
Arabidopsis thaliana
, respectively (Figure 1). In ad-
dition, using Rapid Amplification of cDNA Ends
(RACE) method, we have isolated a
KAS
Ⅱ
gene (ace-
ssion number FJ358425) containing a 1 521 bp com-
plete open reading frame (ORF) from peanut seedling,
named
AhKAS
Ⅱ
, which was presumed to be located
in chloroplast. The plastid-localized AhKAS
Ⅰ
and
AhKAS
Ⅱ
are 50.3% identical in this study, com-
pared with 38% in
E. coli
. AhKAS
Ⅰ
shared 28.8% and
38.5% identity with EcFabB and EcFabF, respectively,
whereas AhKAS
Ⅱ
shared 26.7% and 35.8% identity.
1.2 Sequence and phylogenetic analysis of
AhKAS
Ⅰ
gene
A subgroup of β-ketoacyl-ACP synthases, including
mitochondrial β-ketoacyl-ACP synthase, bacterial plus
plastid β-ketoacyl-ACP synthases
Ⅰ
and
Ⅱ
, and a
domain of human fatty acid synthase, have a Cys-
His-His triad and also a completely conserved Lys in
the active site, which are referred to as the CHH group
(von Wettstein-Knowles et al., 2006). Another group
of decarboxylating condensing enzymes called CHN
(N for asparagine), represented by KAS
Ⅲ
and certain
polyketide synthases (Qiu et al., 1999; Davies et al.,
2000; Scarsdale et al., 2001) with an active site com-
posed of a cysteine nucleophile, a histidine and an
asparagine. Four conserved residues Cys221
–
His361
–
Lys392
–
His397 of AhKAS
Ⅰ
were revealed by se-
quence alignments (Figure 1). The CHH active site
(Cys–His–His) was highly conserved in KAS
Ⅰ
and
KAS
Ⅱ
in higher plants and
E. coli
(Li et al., 2009).
To examine the relationships among different sources
of
KAS
Ⅰ
genes, the neighbour-joining method was
used to construct the phylogenetic trees (Figure 2). All
tree topologies are highly congruent. As shown in the
phylogenetic tree, all of the
AhKAS
Ⅰ
genes fell into
two subfamilies: the bacteria subfamily and the cyano-
bacteria/green algae/mosses/higher plants subfamily.
The
AhKAS
Ⅰ
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.
1.3 Heterologous expression of
AhKAS
Ⅰ
gene in
E.
coli
cells
The
AhKAS
Ⅰ
enzyme was overexpressed using the