Basic HTML Version
Legume Genomics and Genetics (online), 2010, Vol. 1, No.3, 11-17
http://lgg.sophiapublisher.com
Research Article Open Access
Isolation and Expression Analysis of a β-Ketoacyl-Acyl Carrier Protein Syn-
thase
Ⅰ
gene from
Arachis hypogaea
L.
Xiaoyuan Chi , Mingna Chen , Qingli Yang , Ya’nan He , Lijuan Pan , Yuan Gao , Shanlin Yu
Shandong Peanut Research Institute, Qingdao, 266100
Corresponding author email: yshanlin1956@163.com;
Authors
Legume Genomics and Genetics 2010, Vol.1 No.3 DOI:10.5376/lgg.2010.01.0003
Received: 01 Jul., 2010
Accepted: 10 Sep., 2010
Published: 10 Oct., 2010
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article as:
Chi et al., 2010, Isolation and Expression Analysis of a β-Ketoacyl-Acyl Carrier Protein Synthase
Ⅰ
gene from
Arachis hypogaea
L., Legume Genomics and
Genetics (online), Vol.1 No.3 pp.11-17 (DOI:10.5376/lgg.2010.01.0003)
Abstract
β-ketoacyl-acyl carrier protein synthase (KAS) plays a pivotal role in
de novo
fatty acid biosynthesis, joining short
carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction in plants and bacteria. A putative
KAS
Ⅰ
cDNAs was isolated from peanut by searching a peanut seedling full-length cDNA library. The AhKAS
Ⅰ
contains four strictly
conserved residues Cys221
–
His361
–
Lys392
–
His397 in the active site, which is an important characteristic of KAS Is in plants
and bacteria. The
AhKAS
Ⅰ
gene containing 1 912 bp cDNA sequence with a 1 413 bp ORF, encodes 470 amino acids. The predicted
amino acid sequences translated from the
AhKAS
Ⅰ
gene shared 90.2% sequence identity to the corresponding protein in
Glycine
max
. The cDNA was cloned into plasmid pET-28a and expressed in
Escherichia coli
BL21. Quantitative real-time PCR analysis
suggested
AhKAS
Ⅰ
was expressed with higher levels in leaf and seed than those in other tissues. In addition,
AhKAS
Ⅰ
RNA was
found in high abundance at 45 and 65 DAP (days after pegging) during seed development. This work may serve as a foundation for
further studies on the mechanisms regulating the expression of
KAS
Ⅰ
gene and provide candidate genes for modifying oil quality via
transgenic plants.
Keywords
Fatty acid biosynthesis; β-ketoacyl-ACP synthase
Ⅰ
; Expression analysis; Peanut (
Arachis hypogaea
L.)
Background
The
de novo
fatty acid biosynthesis is a very important
primary metabolic pathway, producing palmitic acid
(16:0) and stearic acid (18:0) that serve as the precur-
sors for other fatty acids with different lengths and
saturation levels (Ohkrigge and Browse, 1995). Fatty
acid biosynthesis in higher plants is mainly catalyzed
by a suit of enzymes in plastids and the two-carbon
elongation reactions are catalyzed by β-ketoacyl-acyl
carrier protein (ACP) synthase (KAS, EC 2.3.1.41)
family. KAS
Ⅲ
initiates the fatty acid synthesis to form
4:0-ACP in plants by catalyzing the condensing reac-
tion of acetyl-CoA and malonyl-ACP. KAS
Ⅰ
catalyzes
the condensations of acetate unites to a growing acyl-
ACP leading to the synthesis of palmitoyl-ACP (16:0
-
ACP). KAS
Ⅱ
is responsible for the elongation of
16:0
-
ACP to 18:0
-
ACP (Shimakata and Stumpf, 1982).
A fourth KAS enzyme (KAS
Ⅳ
) located in plastid
specific for the synthesis of medium-chain acids has
also been reported (Siggaard-Andersen et al., 1994;
Dehesh et al., 1998). In addition, the mitochondrial
condensing enzyme (mtKAS), which catalyzes all the
condensation reactions in mitochondrial fatty acid
synthesis, has been characterised from
A. thaliana
(Yasuno et al., 2004) and the crystal structure of this
enzyme has been determined (Olsen et al., 2004).
11
By now, several species of β-ketoacyl-ACP synthases
in plants and bacteria have been identified, distinct in
amino acid sequence, chain length specificity for their
substrates and sensitivity to cerulenin, an inhibitor of
condensing enzymes (Vance et al., 1972; Kauppinen et
al., 1988). The KAS family (fabB, fabF and fabH)
have been most extensively studied in
E. coli
and their
crystal structures have been determined (White et al.,
2005). In contrast to the well-studied
E. coli
KAS fa-
mily, plant KAS family is largely uncharacterized
except in
A. thaliana
(Li et al., 2009). KAS
Ⅲ
was
first purified to homogeneity from spinach (Clough et
al., 1992) and its cDNAs have been cloned from se-
veral plant species (Tai and Jaworski, 1993; Tai et al.,