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Genomics and Applied Biology, 2010, Vol.1 No.2
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Research Article Open Access
Cloning and Expression Analysis of β-ketoacyl-ACP Reductase, β-hydroxyacyl-
ACPDehydrase and Enoyl-ACPReductase from
Arachis hypogaea
L.
Xiaoyuan Chi Qingli Yang Lijuan Pa Yanan He Mingna Chen Yuan Gao Shanlin Yu
Shandong Peanut Research Institute, Qingdao, 266100, P.R. China
Corresponding author, yshanlin1956@163.com;
Authors
Genomics and Applied Biology 2010, Vol. 1 No. 2 doi: 10.5376/gab.2010.01.0002
Received: Aug. 15, 2010
Accepted: Sep. 19, 2010
Published: Sep. 28, 2010
This is an Open Access article published 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:
Chi et al., 2010, Cloning and Expression Analysis of β
-
ketoacyl-ACP Reductase, β
-
hydroxyacyl-ACP Dehydrase and Enoyl-ACP Reductase from
Arachis
hypogaea
L., Genomics and Applied Biology, 2010, Vol. 1 No. 2 (doi: 10.5376/gab.2010.01.0002)
Abstract
Fatty acid biosynthesis is catalysed in most bacteria and plants by a group of highly conserved proteins known as the
Type II fatty acid synthase (FAS) system. In this study, genes for β
-
ketoacyl-ACP reductase (KR), β
-
hydroxyacyl-ACP dehydrase
(HD), and enoyl-ACP reductase (ENR) of Type II FAS have been cloned from peanut (
Arachis hypogaea
L.). The results showed that
the ORF of the three genes were 972 bp, 651 bp and 1 170 bp in length, encoding 323, 216 and 389 amino acids, respectively. The
predicted amino acid sequences of
AhKR, AhHD
,
AhENR
shared high sequence identity of 86.3%, 81.2% and 87.2% to the
corresponding ones in
Glycine max
, respectively. Further investigation of quantitative real-time RT-PCR analysis suggested that
AhKR, AhHD
and
AhENR
were expressed with higher levels in leaf and seed than those in root and stem tissues. Moreover,
AhKR
and
AhENR
genes reached a maximum expression level at 25 DAP (days after pegging) and showed a downward trend thereafter.
In
contrast,
AhHD
gene expressed in an irregular course during seed development. Overall, the information generated by this study will
facilitate the manipulation of the quality of oils produced in seeds of oil crops.
Keywords
Peanut (
Arachis hypogaea
L.); Fatty acid biosynthesis; β
-
ketoacyl-ACP reductase (KR); β
-
hydroxyacyl-ACP
dehydrase (HD); Enoyl-ACP reductase (ENR); Expression analysis
Background
Fatty acid synthesis is essential for the formation of
membranes and hence for the viability of all
organisms (Massengo-Tiassé and Cronan, 2007). A
complex enzyme system, fatty acid synthetase (FAS),
is used throughout nature for the
de novo
biosynthesis
of fatty acids from acetyl-CoA and malonyl-CoA
(Fisher et al., 2000). In general, FAS is organized into
two di
ff
erent systems (FAS I and FAS II) based on the
architecture of the enzymes involved. In FAS I system,
the involved synthases found in fungi and mammals
are large multifunctional enzymes with multiple
domains that catalyze various reactions of FAS (Smith
et al., 2003; Schweizer and Hofmann, 2004). In
contrast, the FAS of bacteria, chloroplasts, apicoplasts
and mitochondria belongs to the FAS II system, where
the acyl chain covalently attached to the acyl carrier
protein (ACP) is elongated with four enzymes
catalyzing consecutively (Campbell and Cronan, 2001;
Olsen et al., 2004).
In bacteria and plants, the enzymes which catalyze the
four successive steps of the pathway are as follows:
β
-
ketoacyl-acyl carrier protein (ACP) synthetases I, II
and III; β
-
ketoacyl-acyl carrier protein (ACP)
reductase; β
-
hydroxyacyl-acyl carrier protein (ACP)
dehydratase; and enoyl-acyl carrier protein (ACP)
reductase (Rafferty et al., 1995). Chain termination is
mediated by the hydrolysis of acyl-ACP, catalyzed by
an acyl-ACP thioesterase. The fatty acyl chain lengthens
by two carbons each time it cycles through the
enzymatic reactions (Tai et al., 2007). Most fatty acids
reach a chain length of 16 or 18 carbons and then are
targeted for glycerolipid synthesis in the plastid or in
the endoplasmic reticulum (Browse and Somerville,
1991).
β
-
ketoacyl-ACP reductase (KR) catalyzes the
pyridine-nucleotide-dependent reduction of a β
-
oxoacyl
form of acyl carrier protein (ACP), the second step in
de novo
fatty acid biosynthesis and a reaction often