Page 5 - Genomics and Applied Biology

Genomics and Applied Biology, 2010, Vol.1 No.2
http://gab.sophiapublisher.com
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performed in polyketide biosynthesis (Fisher et al.,
2000). Two isoforms of the enzyme, one NADPH-
linked and the other NADH-linked, have been
reported (Caughey and Kekwick, 1982). The function
of the NADH-linked enzyme is unknown. It is the
NADPH-linked β-ketoacyl-ACP reductase that
functions in fatty acid biosynthesis (Slabas et al.,
1992). KR is a member of the Short-chain Dehydro-
genase/Reductase (SDR) superfamily (Price et al.,
2001), and thereby displays the amino acid signature
of this family, Ser-X12-Tyr-X3-Lys (Persson et al.,
2003).
The third step in the elongation cycle is catalyzed by
β-hydroxyacyl-ACP dehydratase (HD). There are two
isoforms. FabZ, which catalyzes the dehydration of
(3R)-hydroxyacyl-ACP to trans-2- acyl-ACP, is a
universally expressed component of the type II system.
FabA, the second isoform, as has more limited
distribution in nature and, in addition to dehydration,
also carries out the isomerization of trans-2 - to
cis- 3-decenoyl-ACP as an essential step in
unsaturated fatty acid biosynthesis (Heath and Rock,
1996; Kimber et al., 2004). The catalytic site is
hydrophobic except for a histidine and a glutamine
(aspartic acid), which together are proposed to
catalyze the reactions (Leesong et al., 1996).
The enoyl-ACP reductase (ENR) catalyses the last
step in the fatty acid elongation cycle (Marrakchi et al.,
2003). Four types of ENR, namely, FabI, fabK, FabL
and FabV, have been reported (Bergler et al., 1994;
Heath et al., 2000; Marrakchi et al., 2003;
Massengo-Tiassé and Cronan, 2007). Most ENRs
(FabI, FabL and FabV) are distant members of the
SDR superfamily. Other classes of ENRs are the TIM
barrel flavin containing enzyme, FabK, the ENRs
found in mitochondria and the ENR domains of the
mammalian and fungal megasynthases. Both NADH
and NADPH are used as the hydride source in the
reactions of SDR-type ENRs (Massengo-Tiassé and
Cronan, 2007). FabI and FabL are atypical in that the
key residues are a diad consisting of a Tyr-X6-Lys
motif in contrast with the Tyr-X3-Lys in prototypical
SDRs (Baker, 1995; Parikh et al., 1999). FabV is
considerably larger than either FabI or FabL, and
contains a Tyr-X8-Lys active site motif (Massengo-
Tiassé and Cronan, 2007).
Cultivated peanuts (Arachis hypogaea L.) are
important oilseed crops worldwide, because these
allotetraploids (2n=4x=40) typically contain 50% oil
in the seed. The flavor and quality of either the seed or
the oil is immensely dependent on the fatty acid
composition of the extracted oil (Andersen and Gorbet,
2002; Jung et al., 2000b; Lopez et al., 2000; Yu et al.,
2008). It would be of great importance to study the
fatty acid biosynthesis pathway for improving oil
quality and increasing oil content of peanut. In this
study, we isolated and characterized cDNAs
containing the complete coding region of AhKR,
AhHD and AhENR genes, and analyzed their
expression in different organs and at different
development stages of seeds. The identification of
these novel genes in this study will be helpful for the
reconstruction of the pathways involved in fatty acid
biosynthesis and the metabolic engineering of fatty
acid synthesis in peanut.
1 Results
1.1 Isolation and analysis of AhKR, AhHD and
AhENR genes
Three full-length genes namely β-ketoacyl-ACP
reductase (KR), β -hydroxyacyl-ACP dehydrase (HD),
and enoyl-ACP reductase (ENR) were isolated from a
peanut seedling full-length cDNA library (unpublished
data). 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 (Table 1). Prediction of
subcellular location by two programs, TargetP Server
and Predotar, suggested that these three proteins
probably located in chloroplast. The first 73, 43 or 70
amino acids at the N-terminal end of the deduced
protein for AhKR, AhHD or AhENR had a high
proportion of hydroxylated and small, hydrophobic
amino acids, which was typical of chloroplast transit
peptide. A Blast search revealed that the primary
structure of AhKR , AhHD, AhENR shared high
sequence identity of 86.3%, 81.2% and 87.2% to the
corresponding ones in Glycine max, respectively. The
deduced amino acid sequences of AhKR, AhHD,
AhENR showed 37.3%, 30.2%, and 20.9% identity
with EcFabG, EcFabZ, and EcFabI, respectively.