Journal of Environmental Biology �January, 2009�
© Triveni Enterprises, Lucknow (India) J. Environ. Biol.
ISSN : 0254-8704 30(1), 93-98 (2009)
http : //www.jeb.co.in [email protected]
Cloning and expression analysis of an aldehyde oxidase gene in
Arachis hygogaea L.
Lixia Yang, Jianhua Liang, Haihang Li and Ling Li*
Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Science,
South China Normal University, Guangzhou - 510631, P.R. China
(Received: October 16, 2007; Revised received: February 10, 2008; Accepted: March 05, 2008)
Abstract: Aldehyde oxidase (AO) plays important role in plant hormone biosynthetic pathways, such as abscisic acid (ABA) and indole-
3-acetic acid (IAA). The enzyme catalyzes the last step of the pathways. In this study, a full-length cDNA encoding an aldyhyde oxidase
was cloned and sequenced from leaves of peanut by RT-PCR, RACE-PCR and genomic DNA walking methods. The full-length cDNA,
designated as Arachis hygogaea L. aldehyde oxidase1 (AhAO1), consists of an open reading frame of 4131bp, a 326 bp 5′ untranslated
region and a 128 bp 3′ untranslated region including a poly (A) tail of 21 nucleotides. The gene encodes a polypeptide of 1377 amino acids
with a calculated molecular weight of 150 kDa and an isoelectric point (pI) of 6.99. Analysis of amino acid sequence of AhAO1 shows that
it had 61%, 59% and 55% identity with the AOs from tomato, Arabidopsis and maize, respectively. The peanut AO polypeptide contains
consensus sequences for iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. Semi-quantitative RT-PCR analysis
showed that AhAO1 expression was higher in leaves than in roots of peanut.
Key words: Cloning, Expression, Aldehyde oxidase, Arachis hypogaea L.
PDF of full length paper is available with author (*[email protected])
Introduction
Aldehyde oxidase (AO) catalyzes the oxidation of a variety
of aldehydes and N-containing heterocyclic compounds in the
presence of O2 or certain redox dyes (Hall and Krenitsky, 1986).
AOs belong to the family of molybdenum-containing protein like
xanthine dehydrogenase (XD) and sulfite oxidase (Hille and Massey,
1985). These enzymes possess a short oxidoreductive chain
characterized by four oxidation centers, two iron-sulfur clusters, a
flavine cofactor, and a molybdopterin cofactor (MoCo) (Barber et al.,
1982). AO has been characterized extensively in animals and
microorganisms, where it has been implicated in the detoxification of
environmental pollutants and xenobiotics (Bauer and Howard, 1991).
In higher plants, much of the interest in AO stems from its involvement
in the biosynthesis of abscisic acid (Leydecker et al., 1995) and
indole acetic acid ( Koshiba and Matsuyama, 1993).
ABA is a key regulator in seed development, root growth,
stomatal aperture in higher plants (Zeevaart and Creelman, 1988).
It is also involved in adaptation of plants to various stresses (Zeevaart
and Creelman, 1988), possible the same as other plant growth
regulators (Kim et al., 2006; Chandra et al., 2007; Tilki, 2008). The
regulation of these processes by ABA is partially due to de novo
synthesis of ABA. Endogenous ABA increases upon stress conditions,
most notably under drought and salinity (Munns and Cramer, 1996).
Application of exogenous ABA has similar effects to plants as stress
treatments and increases plant tolerance to environmental stresses
(Fedina et al., 1994; Welbaum et al., 1997). Elucidation of ABA
biosynthetic pathway, related genes and enzymes are important for
the control and regulation of ABA-mediating plant responses.
Two enzymes, involved in the early steps of ABA synthesis in
chloroplasts, have attracted much attention. Zeaxanthin epoxidase
(ZEP) converts zeaxanthin to violaxanthin by a two-step epoxidation
reaction (Marin et al., 1996), and 9-cis-epoxycarotenoid dioxygenase
(NCED) catalyses the oxidative cleavage of 9-cis-xanthophylls to
xanthoxin (Cutler and Krochko, 1999). Over-expression of ZEP in
transgenic tobacco resulted in enhanced seed dormancy (Frey et
al., 1999). Over-expression of the AtNCED3 in Arabidopsis resulted
in increased ABA levels (Iuchi et al., 2001). It was suggested that
NCED catalyses the critical step in the regulation of ABA biosynthesis
(Qin and Zeevaart, 1999; Iuchi et al., 2000).
AO catalyses the final step of ABA biosynthesis. Plant AO
has only been purified from coleoptiles of maize (Koshiba et al.,
1996). Based on the amino acid sequence of the purified AO, two
cDNAs were cloned from maize (Sekimoto et al., 1997). Four cDNAs
(Arabidopsis aldehyde oxidase, AAO1, 2, 3 and 4) from Arabidopsis
(Sekimoto et al., 1998), three cDNAs from pea (Zdunek-Zastocka,
2008) three putative AO genes and two AO pseudogenes from
tomato (Ori et al., 1997, Min et al., 2000) have been reported. Plant
AO is a protein family with similar amino acid sequence, but different
in electrophoretic mobility, substrate specificity, and subunit
composition. An AO polymorphism has been observed in potato
bubers (Rathe, 1974), maize coleoptiles (Koshiba et al., 1996),
Arabidopsis (Akaba et al., 1999), ryegrass (Sagi et al., 1998),
barley (Omarov et al., 1999) and in pea (Zdunek and Lips, 2001).
Peanut (Arachis hypogaea L.) is an important crop. Drought
is one of the major abiotic stresses that limit its growth and production.
The role of AO in the ABA biosynthesis pathways was only well
Journal of Environmental Biology �January, 2009�
Yang et al.
investigated in Arabidopsis. In the present paper, we describe the
molecular cloning of a full-length Arachis hygogaea L. aldehyde
oxidase1 (AhAO1) in attempt to clone an AO gene involved in
ABA biosynthesis, by reverse transcription-polymerase chain
reaction (RT-PCR), rapid amplification of cDNA ends (RACE)
PCR and genomic walking methods. Its expression in peanut
leaves and roots is tested by semi-quantitative RT-PCR to
understand the function and role for peanut during growth and
development.
Materials and Methods
Plant material: Seeds of peanut (Arachis hypogaea L. cv. YueYou
7) were planted in culture medium (vermiculite: perlite: soil, 1: 1: 1)
and grown in a growth chamber with 16 hr of light (200 µmolm-2s-1)
at 26oC and 8 hr of darkness at 22oC. Plants were irrigated daily
with half-strength Murashige and Skoog nutrient solution (Murashige
and Skoog, 1962).
RNA extraction: Total RNA was extracted from peanut leaves (10
days after planting) using a modified phenol–chloroform method
(Vanlerberghe and McIntosh, 1994). 80 mg tissue was ground in
liquid nitrogen to a fine powder using a mortar and pestle. The
ground tissue was transferred to a tube containing 800 µl extraction
buffer (4 mol l-1 guanidine isothiocyanate, 25 mmol l-1 sodium citrate,
200 mmol l-1 sodium acetate(pH4.0), 0.5% (w/v) lauroyl sarcosine,
and 1%(v/v) b-mercaptoethanol). The homogenate was extracted
with phenol/chloroform (1: 1, v/v) on ice for 30 min. After centrifugation
at 12 000 g at 4oC for 20 min, the aqueous phase was transferred
to a fresh RNase-free tube, mixed with equal volume of isopropyl
alcohol and kept on ice for 1 hr. RNA was pelleted and washed
twice with 75% (w/w) cold ethanol. The pellet was resuspended in
diethypyrocarbonate (DEPC) water.
Each RNA sample was quantified with a spectrophotometeric
method (Sambrook et al., 1989). The A260/A280 values of the
RNA samples were all greater than 1.8.
RT-PCR amplification of the conserved region: cDNA was
synthesized from total RNA (2 µg) using SuperScriptTM III RNase
H- Reverse Transcriptase (Invitrogen) and an oligo dT18
primer.
For amplification of a specific fragment of AO gene from peanut, two
primers (AOF3: 5′ -GGI GGI GG/CI TTT/C GGI GGI AA-3′ and
AOR3: 5′ -CAT T/CTG T/CTT IAC T/CTT IGT CCA-3′ ) were
synthesized based on the conserved regions of reported AO genes
(Sekimoto et al., 1998). The fragment between the two conserved
domains was amplified by PCR using the two primers (PCR
conditions: 94oC for 1 min, 52oC for 1 min and 72oC for 2 min, 40
cycles). PCR amplified fragment was cloned into pMD 18-T vector
and confirmed by sequencing.
RACE-PCR amplification for 3′′′′ -end: The 3′ -end of the gene was
obtained using the GeneRacer kit (Invitrogen) according to the
manufacture’s instructions with a gene-specific primer (GSP) of 3AO-
outer (5′ -GGA TTA TCT CGT CTA CCA GTT GTG-3′ ) and a nested
primer of 3AO-inner (5′ -CTC GGA TGG ATC GGT TGT TGT TG-3′ ).
PCR amplification 5′′′′ -end: The first 5′ extended cDNA (RACE-
PCR1) was obtained by amplification with a forward degenerate
primer (AOF, 5′ -CAA/G TGC/T GGA/G/C/T TTC/T TGC/T ACA/
G/C/T CCI GGA/G/C/T ATG-3′ ) and a specific primer (yang 1, 5′-CAC TGT AAG TAA TCT TCA TGG G-3′ ). AOF corresponded to
the conserved peptide QCGFCTPGM in all known plant AOs was
designed. The second 5′ extended cDNA (RACE-PCR2) was
obtained with the forward degenerate primer (AOF4, 5′ -GAG/A
GGA/G/C/T GG A/G/C/T TGC/T GGA/G/C/T GCI TGC/T-3′ ) and
a specific primer (yang 2, 5′ -TTT GGA GAA TCC GGA AGG CGG
ATC C-3′).
Genomic walking for the 5′′′′ -end: The 5′ end of the sequence
was obtained from genomic DNA using Genome Walker kit
(Clontech). The genomic DNA was isolated according to DNA
Extraction kit (Tiangen, China). The DNA was digested with restriction
enzyme (Dra I) and ligated to short adaptor sequences. The first
PCR with primers AP1 (5′ -GTA ATA CGA CTC ACT ATA GGG C-3′)and AOP1 (5′ -CAC CTT CAA GTT CAT TGA AAA CCC CAT
CAG-3′ ) was performed as follows: 94oC, 2 min, 7 cycles of 30 s at
94oC, 3 min at 62oC, followed by 33 cycles of 30 s at 94oC, 3 min at
58oC, and another period of 58oC for 10 min after the final cycle.
The 1 µl genome-walking library was used as template in the first
PCR. The nested PCR with primers AP2 (5′ -ACT ATA GGG CAC
GCG TGG T-3′ ) and AOP2 (5′ -ACC AGA ACA AGG TCC TTT
ATC CTT ATG CCT-3′ ) was performed in the same conditions as
in the first PCR and 1µl product of the first PCR was used as
Table - 1: Identity (% ) among aldehyde oxidases and xanthine dehydroginase
AhAO1 AAO1 AAO2 AAO3 AAO4 TAO1 TAO2 TAO3 ZmAO1 ZmAO2
AAO1 58.7
AAO2 60.9 65.5
AAO3 60.1 60.4 61.0
AAO4 59.4 60.4 60.4 73.3
TAO1 59.7 57.2 58.4 58.5 58.1
TAO2 60.2 58.2 60.1 59.2 59.5 82.3
TAO3 60.9 58.8 60.8 59.5 59.8 83.2 85.0
ZmAO1 54.6 55.2 55.5 55.2 54.0 54.9 57.6 57.3
ZmAO2 54.5 55.4 56.0 54.8 54.2 55.7 56.6 56.9 83.8
AXD1 29.2 28.3 28.7 28.4 28.0 28.9 28.5 29.0 28.6 29.6
94
Journal of Environmental Biology �January, 2009�
Fig. 1: Structural organization of AhAO1 cDNA. The shaded and white boxes indicated the protein coding regions and the 5′ - or 3′ - untranslated regions,
respectively. The thin lines shown in the lower part of figure represent reverse transcription-polymerase chain reaction, RACE-polymerase chain reaction
or genome walking products
Cloning and expression of an aldehyde oxidase gene
template. For amplification of the 5′ cDNA sequence of AhAO1, PCR
was performed with the primers yang 3 (5′ -ATG GAA GTG AAG
AAC AAC ATC-3′) and yang 4 (5′ -TCT TCC GCC TCT GCT
GCT GTC-3′).
Cloning, DNA sequencing, and sequence analysis: All the
amplification products were cloned into a TA cloning vector PMD
18-T (TaKaRa), and sequenced in a 3730 DNA analyzer by
Invitrogen (Guangzhou, China). Multiple alignment of the amino
acid sequences was carried out using the Clustal W program in
BioEdit software. For sequence identity and phylogenetic analysis,
we used megalign software (DNASTAR).
Semi-quantitative RT-PCR analysis: To estimate AhAO1
expression in peanut organs, semi-quantitative RT-PCR analysis
was performed. Extraction of total RNA is the same as described
above. To avoid amplification of peanut AhAO1 genomic DNA in
semi-quantitative RT-PCR analysis, the isolated RNA was treated
with RNase-free DNase I (TaKaRa) at 37oC for 15 min. The reaction
mixture was extracted with phenol: chloroform: isoamyl alcohol (25:
24: 1, v/v) followed by ethanol precipitation. The RNA samples
were also visualized by ethidium bromide (EtBr) staining after
agarose gel electrophoresis. The first-strand cDNA was synthesized
from 20 ng of total RNA using reverse transcriptase (superscriptTM
III, Invitrogen). For gene-specific primers, yang 5 (5′ -AGT TGC
AGA AGG AAA TGG GAT C-3′) and yang 6 (5′ -ATC CGT TCC ATC
TTT GTT GCT C-3′) were used to amplify a 548 bp fragment. As an
internal loading control, primers 18S-F (5′ -ATT CCT AGT AAG
CGC GAG TCA TCA G-3′) and 18S-R (5′ -CAA TGA TCC TTC
CGC AGG TTC AC-3′) specific to a peanut 18S rRNA gene (Bhagwat
et al., 2001) were used to amplify a fragment of 226 bp. The PCR
conditions for AhAO1 was 94oC for 5 min followed by 28 cycles of
94oC for 30 s, 52oC for 45 s, and 72oC for 1 min. The PCR reaction
for 18S was performed at 94oC for 5 min followed by 25 cycles of
94oC for 30 s, 60oC for 45 s, and 72oC for 1 min. PCR products were
visualized by EtBr staining after agrarose gel electrophoresis.
Results and Discussion
Cloning and characterization of AhAO1: Fig.1 illustrated the
PCR amplification strategy for the cloning of the peanut AO gene.
Amino acid sequence analysis of AOs of Arabidopsis, maize and
tomato reveals that two domains, GGGFGGK and WTKVKQM, are
highly conserved in these genes. They were used to design primers,
AOF3 and AOR3, for PCR amplification of the peanut AO gene. A
759 bp fragment was amplified from peanut leaf cDNA and was
confirms to be part of the AO gene by DNA sequencing. The 3′ end
was obtained by RACE-PCR. To obtain the 5′ end, two PCR
reactions were tried, with two degenerate primers and two gene
specific primers. However, we failed to obtain the full-length sequence
from cDNA using the Gene Racer kit and SMART RACE cDNA
Amplification kit (Clontech). A genomic walking method was employed
to clone the 5′ end sequence of AhAO1 from genomic DNA. By
using two gene specific primers (yang 3 and yang 4), we
successfully cloned the 5′ end sequence and got a full-length
sequence of the AhAO1. All primers used, except for yang AOP1,
AOP2, AP1 and AP2, are indicated in Fig. 2. The full sequence in
amino acid of the gene is shown in Fig. 2.
The full-length of AhAO1 cDNA is 4585 bp and contains a
4131 bp open reading frame (ORF), a 5′ untranslated region (5′UTR) of 326 bp and a 3′ UTR of 128 bp. AhAO1 encodes a
polypeptide of 1377 amino acids with a calculated molecular weight
of 150 kDa and an isoelectric point (pI) of 6.99.
Analysis of the deduced amino acid sequences of AhAO1:
Sequence analysis shows that AhAO1 has 61%, 59% and 55%
identity in amino acid sequence with the AOs from tomato, Arabidopsis
and maize, respectively (Table 1). Alignment of amino acid sequences
of AhAO1 with 9 AOs registered in Genebank are shown in Fig. 2
and their phylogenetic relationship was shown in Fig. 3. Predicted
protein for AhAO1 has a high level of similarity to plant AOs and can
be aligned along their entire protein sequences (Fig. 2). As in
other plant and animal AOs (Koshiba et al., 1996), three special
domains for binding cofactor or prosthetic groups could be identified
in the peanut AhAO1, two N-terminal iron-sulfur centers and a
molybdopterin binding domain. One of the two iron-sulfur centers
with consensus sequences (CX4CX
2CXnC) is located between
aa 54-85. The second putative iron-sulfur center is between aa
123-178. Each of the two centers has four cysteines, which may
contribute to the structure and functions of the iron-sulfur center.
95
Journal of Environmental Biology �January, 2009�
Yang et al.
Fig
. 2: A
lignm
ent o
f ded
uced
am
ino
acid
seq
uenc
e of
the
pean
ut a
ldeh
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and
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e ac
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Edi
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ansl
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n nu
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re: A
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m p
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AO
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), A
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AO
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6602
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O4
(AB
0372
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rom
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s; T
AO
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F25
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AO
2 (A
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O3
(AF
258
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fro
m to
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mA
O1
(D88
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) an
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(D88
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he th
in li
nes
repr
esen
ted
the
prim
ers
and
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tive
activ
e m
otifs
. a, b
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n o
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n-su
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Mo
Co
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. h: p
ropo
sed
pept
ide
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as
subs
trat
e bi
ndin
g
96
Journal of Environmental Biology �January, 2009�
The consensus motifs of the two iron-sulfur centers were also
found in the corresponding regions of AOs from tomato (Ori et al.,
1997), maize (Sekimoto et al., 1997) and Arabidopsis (Sekimoto
et al., 1998).
Although the presence of flavin adenine dinucleotide (FAD)
had been demonstrated in purified AO from coleoptiles of maize
(Koshiba et al., 1996), the FAD-binding domain described by Correll
et al. (1993) cannot be identified in AhAO1. The same results were
reported in Arabidopsis AOs (Sekimoto et al., 1998).
The C-terminal domain participates for the MoCo and
substrate binding (Sekimoto et al., 1998) were also identified in the
AhAO1. Five motifs for MoCo binding sites, MoCo 1 (aa 839-845),
MoCo 2 (952-961), MoCo 3 (1080-1082), MoCo 4 (1124-1129)
and MoCo 5 (1302-1307), are found in the sequence of AhAO1 (Fig.
2 c, d, e, f and g). A substrate binding site in plant AOs was proposed
by Sekimoto et al. (1998). A domain (IMPARLVSG, aa 919-927) with
similar sequences to Arabidopsis AAO3 (IMPRNIMGP, aa 878-886)
and tomato TAO1 (IIPSYLMNT, aa 906-914) was considered to be
the substrate binding sequence in AhAO1 (Fig. 2, h).
Gene expression analysis of AhAO1 in peanut roots and
leaves: Semi-quantitative RT-PCR analysis showed that AhAO1
was expressed both in roots and leaves (Fig. 4). The expression
level of AhAO1 is slightly higher in leaves than in roots.
Four AOs isoforms (AOα, AOβ, AOγ, AOδ) were identified
in Arabidopsis thaliana and are all in dimmers. AOα, AOβ and AOδare homodimers of AAO1, AAO2 and AAO3 products, respectively,
while AOβ is a heterodimer of AAO1 and AAO2 products (Akaba et
al., 1999; Seo et al., 2000). Studies on Arabidopsis mutant superroot1
(sur1), an auxin-overproducing mutant, have shown that AAO1
has a higher substrate preference for indole-3-aldehyde. AAO1
activity is significantly higher in sur1 mutant seedlings than in wild
type (Seo et al., 1998). AO isoforms can efficiently oxidize abscisic
aldehyde to abscisic acid in Arabidopsis rosette leaves (Seo et al.,
2000), in roots of barley (Omarov et al., 2003) and in pea (Zdunek-
Zastocka et al., 2004). Drought-induced increase of abscisic
aldehyde oxidase activity is observed in leaves of Arabidopsis by
Bittner et al. (2001). When the Arabidopsis rosette leaves are
detached and exposed to dehydration, the expression of AAO3
mRNA increases rapidly, but AOδ activity is unaffected (Seo et al.,
2000). An ABA-deficient Arabidopsis mutant, aao3, was maps at the
AAO3 locus. A wilty phenotype of aao3 mutant could be restored to
wild type when AAO3 gene was introduced into it (Seo et al., 2000).
It was suggested that AAO1 may be responsible for IAA biosynthesis,
while AAO3 for ABA biosynthesis in Arabidopsis. The gene AhAO1
we cloned from peanut has 58.7, 60.9, 60.1 and 59.4% identities,
in amino acid sequence, with Arabidopsis AAO1, AAO2, AAO3 and
AAO4, respectively (Table 1). Although it showed slightly higher
identity with AAO2 and AAO3, compared to AAO1 and AAO4, we
are unable to identify the type and function of AhAO1 at this point.
Local sequence analysis for all possible function motifs could not
distinguish them, too. AhAO1 belongs to a multigene family coding
for AOs which act on variable substrates. Production of transgenic
Arabidopsis with AhAO1 is progressing and will be useful for
understanding the function of the gene of peanut.
Acknowledgments
This research was supported by a grant (03098) of the
key project on scientific and technological research from Ministry of
Education of P. R. China and the national natural sciences foundation
of China (grant nos. 30771297 to L. Li).
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