Download Characterization of a new stearoyl-acyl carrier protein desaturase

 Springer 2006
Biotechnology Letters (2006) 28: 657–662
DOI 10.1007/s10529-006-0034-3
Characterization of a new stearoyl-acyl carrier protein desaturase gene
from Jatropha curcas
Luo Tong1,2, Peng Shu-Ming1, Deng Wu-Yuan1, Ma Dan-Wei1, Xu Ying1, Xiao Meng1
& Chen Fang1,*
1
College of Life Sciences, Sichuan University, Chengdu 610064, China
Department of Bioengineering, Yibin University, Yibin 644007, China
*Author for correspondence (Fax: +86-028-8541-7281; E-mail: [email protected])
2
Received 25 November 2005; Revisions requested 16 December 2005; Revisions received 6 February 2006; Accepted 7 February 2006
Key words: fatty acid, Jatropha curcas, prokaryotic expression, stearoyl-acyl carrier protein desaturase
Abstract
A new full-length cDNA of stearoyl-acyl carrier protein desaturase was obtained by RT-PCR and RACE
techniques from developing seeds of Jatropha curcas. Sequence alignment showed that its deduced amino acid
sequence had high similarity with other stearoyl-acyl carrier protein desaturases. The gene was functionally
expressed in E. coli and the desaturating activity of recombinant protein was easily detected when assayed in
vitro with added spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small
gene family. Northern blot analysis revealed it was highly expressed in developing fruits of J. curcas.
Introduction
Stearoyl-acyl carrier protein (ACP) desaturase
(SAD, EC 1.14.99.6) is an important enzyme of
fatty acid biosynthesis in higher plants. Located
in plastid stroma, SAD catalyzes the desaturation of stearoyl-ACP to oleoyl-ACP. SAD plays
a key role in determining the ratio of saturated
fatty acids to unsaturated fatty acids in plants
(Lindqvist et al. 1996) and this ratio is closely
related to many functions of plants, especially to
acclimation to low-temperature (Kodama et al.
1995). Many genes coding for SAD have been
cloned from different plants and the structures
and functions of several SAD have been studied
(Davydov et al. 2005, Lindqvist et al. 1996).
Antisense expression of Brassica rapa SAD gene
in Brassica nupus led to dramatically increased
stearate levels (up to 40%) in the seeds of transgenic B. nupus (Knutzon et al. 1992). In the
reverse, when the SAD gene from Lupinus luteus
was over-expressed in tobacco, the transgenic
tobacco contained very high level of oleic acid
(up to 60%) in comparison with control plants
(Zaborowska et al. 2002). These imply that it
promises to modify the composition of plant fatty acids by manipulating SAD gene.
Jatropha curcas, which belongs to Euphorbiaceae and thrives in many parts of the tropics and
sub-tropics, may be used to reclaim land and
simultaneously produce feedstuff, soap, cosmetics, pesticide and anti-cancer medicine. Recently,
J. curcas received much attention for its high
content (5060%) of seed oil (Openshaw 2000).
The seed oil contains approximately 26% saturated fatty acids and 74% unsaturated fatty acids
of which 4249% is oleic acid. Thus, developing
seeds of J. curcas should have a high activity of
SAD.
In this study, we describe the molecular cloning and nucleotide sequence of the gene of SAD
from J. curcas (JSAD). The gene encodes a functional SAD as demonstrated by the expression of
active enzyme in E. coli.
658
Materials and methods
Materials
Young seeds, flowers, seedcases, leaves, stems and
roots of J. curcas were collected in summer from
Panzhihua city of Sichuan Province and instantly
frozen in liquid N2 and stored at )70 C until
use. Spinach ferredoxin, ferredoxin:NADP+ oxidoreductase, catalase and NADPH were from
Sigma. [9,10(n)-3H]Stearic acid and [9,10(n)-3H]
stearoyl-ACP were prepared following the procedure of Thompson et al. (1991).
RNA extraction and amplification of a full-length
cDNA
Total RNA was extracted from the developing
seeds of J. curcas according to the method of
Zhang et al. (2004). RT-PCR was performed as
the Bca BEST RNA PCR Kit (TakaRa Biotechnology Co. Ltd.) with degenerate primers
DP1 (5¢-AYCTYCTBAATAARTAYCTYT-3¢)/
DP2 (5¢-HTCMACCTTCCAYCTSCC-3¢) derived from GenBank according to conserved
sequence of SAD. The specific 5¢-RACE (rapid
amplification of cDNA end) and 3¢-RACE primers were designated as follows: 5¢RT-P [5¢-(P)GC
CAACTTTATGTC-3¢], A1 (5¢-CATCATTCCA
AGAAAGGG-3¢)/S1(5¢-GATGAATCCAAGAT
AGGG-3¢), A2 (5¢-CAGACTTGCCAAAGA
ACA-3¢)/S2 (5¢-GTCCAGACAGATAGAGATA
C-3¢) and 3¢GSP (5¢-ACAATCTTTTTGACCA
C-3¢). The 3¢ and 5¢ sequences of JSAD cDNA
were obtained by RACE with the 3¢- and 5¢-Full
RACE Core Set (TaKaRa). The PCR products
were cloned to pMD18-T vector and sequenced.
Based on the nucleotide sequences of the 5¢- and
3¢-RACE products, primers GSP1 (5¢-AACAA
TGGCTCTCAAGCTCAATC-3¢)/GSP2 (5¢-TTT
TGCCACAAGGATTGTATGC-3¢) were used
for the amplification of full-length cDNA sequence of JSAD.
Southern blot analysis
Genomic DNA was prepared from young leaves
using the method of CTAB. A 10 lg portion of
DNA was completely digested by EcoRI, XhoI,
SalI and EcoRV separately. Digested products
were loaded on 0.7% agarose gel and transferred
to nylon membrane according to manufacturer’s
directions (Sambrook et al. 1989). The probe for
Southern blot was the mature peptide gene fragment recovered from PCR products and labeled
using DIG High Prime DNA Labeling and Detection Starter KitII (Roche Ltd.). Hybridization was
conducted according to manufacturer’s directions.
Northern analysis
Total RNA was extracted from young roots,
stems, flowers, seedcases, seeds, leaves and
mature leaves as above. Total RNA (25 lg per
lane) was electrophoresed through agarose gel
containing formaldehyde according to established
procedures (Sambrook et al. 1989). Transfer,
hybridization, detection conditions and probe
were as described for Southern blots.
E. coli expression assay
A primer pair (5¢-GCGGCATGCGCTTCTACC
CTCAAGTCCGG-3¢)/(5¢-GCGTCGACTTACA
GCTTCACTTCTCTATC-3¢) was used to clone
the mature protein gene. Purified PCR products
were digested with restriction enzymes SphI and
SalI and cloned into the corresponding sites of
E. coli vector pQE-30, resulting in a recombinant
plasmid pQESAD.
The recombinant plasmids, pQESAD, were
introduced in E. coli strain M15 (QIagen Co.).
E. coli cells harboring pQESAD were grown at
37 C to an OD595 of about 0.7 in NZYM broth
(Sambrook et al. 1989) containing 0.4% glucose
and 300 mg penicillin/l, and then induced for 3 h
with 0.4 mM IPTG. Cells slang from 1 ml
culture, dissolved in 125 ll SDS sample buffer,
and heated to 100 C for 10 min. Samples were
analyzed by SDS-PAGE.
For activity assays, cells were centrifuged at
14,800g for 15 min, resuspended in 20 mM
potassium phosphate buffer at pH 6.8, and
broken in a French press apparatus at 110 MPa.
Debris was pelleted at 5000g for 5 min. The
supernatant fraction was applied to a Sephadex
G-25 centrifugal gel filtration column (Boehringer Mannheim) equilibrated with 20 mM potassium phosphate buffer at pH 6.8. Columns were
centrifuged for 4 min at 5000g. The effluent
was collected and used as enzyme source in activity assay.
659
Activity assay was prepared by mixing 5 ll
dithiothreitol (100 mM), 10 ll bovine serum
albumin (10 mg/ml), 15 ll NADPH (25 mM,
freshly prepared in 0.1 M Tricine/HCl, pH 8.2),
25 ll spinach ferredoxin (2 mg/ml), 3 ll ferredoxin:NADP+ oxidoreductase (2.5 units/ml),
1 ll catalase (800,000 units/ml), and 150 ll
water. After 10 min at room temperature, this
mixture was added to a 13100 mm screw-cap
test tube containing 250 ll Pipes buffer (0.1 M,
pH 6.0), and 10 ll enzyme preparation. The
reaction was started by adding substrate, 30 ll
[9,10(n)-3H]stearoyl-ACP
(10 lM
in
0.1 M
PIPES, pH 5.8) and, after the tube had been
sealed with a cap, allowed to proceed for
10 min with shaking at 23 C. The reaction was
terminated by adding 1.2 ml trichloroacetic acid
(5.8%, v/w), and the resulting precipitated acylACP was removed by centrifugation. About
500 ll of the aqueous fraction was neutralized
with 1 M Tris base, and the tritium (3H) eleased
from the desaturease reaction was measured by
liquid scintillation spectrometry (Thompson
et al. 1991).
plastid stroma. Similar secretary signal peptides
of SAD were also reported in castor and
safflower (Knutzon et al. 1991, Thompson et al.
1991).
Results and discussion
Southern hybridizing
Gene cloning and sequencing
Hybridization at high stringency revealed several
hybridizing bands, demonstrating a very limited
potential number of genes. Two hybridizing
bands were observed in each lane which suggested that other closely related isoenzyme genes
encoding SAD families may occur in the whole
genome (Figure 3). This result was consistent
with one other study (Shah et al. 2000).
A 491 bp segment of JSAD gene was amplified
by RT-PCR firstly. Then by 3¢- and 5¢-RACE,
580 bp and 578 bp segments were obtained
respectively. Finally, the full-length cDNA of
JSAD was amplified with primers GSP1 and
GSP2. Sequence analysis showed that the cDNA
was 1491 bp in length including an 1191 bp complete ORF (open reading frame), a 4 bp 5¢- and
a 296 bp 3¢-untranslated sequence (GenBank
accession No. DQ084491, Figure 1). Analyzed by
DNA Tools 5.1, the gene encoded a 396-amino
acids precursor. According to Knutzon et al.
(1991) and Thompson et al. (1991), the putative
protein contains a 33-amino acids signal peptide
and a 363-amino acids mature peptide with Mw
41.7 kDa and isoelectric point 5.74. The signal
peptide was a secretary sequence containing high
content of hydrophobic amino acids (63.6%),
which was expected to transport the nascent
polypeptide chain across plastid membrane into
Comparison of the amino acid sequence of JSAD
with others
When the putative amino acids sequence of
JSAD was compared with the amino acids sequences of other SAD in the NCBI database
using BLAST program, a high degree of identity
was found. Among the SAD in GenBank, Ricinus communis SAD has the highest identity with
JSAD (96.2%), and great majority of SAD have
more than 60% identity with JSAD. The polypeptide has two conserved domains: one belongs
to acyl-ACP desaturase family with considerable
homology in a number of highly conserved
blocks (Figure 2a) and another belongs to a ferritin-like family (Figure 2b). Regions candidated
for the catalytic and substrate-binding sites of
the enzymes were much higher in the conserved
domains and have been detected in other SAD
(Fox et al. 1994, Lindqvist et al. 1996).
Northern hybridizing
The analysis of Northern blot indicated clearly
that JSAD was expressed in each organ detected
(Figure 4). The levels of JSAD mRNA in young
seedcases and seeds were highest, followed by
the mRNA level in young leaves, implying
developing fruits were the organs where SAD
was largely expressed. The mRNA levels of
young roots, stems, flowers and mature leaves
were relatively low and this was presumably
related to low transcriptional activity of SAD
gene in these organs.
660
1 AACAATGGCTCTCAAGCTCAATCCTTTCATTTCTCAATTTCACAAGTTGCCTACTTTCGCTCTCCCACCA ATGGCC
1
M
A
L
K
L N
P
F I
S
Q
F
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A
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M A
7 AATCTCAGATCTCCCAAGTTCTATATGGCTTCTACCCTCAAGTCCGGTTCCAAGGAGGTGGAGAATCTTAAGAAG
5
N L
R
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F Y M
A
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V
E N
L K
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152 CCTTTTATGCCTCCTCGGGAGGTGCATGTTCAGGTTACTCATTCTATGCCACCCCAGAAGATTGAGATATTTAAA
50 P F
M
P P
R
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V H
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Q
V T H S
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P
P
Q K
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F K
227TCTCTAGATGAATGGGCTGAGCAGAACATTCTTGTTCATCTGAAACCAGTTGAGAAGTGTTGGCAACCACAGGAT
75 S L
D E
W A
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Q
N
I
L
V H
L
K
P
V
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C
W Q
P Q
D
302TTTTTGCCTGATCCTTCCTCTGATGGATTTGATGAACAAGTCAGGGAACTTAGGGAGAGAGTGAAGGAGATTCCA
100 F
L P
D
P
S
S
D
G
F
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R E
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377 GATGACTACTTTGTTGTTTTGGTTGGAGACATGATCACAGAAGAAGCCCTTCCCACTTATCAAACAATGCTGAAC
125 D
D
Y F
V V
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M I
T
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452ACTTTAGATGGAGTTCGGGATGAAACTGGTGCTAGCCTTACTTCTTGGGCAATTTGGACAAGGGCATGGACTGCT
150
T L D
G
V
R D
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G
A S
L
T S
W A
I
W
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527GAAGAGAATAGACATGGCGACCTTCTCAATAAGTATCTCTATCTGTCTGGACGAGTGGACATGAGGCAAATTGAG
175 E E
N
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H G
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L N
K
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L Y
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602 AAGACAATTCAATATTTGATTGGATCAGGAATGGATCCACGGACTGAAAACAGTCCCTATCTTGGATTCATCTAC
200 K
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677ACATCATTCCAAGAAAGGGCAACGTTCATCTCGCATGGAAACACTGCCAGACTTGCCAAAGAACATGGAGACATA
225
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752AAGTTGGCTCAAATATGTGGTACAATTGCTGCAGATGAGAAGCGACATGAGACAGCATACACAAAGATAGTGGAA
250
K L
A
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827 AAGCTCTTCGAGATTGATCCTGATGGAACTGTGTTGGCTTTTGCTGACATGATGAGAAAGAAAATTTCCATGCCG
275 K
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902 GCACACTTGATGTATGATGGTCGTGATGACAATCTTTTTGACCACTTTTCAGCTGTTGCACAGCGGCTTGGTGTC
300 A H
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F D
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F
S
A
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A Q
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977 TATACTGCAAAGGACTATGCAGATATATTGGAGTTCTTGGTGGGCAGATGGAAGGTGGATAAGCTAACAGGACTT
325
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A
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Y A
D
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1052TCAGCTGAGGGGCAAAAGGCTCAGGATTACGTTTGTCGGCTACCTCCAAGAATTAGAAGGCTGGAAGAGAGAGCT
350 S
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1127 CAAGGACGAGCCAAGGAAGGACCCACAATTCCTTTCAGTTGGATTTTTGATAGAGAAGTGAAGCTGTAAGTGCAG
375 Q G
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1202 AATGAAACACAAATAGTCAGTTTGGCCCTTTTTCATGTCCCTTCCTGCAGAATCAGAAGCGGGGCAGAATTTTGT
1277 AGTTTCATTTTGTTTACAAATCCAGTTTAGTTTTATGTCTGGGAAAGGGGAGTGAATTTGGTAAATTGTAGATTC
1352 AGTTGGGTCTTGTGTGTTTTCTTGAGTATGCTGATAGGGGGCATCTGTAGTTTTGGTATTGTGTTCTTTTACATGG
1428TCTCTTCTATTAGTTTGTTTGTCTTTAGTTTCTTGAGTATAAGCATACAATCCTTGTGGCAAAA
Fig. 1. cDNA sequence and putative amino acid sequence of Jatropha curcas stearoyl-ACP desaturase. The start codon and stop
codon are bold. Signal peptide sequence is shaded; Vir box is doubly underlined; archB boxes are underlined by broken-lines; bra
boxes are underlined; gluco sequence is underlined by wave-line; glycol sequence is in box (GenBank accession No. DQ084491).
Prokaryotic expression and activity
of recombinant protein
For prokaryotic expression, the mature portion
of the JSAD-coding sequence was engineered by
PCR to have the amino-terminal sequence G-SA-C-A-S-T-L-K-S..., where the A and C residues
resulted from the introduction of a SphI restriction site. The gene was transferred to an E. coli
strain M15 vector expression behind the T5
661
Fig. 4. Northern hybridizing analysis. Upper part, the ethidium bromide stained gel of total RNA (25 lg per lane); Lower
part, corresponding spots of Northern blot; M, DL2000
DNA marker; 1, young root; 2, young stem; 3, flower; 4,
young seedcase; 5, young seed; 6, mature leaf; 7, young leaf.
Fig. 2. Comparisons of putative amino acid sequence of
Jatropha curcas stearoyl-ACP desaturase with NCBI searched
domain of acyl-ACP-desaturase family (a), and with NCBI
searched domain of ferritin-like family (b). Identical amino
acids are underlined. Abbreviation: J, Jatropha curcas stearoyl-ACP desaturase; C, acyl-ACP desaturase family; F, ferritin-like family.
Fig. 3. Genomic Southern hybridizing analysis of Jatrohpa
curcas. 1, Digestion with EcoRI produced 6.5 kb and 2.4 kb
fragments; 2, XhoI produced 7.2 kb and 5.4 kb fragments; 3,
SalI produced 4.1 kb and 6.2 kb fragments; 4, EcoRV produced 6.2 kb and 3.1 kb fragments.
Fig. 5. SDS-PAGE analysis of Jatropha curcas stearoyl-ACP
desaturase expressed in E. coli. 1, protein marker; 2, lysate
from E. coli M15 harboring pQE-30 with IPTG; 3, lysate
from E. coli M15 harboring pQESAD without IPTG induction; 4, lysate from E. coli M15 harboring pQESAD with
IPTG induction. 6His-stearoyl-ACP desaturase is indicated
by the arrow (about 43 kDa).
promoter-QIAexpress System pQE-30 (which allows the fast and efficient production and purification of heterologously expressed 6His-tagged
proteins).
The extracts of expressing E. coli were subjected to SDS-PAGE (Figure 5). The recombinant
gave rise to an about 43 kDa protein in response
to the IPTG induction; its content was about 19%
among total cell protein by Gene Genius Bio
Imaging System (Syngene, a division of Synoptics
Ltd.).
When spinach ferredoxin was added to in vitro
enzyme assay, significant amounts of desaturase
activity were clearly demonstrated in protein extracts from expressing E. coli cells harboring
pQESAD (Table 1), even in the absence of IPTG
662
Table 1. Expression of stearoyl-ACP desaturase activity in
extracts from E. coli M15 transformed with pQE-30 or
pQESAD.
Plasmid
pQE-30
pQESAD
Uninduced
IPTG-induced
Without
Fd
With
Fd
Without
Fd
With
Fd
0
3±1.96
1±1.96
1570±272
2±1.96
82±13
0
7950±541
Activity (pmol/min.mg protein): a unit of activity was defined as
the release of 4 lM 3H per min, equivalent to the conversion of
1 lM stearoyl-ACP to oleoyl-ACP; each individual determination was done for three times. Fd, reduced spinach ferredoxin.
for full induction of T5 polymerase. These results
verified not only that the cloned insert of pQE-30
does encode the stearoyl-ACP desaturase, but
also SAD requires spinach ferredoxin for its
activity.
As stated above, recombinant plasmid pQESAD provides the means to produce native active
enzyme. This study will be essential to understand JSAD regulating mechanism and the
relationship of its structure to function. In addition, cloned genes will allow alteration of in vivo
levels of enzyme activity in transgenic plants for
examination of SAD role in determining total
unsaturated fatty acid content.
Acknowledgments
Dr Yang Yi, Zhang Nian-Hui, and Sun Qun are
acknowledged for their generous help during the
preparation of the manuscript. This work was
supported by ‘‘Tenth Five Years’’ Key Program
of the State Science and Technology Commission
on China (2002BA901A15 and 2004BA411B01).
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