全 文 ：Foundation Program: This research were supported by National Natural Science Foundation of China (30370902).
分子植物育种，2007，第 5卷，第 1期，第 15-20页
Molecular Plant Breeding, 2007, Vol.5, No.1, 15-20
Research Report
研究报告
Molecular Cloning and Expression Analysis of an Oleate Desaturase Gene
DsFAD6 from Descurainia sophia
Tang Sanyuan Huang Ji Zhang Hongsheng Guan Rongzhan*
National Key Lab of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095
﹡Corresponding author, guanrzh@njau.edu.cn
Abstract DsFAD6 encoding a plastidial oleate desaturase, a key enzyme for catalyzing oleic into linoleic
acid, was isolated from Descurainia sophia using RACE-PCR approach. The full-length cDNA of DsFAD6
with a complete open reading frame of 1 344 bp, encoded a peptide of 447 amino acid residues with a predicted
molecular mass of 51.16 kD and a PI of 9.27. Sequence analysis showed the three histidine boxes characteristic of
all membrane-bound desaturases and a N-terminal plastidial signal peptide. Sequence alignment and phylogenetic
tree analysis showed that DsFAD6 was more like FAD6s from cruciferous species. Expression analysis by RT-PCR
showed that DsFAD6 gene was expressed in all tissues detected, but higher in stems, leaves and young siliques. In
addition, the expression of DsFAD6 gene was induced by wounding, but inhibited by cold stress in leaves.
Keywords Descurainia sophia, Plastidial oleate desaturase, Oleic acid, Linoleic acid
播娘蒿油酸脱氢酶基因 (DsFAD6)的克隆和表达分析
唐三元 黄骥 张红生 管荣展 *
南京农业大学作物遗传与种质创新国家重点实验室 , 南京 , 210095
﹡通讯作者 , guanrzh@njau.edu.cn
摘 要 采用 RACE-PCR的技术克隆了播娘蒿的油酸脱氢酶基因 (DsFAD6)，它是催化油酸转化为亚油酸
的一个关键酶基因。DsFAD6 cDNA全长序列的开放阅读框长 1 344 bp，编码一个由 447个氨基酸残基组
成的蛋白质，推测的蛋白质相对分子质量为 51.16 kD，等电点为 9.27。序列分析表明 DsFAD6的氨基酸序
列具有 3个高度保守的组氨酸盒子，该结构在去饱和酶中是高度保守的，且在 N端预测到信号肽，初步
认定该酶定位于质体。序列比对和系统发生分析显示 DsFAD6与十字花科植物 FAD6基因具有较高一致性。
RT-PCR分析表明，DsFAD6在根、茎、叶、花蕾、花及幼嫩角果中均表达，但在茎、叶和幼嫩角果中表
达较高。胁迫诱导表达中，DsFAD6在播娘蒿叶片中表达明显受伤害胁迫诱导，但在冷胁迫下呈负相关。
关键词 播娘蒿 , 质体油酸脱氢酶 , 油酸 , 亚油酸
Cruciferous wild species Descurainia sophia has
two properties useful. One property is high content
of linolenic acid of around 40% in its seeds (Dong et
al., 2005; 2006), showing characteristics of functional
oil; another is that it can tolerate to extreme ecological
conditions, specially to extremely low temperature. As
a plant resource, its key genes related to its important
traits may be listed as one research target.
Plant unsaturated fatty acid mainly of 18 carbon
atoms, oleic acid (18:1), linoleic acid (18:2) and
linolenic acid (18:3), are essential for human diet, major
structural components of membrane lipids, provide
a substantial reserve of free energy and serve as key
precursors for the biosynthesis of messengers in signal
transduction mechanisms that influence plant growth,
development and responses to various environmental
cues (Weber, 2002; Nandi et al., 2003). From view of
functionality of unsaturated fatty acid, plastidial oleate
16 分子植物育种
Molecular Plant Breeding
desaturase gene is very important because it initiates
the biosynthesis process and cloning and characterizing
this gene have become targets of researchers. Up to
now, cDNAs of FAD6 have been isolated from several
plant species, such as Arabidopsis thaliana (Falcone
et al., 1994), Spinacia oleracea (Schmidt et al., 1993),
Glycine max (Schmidt et al., 1993), Brassica napus
(Hitz et al., 1994), Olea europaea L. (Banilas et al.,
2005), Zea mays (Mikkilineni and Rocheford, 2003)
and Brassica rape (Hitz et al., 1994). Here we reported
the isolation of DsFAD6 encoding a plastidial oleate
desaturase from D. sophia and its mRNA expression
was investigated.
1 Materials and Methods
1.1 Plant materials
Descurainia sophia was planted in soil under
natural conditions. Roots, stems, leaves, floral buds,
flowers and siliques were harvested at different
developmental stages. For expression analysis of
DsFAD6 under wounding stress, the 6-month-old D.
sophias were wounded with a sterile razor blade across
the midrib of the lower leaves for 1 cm long fi ssures.
Wounded plants were incubated for 24 h under natural
growth conditions. For expression analysis of DsFAD6
under cold stress, the 6-month-old D. sophias were
placed at 4 ℃ for 48 h. The wounded and cold leaves
were harvested at different times respectively. All
harvested samples were immediately frozen in liquid
nitrogen and stored at -80℃ for RNA extraction.
1.2 RNA extraction and RT reactions
Total RNA was isolated from different D. sophias
tissues using a RNA extraction kit (Beijing Tianwei
Corporation). The first-strand cDNA was synthesized
with 2 µg of purifi ed total RNA (pre-treated with DNase
Ⅰ ) using the RT-PCR system (Promega) according to
the manufacturer’s protocol. The oligo (dT)18 was used
as a primer and the reverse transcription reaction was
incubated at 42℃ for 1 h in a total volume of 20 μL.
1.3 Cloning of DsFAD6 cDNA fragment
To amplify conservative cDNA fragment of
DsFAD6, two primers (sense: 5′-ATCGCCGTGGTA
TCTGC-3′, antisense: 5′-AAAGCGTTGGTGAA
GTGTTA-3′) were designed based on nucleotide
sequences of FAD6 from A.thaliana and other
orthologues. The first-strand cDNA was synthesized
from total RNA from D. sophia young siliques.
Polymerase chain reaction (PCR) was performed in
a 50 μL reaction mixture containing 5 μL 10×PCR
buffer with MgCl2, 1 μL 20 mmol/L dNTPs, 1 μL RT
products, 2 μL DMSO, 1 μL pfu high fidelity DNA
polymerase (Roche), 1 μL 25 mmol/L each primer, and
38 μL ddH2O on a DNA amplification machine (MJ,
USA). The reaction mixture was subjected to an initial
denaturation at 94℃ for 5 min, 30 cycles at 94℃ for
30 s, 52 ℃ for 30 s and 72 ℃ for 80 s followed by a
fi nal incubation at 72 ℃ for 7 min. The PCR product
was run on 1% agarose gel and the target band was
purifi ed with Gel Extraction Kit (HuaShun, Shanghai)
according to the manufacturer’s protocol. The purifi ed
product was cloned into pGEM-T vector (Promega,
USA) and sequenced (SNBC, Shanghai).
1.4 Rapid amplifi cation of cDNA ends (RACE)
For amplifi cation of the unknown 3′- and 5′-ends
of the DsFAD6, the 3′ and 5′ RACE were performed
according to protocols described in GeneRacerTM kit
(Invitrogen). Based on sequence information of the
conservative cDNA fragment, gene specific primers
(GSP) were designed as follows: 3′ FAD6 GST : 5′-C
CTCAAGAAAGCAATGCCGAACTAC-3′, 5′ FAD6
GST: 5′-GTTCCAGTCCACGCCCAAGCCAGC-3′.
To amplify the full-length DsFAD6 cDNA, two primers
were designed: based on the assembled sequence:
ALLFAD6.1: 5′-CTTCACAGCCATTCAATCTC-3′,
ALLFAD6.2: 5′-GGTGAAGTGTTAAGAGAAGC-3′.
The PCR conditions were same to 1.3 but the extension
time is 2 min instead of 80 s.
1.5 Semi-quantitative RT-PCR assay
For further understanding the expression of the
DsFAD6 gene, we performed semi-quantitative RT-
PCR assay using RNA samples from various tissues.
PCR amplification was performed using the primers
sFAD6 and asFAD6, which amplify the conservative
DsFAD6 cDNA fragment. PCRs were performed using
equal amounts of templates and 18S rRNA primers
17
and carried out for different numbers of cycles in order
to optimize reproducibility and ensure that reactions
remained in log-linear range.
1.6 Sequence analyses
The functional domains were analyzed using
online server program SMART 4.0 (http://smart.
embl hei delberg.de/ ). The molecular weight was
calculated using online server program Protscale (http://
au.Exp asy. org/cgi-bin/protscale.pl). Transmembrane
regions were predicted by the TMPRED (http://www.
ch. embnet.org / software/ TMPRED _form.html )
and NetPhosK 1.0 (http://www.cbs.dtu.dk/services/
NetPhosK/). Predictions of subcellular localization
were conducted by using the PSORT (http://www.
psort.nibb.ac.jp/ form.html) and TargetP (http://www.
cbs.dtu.dk/services/TargetP/) algorithms. Multiple
sequence alignment and phylogenetic tree analysis
were performed using ClustalX program (Thompson et
al., 1997).
2 Results and Analyses
2.1 Cloning of DsFAD6
Based on nucleotide sequences of FAD6 from
different plant species, two primers were designed.
PCRs employing the primer pairs generated conserved
fragments with siliques cDNA of D. sophia as a
template. To clone the full-length cDNA, 3′ and
5 RACE reactions were conducted and generated
two fragments of 323 bp and 602 bp (Figure1A
and 1B) of the DsFAD6 gene. These two fragments
are perfect correspondence to the expected gene.
Sequence comparisons of the 5′- and 3′- ends with
the conservative parts of the gene indicated that
the overlapping regions matched perfectly. BLAST
searches of the deduced amino acid sequences revealed
that these sequences represented the missing parts
of DsFAD6 gene. Based on the above sequence data,
primers were designed from the 5′- and 3′- UTRs of
DsFAD6 and the full-length cDNAs was amplified,
cloned and sequenced (Figure 1C).
2.2 Sequence analysis of DsFAD6
The full-length cDNA DsFAD6 is 1 521 bp with
an open reading frame of 1 344 bp. It encoded a protein
of 447 amino acid with a predicted molecular mass of
51.16 kD and an isoelectric point of 9.27. The DsFAD6
showed high similarities (70%~89% identity) in
amino acid sequences with several orthologues in the
GenBank through Blast searches. There are three trans-
membrane regions at amino acid residues 146~168,
182~201, 276~294 and three histidine-boxes (HXXXH,
HXXHH, and HXXHH) with conserved inter-spaces in
the DsFAD6 (Figure 2). The eight histidines arranged
into three histidine-boxes in the oleate desaturase
sequences have been shown essential for desaturase
activity (Chapman, 1975). It was also predict a putative
chloroplast transit peptide sequence of 64 amino acids
at the N-terminus of DsFAD6 protein (Figure 2). This
N-terminal sequence also had several characteristrics of
plastidial transit peptides, including a high content of
hydroxylated residues (Ser, Thr and Tyr), a low content
of acidic residues and the conserved N-terminal Met-
Ala dipeptide (Banilas et al., 2005).
The neighbor-joining phylogenetic tree (Figure 3)
showed the oleate desaturases was divided into two
major groups corresponding to the classical botanical
division of plants. DsFAD6 and AtFAD6 were clustered
into cruciferous species subgroups.
2.3 Expression analysis of DsFAD6
The expression of DsFAD6 in various tissues
by semi-quantitative RT-PCR revealed that DsFAD6
was constitutively expressed in roots, stems, leaves,
floral buds, flowers and young siliques. The higher
transcript level of DsFAD6 was observed in green
tissues, i.e. leaves, stems and young siliques. The
expression of the gene was the lower in roots, floral
Figure 1 Cloning of DsFAD6
Note: A~C indicated the PCR results of 3′ RACE, 5′ RACE and
full-length DsFAD6 gene; M: DL-2000 marker; 1: PCR product
Molecular Cloning and Expression Analysis of an Oleate Desaturase Gene DsFAD6 from Descurainia sophia
播娘蒿油酸脱氢酶基因 (DsFAD6)的克隆和表达分析
18 分子植物育种
Molecular Plant Breeding
Figure 2 Alignment of the deduced amino acid sequences from DsFAD6 and other plant FAD6
Note: The fatty acid desaturases were from Descurainia sophia (DQ904566), Arabidopsis thaliana (NM-119243), Olea europaea
(AY772187), Brassica napus (L29214), Spinacia oleracea (X78311), Glycine max (L29215), Oryza sativa (XM482619); Numbers in
parentheses refer to GenBank accession No. Identical or similar amino acids are shaded black or grey respectively; The black arrow
indicates a putative signal peptide cleavage site; The eight histidines grouped in three different boxes characteristic for desaturases are
indicated by asterisks; The three trans-membrane region are underlined
buds and fl owers, and was more accumulated in fl oral
buds, fl owers and young siliques (Figure 4). The result
reflected the crucial roles of this enzyme in the fatty
acid polyunsaturation pathway to fulfill the flower’ s
developmental needs, such as for the production of
alinolenate for pollen development or as precursor for
jasmonic acid and oxylipin biosynthesis (McConn and
Browse, 1996; McConn et al., 1997). These results are
consistent with former research results in fl ax and olive
(Banilas et al., 2005; Fofana et al., 2004).
19
3 Discussion
The plastidial oleate desaturase (FAD6) is the
enzyme responsible for converting oleic acid to linoleic
acid during the plant fatty acid desaturation pathway
(McConn and Browse, 1996; McConn et al., 1997).
We isolated and characterized an oleate desaturase
gene from D. sophia, containing the three histidine
boxes typical of all membrane-bound desaturases.
These invariant residues were arranged in three
histidine boxes (HXXXH, HXXHH, and HXXHH)
with conserved spaces between it (Figure 2).This
feature was the characteristics of all membrane- bound
desaturases. These histidine boxes were thought to
comprise the catalytic centre of the enzyme, since
they formed ligands to a diiron cluster in the catalytic
site (Shanklin and Cahoon, 1998). Multiple alignment
of deduced amino acid sequences and phylogenetic
analysis showed that the DsFAD6 was highly similar
to FAD6 from other plants. The bioinformatics analysis
predicted that DsFAD6 possessed a putative N-terminus
transit peptide of 64 amino acids capable of plastid
transportation.
Membrane lipids of plant cells are characterized
by a high content of polyunsaturated fatty acids.
An increase in polyunsaturated fatty acids in lipids
has been thought to increase the membrane fluidity
by decreasing the scope for orderly packing of acyl
chains within the membrane interior (Chapman,
1975). Increasing and decreasing in trienoic fatty acids
levels have been observed in a variety of plant species
exposed to low and high temperatures, respectively
(Smolenska and Kuiper, 1977; Horiguchi et al.,
1996). The abundance of trienoic fatty acids relative
to dienoic fatty acids changes more sensitively in
accordance with environmental conditions. FAD6
mutant plants are characterized, at low temperatures,
by leaf chlorosis, a reduced growth rate, and changes
in chloroplast morphology, such as a decrease in size
and appressed regions of thylakoid membranes (Hugly
and Somerville, 1992). The expression of DsFAD6 was
down- regulated by cold stress, indicating DsFAD6
might play a negative role in cold stress response.
However, this down-regulation phenomena different
Molecular Cloning and Expression Analysis of an Oleate Desaturase Gene DsFAD6 from Descurainia sophia
播娘蒿油酸脱氢酶基因 (DsFAD6)的克隆和表达分析
Figure 3 Phylogenetic relationships of oleate desaturases from
the plant microsomals and plastidials
Note: The tree was constructed using the Neighbor-Joining
algorithm program; The scar bar represents branch distance.
The organisms and accession numbers are as follows:
Descurainias sophia (DsFAD6), Arabidopsis thaliana (At-
FAD6, NM-119243), Olea. europaea (OeFAD6, AY772187),
Brassica napus (BnFAD6, L29214), Spinacia oleracea
(SoFAD6, X78311), Glycine max (GmFAD6, L29215),
Oryza sativa (OsFAD6, XM482619), Medicago truncatula
(MtFAD6, ABE88706), Chlamydomonas. reinhardtii (CrFAD6,
BAA23881), Trichodesmium erythraeum (TeFAD6, ABG49637),
Gloeobacter violaceus (GvFAD6, AAF61413), Arthrospira
platensis (ApFAD6, CAA60415)
The transcript levels of DsFAD6 rapidly increased
in the wounded leaves and reach peak at 3 h, then
gradually decreased (Figure 5A). Whereas the
transcript levels of DsFAD6 rapidly decreased in the
leaves treated by cold and reach the lowest level at 3 h,
gradually increased thereafter (Figure 5B).
Figure 4 Expression of DsFAD6 in D. sophia tissues
Note: 1~6 indicated the results of roots, stems, leaves, floral
buds, fl owering and young siliques
Figure 5 Expression of DsFAD6 in the leaves wounded (A) and
cold-treated (B)
20 分子植物育种
Molecular Plant Breeding
from research with mutation, needs to be furthered.
Expression of DsFAD6 was induced in the D.
sophia leaves upon mechanical wounding. Transcript
levels of DsFAD6 increased rapidly to peak between
1~3 h after wounding, then gradually decreased.This
result is consistent with the report of tobacco and
Arabidopsis ω-3 FAD genes whose expression levels
started to increase after 1 h treatment by wounding
and the high levels were maintained more than 6 h
(Hamada et al., 1996; Nishiuchi et al., 1997). Wounding
activates the octadecanoid pathway in which linolenic
acid is converted to Jasmonic acid (JA), resulting in
a significant accumulation of this hormone. Jasmonic
acid (JA), a fatty acid-derived hormone, is one of
several candidate molecules for wounding signaling and
is thought to play a pivotal role in the transcriptional
activation of wound-inducible genes (Farmer and Ryan,
1992; Farmer et al., 1998).
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