全 文 ：Received 10 Dec. 2003 Accepted 29 Apr. 2004
Supported by the National Natural Science Foundation of China (370100100).
* Co-first authors.
** Author for correspondence. Tel: +86 (0)21 62932002; Fax: +86 (0)21 62824073; E-mail: or .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1226-1233
Molecular Cloning and Characterization of Enolase from
Oilseed Rape (Brassica napus)
ZHAO Jing-Ya 1*, ZUO Kai-Jing1*, QIN Jie1, TANG Ke-Xuan 1, 2**
(1. Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Plant Biotechnology Research Center, School of Agriculture and
Biology, Shanghai JiaoTong University, Shanghai 200030, China;
2. Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, State Key Laboratory of Genetic Engineering, School of Life
Sciences, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, China)
Abstract: An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene
(accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and
encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced
amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human
beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and
human beings. Comparison of its primary structure with those of other enolases revealed the presence of
an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated
that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase
gene increased under salt stress, but decreased in response to low temperature. Our studies suggested
that the cloned gene was a new member of plant enolase gene family, which contributed to the energy
supply in stress-treated tissues.
Key words: enolase; oilseed rape (Brassica napus); cDNA cloning
The Embden-Meyerhof-Parnas pathway of glycolysis
is the backbone of energy metabolism (ATP synthesis) in
eukaryotes. Enolase (2-phospho-D-glycerate hydrolyase,
EC 4.2.1.11) is an essential and ubiquitous enzyme that cata-
lyzes the Mg2+-dependent dehydration of 2-phosphoglyc-
erate (2-PGA) to phosphoenolpyruvate (PEP), which is con-
verted by pyruvate kinase into pyruvate with the concomi-
tant generation of ATP in the subsequent final step of gly-
colysis (Hannaert et al., 2000). Like other enzymes involved
in glycolysis and gluconeogenesis, enolase is one of the
most conservatively evolved glycolytic enzymes (Fothergill-
gilmore and Michels, 1993). Highly conserved amino acid
residues that are the ligands for “conformational” Mg2+
(Lebioda et al., 1989) that participate in substrate binding,
function in enolase catalysis (Brewer et al., 1993) or partici-
pate in the catalytic region of the enzyme in yeast (Lebioda
et al., 1989) have been documented.
The enzyme has been cloned from diverse sources such
as Escherichia coli (Weng et al., 1986), Bacillus subtilis
(Verma, 1989), Saccharomyces cerevisiae (Chin et al., 1981),
Arabidopsis thaliana, Lycopersicon esculentum (van Der
Straeten et al., 1991), Zea mays (Lal et al., 1991) and human
beings (Giallongo et al., 1986). The yeast enolase is by far
the best characterized and its crystal structure has been
determined at high resolution (Lebioda et al., 1989).
Moreover, enolase is found to be identical to the heat shock
protein HSP48 and, hence, is important in thermal tolerance
and growth control of yeast (Iida and Yahara, 1985).
Environmental stresses can cause severe effects on plant
cells sufficient to cause cell death. Under anaerobic stress,
most plants have been shown to shift their carbohydrate
metabolism from the oxidative pathway to a fermentative
pathway. It has been demonstrated that the synthesis of
several proteins engaged in the glycolysis and fermenta-
tion process is induced in plants exposed to anaerobic
conditions. The anaerobic polypeptides include enolase
(Forsthoefel et al., 1995), aldolase (Kelley and Tolan, 1986),
pyruvate decarboxyla (Kelley, 1989), and alcohol
degydrogenase (Dennis et al., 1987).
It is generally accepted that fermentative or anaerobic
metabolism is not prevalent throughout the normal life cycle
of photosynthetically active cells, but in certain nongreen
and anaerobically treated tissues which are dependent on
glycolysis and oxidative phosphorylation for their energy
supplies (Goodwin and Mercer, 1983), this enzyme is
important. Increased expression of enolase might be ex-
pected under conditions such as hypoxia or anoxia that
limit oxidative phosphorylation of ADP and favor ATP
ZHAO Jing-Ya et al.: Molecular Cloning and Characterization of Enolase from Oilseed Rape (Brassica napus) 1227
regeneration via glycolysis and the ethanolic fermentative
pathway. Enolase has been shown to be rapidly induced
by hypoxia stress in both anoxia-tolerant and -intolerant
Echinochloa (barnyard grass) spp.(Fox et al., 1993; Zhang
et al., 1994). Enolase transcripts in rice seedlings increased
approximately 8-fold after 24 h of submergence in distilled
water without aeration. In maize subjected to anoxia for up
2 d, enolase mRNA in root tips increase 2- to 5-fold and
activity increased 200% over aerobic levels (Umeda and
Uchimiya, 1994).
Oilseed rape (Brassica napus) is one of the most impor-
tant industrial and food oil crops worldwide, with high pro-
duction in Asia, Europe and North America. The oilseed
rape yield is affected by a host of environmental factors
amongst which drought, flooding, low temperature and
salinity are considered agronomically most significant. In
this study, a full-length cDNA encoding enolase was iso-
lated from the oilseed rape. Its primary structure was com-
pared with Bacillus, yeast, other reported plant and human
counterparts. Both the conformational metal ion-binding
site residues and all of the amino acids required for cataly-
sis were found to be conserved among various enolases.
In addition, the expression profile of the cloned enolase
gene in stressed plant tissues was also studied.
1 Materials and Methods
1.1 Plant material
The seeds of Brassica napus L. cv. Huyou 15, after
being rinsed with distilled water for 12 h at 37 ℃ and steril-
ized with 75% ethanol, were sown in pasteurized vermicu-
lite in pots and cultured in growth chamber with a cycle of
14 h artificial light at 25 ℃ and 10 h dark at 22 ℃ (70%
relative humidity). When having 2-3 leaves, the germinated
seedlings were subjected to stress treatments.
For salt treatment, plants were stressed by the addition
of NaCl to a final concentration of 100 mmol/L. Low tem-
perature stress was imposed by transferring plants to 4 ℃.
The roots of stressed plants were harvested after 0, 30 min,
1, 3, 7, 12, 24, 48 h treatments. All the roots were used to
extract total RNA immediately.
1.2 Cloning and sequencing of the enolase full-length
cDNA from B. napus
Total RNA was extracted from the roots of B. napus
using TRIZOL Reagent (Gibco BRL, USA) according to the
manufacturer’s instructions. RNA was treated twice with
RNase-free DNase (Gene Hunter, Nashville, USA) to re-
move DNA contamination. For the cloning of enolase gene
from B. napus, the following procedure was used including
internal conservative fragment cloning, 3 end fragment
cloning and 5 end fragment cloning.
1.2.1 Internal conservative fragment cloning By align-
ing human and Arabidopsis enolase sequences retrieved
from GenBank, two conservative sequences, ENO1 (5-
AATCCCACNGTTGAGGTTGA-3) and ENO2 (5-
AGCTGGTTGTACTT GGC-3), were synthesized and used
as the pair of primers in the RT-PCR reaction for the ampli-
fication of the internal conservative fragment from B. napus
under the following condition: the template was denatured
at 95 ℃ for 5 min followed by 30 cycles of amplification (1
min at 95 ℃, 1 min at 55 ℃ and 1 min at 72 ℃) and by 10 min
at 72 ℃. The PCR product was purified and cloned into
pGEM-T vector (Promega, Madison, WI, USA) followed
by sequencing.
1.2.2 3 rapid amplification of the cDNA ends (RACE)
The 3 RACE of the enolase cDNA from B. napus was per-
formed with the 3 RACE kit (Invitrogen) according to the
manufacturer’s instructions. The first-strand cDNA was
synthesized from 1 µg of total RNA using a cDNA synthe-
sis primer AP (5-GGCCACGCGTCGACTAGTAC(T)16-3)
provided within the kit. Utilizing primer ENO3 (5-
TGGACAAATCAAGACTGGAG-3) synthesized based on
the cloned internal fragment as the forward primer and the
Abridged Universal Amplification Primer (AUAP, 5-
GGCCACGCGTCGACTAGTAC-3) as the reverse primer, 3
RACE was performed in a total volume of 50 mL containing
2 mL cDNA, 10 pmol each of primer ENO3 and AUAP, 10
mmol dNTPs, 1× cDNA reaction buffer and 5 U Taq
polymerase. PCR reaction was carried out under the follow-
ing condition: template was firstly denatured at 94 ℃ for 5
min followed by 35 cycles of amplification (1 min at 95 ℃, 1
min at 55 ℃ and 1 min at 72 ℃) and by extension at 72℃ for
10 min. The PCR product was purified and subcloned into
pGEM-T Easy vector (Promega, USA), followed by
sequencing.
1.2.3 5 RACE
Based on the sequence of 3 RACE product, three gene
specific primers ENO4 (5- ATCAAGTTCATGGACCATGA-
3), ENO5 (5-GACCATGAAGTTGTCAATAG-3) and ENO6
(5- GTCCTTTCCAATCAACGCTG-3) were subsequently
designed and synthesized for the 5 RACE of the enolase
cDNA from B. napus. The 5 RACE was performed with the
5 RACE kit (Invitrogen) according to the manufacturer’s
instructions. The first-strand cDNA was synthesized us-
ing ENO4 followed by tailing cDNA with oligo (C). The first
round of PCR was performed with ENO4 and Abridged
Anchor Primer (AAP, 5 -GGCCACGCGTCGACTAGTACG-
GGIIGGGIIGGGIIG-3). PCR was carried out by denatur-
ing cDNA at 94 ℃ for 5 min followed by 35 cycles of
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041228
amplification (1 min at 95 ℃, 1 min at 55 ℃ and 1 min at 72
℃) and by extension at 72 ℃ for 10 min. The PCR product
was diluted 50-fold for nested PCR with a second round of
amplification with ENO5 and AUAP and the third round of
amplification with ENO6 and AUAP. Using the same condi-
tion as described in the first round of PCR, the 5 RACE
product was purified and subcloned into pGEM-T Easy
vector followed by sequencing.
1.3 Southern and Northern blotting analyses
Genomic DNA was extracted from young leaves of oil-
seed rape by using the cetyltrimethylammonium bromide
(CTAB) method (Sambrook et al., 1989). Aliquots of DNA
samples (10 µg/sample) were digested overnight at 37 ℃
with BamHⅠ and XhoⅠ, which did not cut within the full-
length cDNA region, respectively, fractionated by 0.8%
agarose gel electrophoresis and transferred onto a posi-
tively charged Hybond-N+ nylon membranes (Amersham
Pharmacia, England). A segment of B. napus enolase cDNA
(345-658 nt) and 3 UTR sequence of enolase were used as
the probe for the detection of copy number of the enolase
gene in the genome of B. napus. The Gene Images random
priming labeling module and Gene Images CDP-Star detec-
tion module were used for probe labeling, hybridization
and detection procedures (Gibco BRL).
For Northern blotting analysis, aliquots of total RNA
(30 mg) were denatured and separated in 1.2% formalde-
hyde-denatured (W/V) agarose gel. After electrophoresis
the RNA was transferred onto a Hybond-N+ nylon mem-
brane and hybridized using the probe mentioned before.
The Gene Images random priming labeling module and Gene
Images CDP-Star detection module were the same as men-
tioned for Southern blotting analysis except that Northern
hybridization was carried out at 50 ℃.
1.4 Semi-quantitative one-step RNA PCR
Total RNA was extracted using TRIZOL kit from roots
of B. napus after the plants were treated for 0, 30 min, 1, 3, 7,
12, 24 and 48 h with salt (NaCl, 100 mmol/L) and low tem-
perature (4 ℃) respectively. One mg of total RNA was used
as template in one-step RNA PCR (One-step RNA PCR Kit,
TaKaRa) with ENO7 (5-GGCAATCCCACGGTTGAGGT-3)
and ENO8 (5-AGAGAGGAATGCCACTGACA-3) as
primers. The template was amplified at 95 ℃ for 5 min, fol-
lowed by 25 cycles of amplification (95 ℃ for 1 min, 55 ℃
for 1 min, 72 ℃ for 1 min). The RT-PCR reaction for the
house-keeping gene (actin gene) using specific primers actF
(5-GTGACAATGGAACTGGAATGG-3) and actR (5-
AGACGGAGGATAGCGTGAGG-3) was performed as de-
scribed above to estimate if equal amounts of RNA among
samples were used in the RT-PCR reaction. The PCR
products (10 µL) were separated on 1% agarose gels stained
with ethidium bromide (10 µg/mL). The quantity of prod-
ucts was analyzed with Gene analysis software package
(Gene company, USA).
2 Results
2.1 Isolation of the enolase full-length cDNA of B. napus
By using the RT-PCR method, a 1 205-bp cDNA frag-
ment was initially isolated by the internal conservative frag-
ment cloning. Nucleotide BLAST search showed that the
isolated cDNA fragment shared high sequence homology
with many known plant enolases, implying that it was prob-
ably a part of an enolase gene. By 3 RACE cDNA cloning,
a 440 bp-cDNA fragment was generated. When the 20 bp
AUAP primer sequence was cut away, a fragment of 420 bp
3 cDNA end resulted, which had 148 bp overlapping se-
quences with the initially isolated 1 205 bp cDNA fragment.
The primary PCR of 5 RACE showed a specific band of
about 340 bp. Nested amplification was carried out directly
using this PCR product as template and a specific band of
about 300 bp was yielded. By cutting away the 20 bp AUAP
and 16 bp poly(dG) sequences, the length of 5 cDNA end
was 265 bp. It has 198 bp overlapping sequences with the
initially isolated 1 205 bp cDNA fragment (Fig.1).
By comparing and aligning the sequences of the inter-
nal fragment, 3 RACE and 5 RACE products, the full-length
cDNA of the B. napus enolase gene was obtained which
was subsequently confirmed by sequencing. The full-
length cDNA was 1 624 bp, which was predicted to have an
initiation codon ATG at position 20 nt and a stop codon
TAG at position 1 352 nt using the lasergene program of
DNASTAR. T he cDNA seq uence a lso had a
polyadenylation signal AATAAA at position 1 438 nt and a
Fig.1. Agarose gel electrophoresis for the cloning of oilseed rape
enolase gene by RACE-PCR. a, b, c and d, the 1 205 bp, 440 bp,
300 bp and 1 335 bp amplified DNA bands of RT-PCR, 3 end, 5
end (nested) and the full-length cDNA (ORF), respectively. M,
molecular marker (DL2000 DNA marker).
ZHAO Jing-Ya et al.: Molecular Cloning and Characterization of Enolase from Oilseed Rape (Brassica napus) 1229
polyA tail. The cDNA contained a 1 335 bp open reading
frame (ORF) encoding a protein of 444 amino acid residues
with isoelectric point (pI) of 5.78 and calculated molecular
weight of about 47.38 kD.
2.2 Sequence analysis of B. napus enolase gene
In the present study, we cloned the enolase gene from
B. napus. A number of deductions could be made from the
inspection of the amino acid alignment, including B. napus
with prokaryotic (B. subtilis) and eukaryotic enolases, such
as A. thaliana, C. reinhardtii. B. napus, L. esculentum, S.
cerevisiae, Z. mays and H. sapiens. The derived consensus
sequence covered all amino acids found in the active site
region of yeast enolase. Higher eukaryotic enolases had an
increased number of cysteines ( > 3), all of which appeared
at positions different from the single cysteine in Bacillus
or yeast enolase. Furthermore, all of the active site residues
and the conformational metal ion-binding site residues were
conserved in the predicted amino acid sequence. Another
striking feature was the occurrence of a 5-amino-acid inser-
tion in enolase of B. napus (EWGWC, residues 107-111).
The pentapeptide EWGWC insertion is only found in
streptophytes (land plants and charophyte green algae)
and alveolates (apicomplexa and ciliates), but not in
chlorophytes, red algae, or any other eukaryotes with the
exception of Chlorarachnion (Keeling and Palmer, 2001).
The high degree of similarity among enolases is shown
in Table 1, where pairwise comparison was presented. The
B. napus enolase protein shared 95.5%, 89.0%, 86.5% and
58.8% identity with other enolase proteins from higher
plants including Arabidopsis, tomato, maize and
Chlamydomonas, respectively. The B. napus enolase pro-
tein also shared 70.2% identity with human enolase protein,
and shared much lower identity with enolase proteins from
Bacillus and yeast. All the plant enolases had a lower simi-
larity when compared with yeast than do human compared
with yeast. By using Swiss-Model and Swiss-PDBViewer,
the three-dimensional model of B. napus enolase was gen-
erated (http: //www.expasy.org / swissmod / SWISS-
MODEL.html) (Guex and Peitsch, 1997). As shown in Fig.2,
DomainⅠ contained some residues (G266-Y272). The pen-
tapeptide EWGWC insertion only found in plants was ob-
served in Domain Ⅱ, where it likely formed a small external
loop, which was recently shown by comparative modeling
with the yeast enolase structure (Dzierszinski et al., 1999).
The residues of B. napus enolase corresponding to the po-
sition of the indel in question were T248, G251, K252 and these
positions were mapped to a small loop as shown in Fig.2.
2.3 DNA and RNA analyses
To determine the copy number of enolase gene in the B.
napus genome, we performed genomic DNA gel blot hy-
bridization analysis. Total genomic DNA from B. napus was
digested with the restriction enzymes, fractionated,
transferred, and hybridized with either the partial-length
cDNA or the 3 untranslated region (UTR) of enolase as a
Table 1 Amino acid similarity between different prokaryotic and eukaryotic enolases (%)
1 2 3 4 5 6 7 8
1 100
2 49.2 100
3 43.8 51.1 100
4 50.3 57.2 58.8 100
5 50.2 57.7 59.9 95.5 100
6 49.2 58.8 58.7 86.5 85.7 100
7 49.7 59.0 61.0 89.0 90.1 86.8 100
8 49.3 63.2 56.9 70.2 70.9 67.9 70.6 100
1, Bacillus subtilis; 2, Saccharomyces cerevisiae; 3, Chlamydomonas reinhardtii; 4, Brassica napus; 5, Arabidopsis thaliana; 6, Zea mays; 7,
Lycopersicon esculentum; 8, Homo sapiens.
Fig.2. The three-dimensional structure of oilseed rape enolase.
The amino acid residues corresponding to the three loops (Ⅰ,
266-272; Ⅱ, 107-111; Ⅲ, 248-252) and amino acid residues
(for example, E178, H389, R390 and K412) constituting the active site
of the enzyme are both indicated according to their positions.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041230
Fig.3. Southern blotting analysis and organ-specific expression of enolase in Brassica napus. A. Genomic DNA (10 µg/sample) was
isolated from leaves of B.napus and digested with BamHⅠ and XhoⅠ，respectively, followed by hybridization with partial-length
enolase cDNA (left) or 3 UTR fragment probe (right). DNA sizes are indicated on the left. B. Organ-specific expression of the enolase
gene. In each lane, 30 µg of total RNAs prepared from leaves and roots was loaded. The blotted membrane was hybridized with the
partial-length enolase cDNA (left) or 3 UTR fragment probe (right). The 28s rRNA was used as internal control paralleling in Northern
blot (lower panel).
Fig.4. Expression profiles of enolase under salt stress and low temperature stress in Brassica napus. Total RNA (1 µg/lane) was isolated
at 0, 0.5, 1, 3, 7, 12, 24 and 48 h after exposure to 100 mmol/L NaCl and 4 ℃ respectively, and subjected to one-step RT-PCR
amplification (upper panel). The entire experiments were repeated twice using total RNA isolated from the roots of oilseed rape as
templates. The actin cDNA was used to normalize the amount of templates added in PCR reactions (lower panel).
probe. Southern blotting analysis was carried out under
conditions of high stringency. As shown in Fig.3A, a small
number of bands were observed for each DNA digest when
the partial-length enolase cDNA was used as a probe. When
the 3 UTR sequence of enolase was used as a specific
probe, similar results were obtained for the corresponding
DNA digest. Since these endonucleases had no cleavage
sites within the enolase cDNA, and introns exist within the
region of genomic sequence corresponding to the enolase
full-length cDNA (data not shown), enolase was likely to
be a low-copy gene in the genome of B. napus.
To monitor the expression pattern of the enolase gene,
we investigated differences in mRNA levels in photosyn-
thetic and nongreen tissue. As shown in Fig.3B, transcripts
corresponding to the enolase gene were detected abun-
dantly in roots and weakly in leaf tissue with either the
partial-length cDNA or the 3 UTR sequence of enolase as
a probe. This result indicated the tissue specificity of
enolase gene expression.
2.4 Expression profiles of B. napus enolase in stress
conditions
In order to examine the influence of various stresses on
enolase expression, total RNA (1 µg) extracted from B.
napus roots was used as template to detect enolase
transcript’s levels at the different time points after expo-
sure to salt and low temperature treatment by one-step
RNA PCR analysis and Northern blot. The results showed
that the enolase gene transcripts were temporarily declined
0.5 h after exposure to 100 mmol/L NaCl, but subsequently
increased and reached the maximum level after 48 h
treatment. However, low-temperature stress (4 ℃) de-
creased the enolase transcript level 1 h after the treatment,
and the enolase transcript level decreased gradually to
the minimum level after 48 h treatment (Fig.4). Northern
blotting analysis further confirmed the one-step RNA PCR
results (Fig.5).
ZHAO Jing-Ya et al.: Molecular Cloning and Characterization of Enolase from Oilseed Rape (Brassica napus) 1231
3 Discussion
In this report, we described the cloning of enolase gene
from B. napus. Like other enzymes involved in glycolysis
and gluconeogenesis, enolase is one of the most conser-
vatively evolved glycolytic enzymes (Fothergill-Gilmore and
Michels, 1993). Highly conserved amino acid residues that
are the ligands for “conformational” Mg2+ (Lebioda et al.,
1989) that participate in substrate binding, that function in
enolase catalysis (Brewer et al., 1993) or that participate in
the catalytic region of the enzyme in yeast (Lebioda et al.,
1989) have been documented.
Primary structure comparison of enolases throughout
evolution showed that the B. napus enolase protein shared
much higher identity with other higher plant enolase pro-
teins than did with Bacillus, yeast, and human beings, which
is consistent with the previous results that animals and
fungi are sister groups while plants constitute indepen-
dent evolutionary lineage (Baldauf and Palmer, 1993). Eu-
karyotic enolases were, on average, 48.8% identical to the
prokaryotic Bacillus counterpart. This high degree of con-
servation is a property of most glycolytic enzymes, which
is also reflected by their exceptionally low rate of evolution,
about as low as cytochrome c (Fothergill-Gilmore, 1986).
Obviously, most of the residues located in or near the ac-
tive site (Lebioda et al., 1989) remain unchanged. Strikingly,
however, yeast enolase has a significantly lower similarity
to its plant counterparts (56.8%) than to its animal counter-
parts (63.2%). This might imply that yeast and plant pro-
genitor genes have diverged earlier than yeast and animal
ancestral genes. This implication is consistent with the
multikingdom tree proposed by Sogin (1989).
Furthermore, one notable indel found in B. napus eno-
lase amino acid sequence, at positions 107-111, underscores
the specificity of a five-amino acid insertion previously re-
ported to be shared by cytosolic enolases from higher
plants. Interestingly, Chlamydomonas enolase sequence
does not share the insertion at positions 107-111. In light-
grown Chlamydimonas, glycolysis is primarily localized in
the plastid, suggesting that the Chlamydimonas enolase is
a plastid isoform, consistent with its shared lacking of the
insertion at positions 107-111 (Hannaert et al., 2000). An-
other notable indel found in B. napus enolase amino acid
sequence is that like the other eukaryotic sequences, it
lacks the insertion at positions 249-250 (PG)，which
might tend to favor an archaebacterial ancestry of the eu-
karyotic enzyme.
To further examine this possibility in detail, we mapped
the corresponding position to the three-dimensional struc-
ture of B. napus enolase. As shown in Fig.2, some residues,
(G266-Y272) and (E107-C111) were observed in Domains Ⅰ
and Ⅱ, respectively and these short fragments of only five
or seven amino acids seemed to be responsible for the in-
creased length of the loop. Insertions and deletions in other
residues such as T248-K252 might be easily tolerated by
the proteins without altering enzyme function.
Enolase is encoded by more than one gene in yeast and
in vertebrates. On the basis of genomic Southern blotting
analysis, it was reported that enolase was represented by
multigene families in all plants examined except A. thaliana,
Z. mays, E. phyllopogon and E. cruspavonis, in which a
single gene encoded enolase (Forsthoefel et al., 1995). In
the present study, Southern blotting analysis revealed that
B. napus enolase gene was likely to be a low-copy gene.
In plants, plant cells rely primarily on photosynthesis
f o r t h e i r e n e r g y s u p p l i e s . H o w e v e r , i n
nonphotosynthetically active cells, glycolysis plays a role
in energy metabolism. In Arabidopsis and tomato, the ex-
pression of enolase in roots is higher than in leaves (van
Der Straeten et al., 1991). Similarly, messenger RNA in oil-
seed rape demonstrated that enolase expression is also more
prevalent in roots than in green tissue. This is consistent
with the higher demand for energy from glycolysis in non-
green tissue.
In the absence of oxygen, there is an energy crisis caused
by the limitation of oxidative phosphorylation of ADP. To
survive, most plants must shift their metabolisms from the
oxidative pathway to a fermentative pathway. This
Fig.5. Northern blotting analysis of oilseed rape enolase gene. Total RNA (30 µg/lane) was isolated from the roots of oilseed rape at
different stages after salt and cold stress, followed by hybridization with partial enolase cDNA fragment as the probe (upper panel). The
28s rRNA was used as internal control paralleling in Northern blot (lower panel).
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041232
metabolic transition is marked by a dramatic induction at
the levels of transcription and translation of a few key fer-
mentation enzymes, such as enolase (Lal et al., 1998), ADH
(Freeling, 1973) and PDC (Kelley, 1989). In our study, we
observed that the transcript of B. napus enolase gene in-
creased after exposure to 100 mmol/L NaCl, and decreased
after low temperature treatment (Figs.4, 5). In maize and ice
plant, steady-state enolase transcript accumulation oc-
curred in roots in response to low temperature (Lal et al.,
1991; Forsthoefel et al., 1995). Moreover, in ice plant, a
transient decrease in enolase expression occurred in leaves
after 12 h of salt stress, and then increased rapidly through-
out 5 d of stress (Forsthoefel et al., 1995). This may have
been the result of a salt shock, which temporarily decreased
enolase gene transcript.
Enolase has been known to be present in all living or-
ganisms since firstly identified in yeast. In our study, we
reported for the first time the cloning and characterization
of a new enolase gene in B. napus. The B. napus enolase
gene was found to be stress inducible. In order to elucidate
more biological roles of enolase in B. napus, further inves-
tigation is required to determine if the stressed expression
of enolase involves phosphorylation or other posttransla-
tional modifications.
References:
Baldauf S L, Palmer J D. 1993. Animals and fungi are each other’s
closest relatives: congruent evidence from multiple proteins.
Proc Natl Acad Sci USA, 90: 11558-11562.
Brewer J M, Robson R L, Glover C V C, Holland M J, Lebioda L.
1993. Preparation and characterization of the E168Q site-
directed mutant of yeast enolase 1. Protein Struct Funct Genet,
17: 426-434.
Chin C C Q, Brewer J M, Wold F. 1981. The amino acid sequence
of yeast enolase. J Biol Chem, 256: 1377-1384.
Dennis E S, Walker J C, Llewellyn D J, Ellis J G, Singh K, Tokuhisa
J G, Wolstenholme D R, Peacock W J. 1987. The response to
anaerobic stress: transcriptional regulation of genes for anaero-
bically induced proteins. Von Wettstein D, Chua N J. Plant
Molecular Biology. New York: Plenum Publishing Corp. 407-
417.
Dzierszinski F, Popescu O, Toursel C, Slomianny C, Yahiaoui B,
Tomavo S. 1999. The protozoan parasite Toxoplasma gondii
expresses two functional plant-like glycolytic enzymes. J Biol
Chem, 274: 24888-24895.
Forsthoefel N R, Mary A F C, John C C. 1995. Posttranscrip-
tional and posttranslational control of enolase expression in
the facultative crassulacean acid metabolism plant mesembry-
anthemum crystallinum L. Plant Physiol, 108: 1185-1195.
Fothergill-Gilmore L A. 1986. The evolution of the glycolytic
pathway. Trends Biochem Sci, 11: 47-51.
Fothergill-Gilmore L A, Michels P A M. 1993. Evolution of
glycolysis. Prog Biophys Mol Biol, 59: 105-235.
Fox T C, Andrews D L, Mujer C V, Drew M C. 1993. Effect of
oxygen level on expression of enolase in flooding tolerant and
intolerant plants. Plant Physiol, 102: 80-89.
Freeling M. 1973. Simultaneous induction by anaerobiosis or 2,4-
D of multiple enzymes specified by two unlinked genes: dif-
ferential Adh1-Adh2 expression in maize. Mol Gen Genet, 127:
215-227.
Giallongo A, Feo S, Moore R, Croce C M, Showe L C. 1986.
Molecular cloning and nucleotide sequence of a full-length
cDNA for human enolase. Proc Natl Acad Sci USA, 83: 6741-
6745.
Goodwin T W, Mercer E I. 1983. Introduction to Plant
Biochemistry. 2nd ed. Oxford: Pergamon Press.
Guex N, Peitsch M C. 1997. SWISS-MODEL and Swiss-
PdbViewer. An environment for comparative protein modeling.
Electrophoresis, 18: 2714-2723.
Hannaert V, Henner B, Ulrich N, William M. 2000. Enolase from
Trypanosoma brucei, from the amitochondriate protist
mastigamoeba balamuthi, and from the chloroplast and cyto-
sol of Euglena gracilis: pieces in the evolutionary puzzle of
the eukaryotic glycolytic pathway. Mol Biol Evol, 17: 989-
1000.
Iida H, Yahara I. 1985. Yeast heat-shock protein of MR 48 000 is
an isoprotein of enolase. Nature, 315: 688-690.
Keeling P J, Palmer J D. 2001. Lateral transfer at the gene and
subgenic levels in the evolution of eukaryotic enolase. Proc
Natl Acad Sci USA, 98: 10745-10750.
Kelley P M, Tolan D R. 1986. The complete amino acid sequence
for the anaerobically induced aldolase from maize derived from
cDNA clones, Plant Physiol, 82: 1076-1080.
Kelley P M. 1989. Maize pyruvate decarboxylase mRNA is
induced anaerobically. Plant Mol Biol, 13: 213-222.
Lal S K, Johnson S, Conway T, Kelley P M. 1991. Characteriza-
tion of a maize cDNA that complements an enolase-deficient
mutant of Escherichia coli. Plant Mol Biol, 16: 787-795.
Lal S K, Lee C, Sachs M M. 1998. Differential regulation of
enolase during anaerobiosis in Maize. Plant Physiol, 118: 1285-
1293.
Lebioda L, Stec B, Brewer J M. 1989. The structure of yeast
enolase at 2.25-Å resolution. An 8-fold b+a-barrel with a
novel bbaa(ba)6 topology. J Biol Chem, 254: 3685-3693.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laboratory Manual. New York: Cold Spring Harbor Labora-
tory Press.
Sogin M L. 1989. Evolution of eukaryotic microorganisms and
ZHAO Jing-Ya et al.: Molecular Cloning and Characterization of Enolase from Oilseed Rape (Brassica napus) 1233
their small subunit ribosomal RNAs. Am Zool, 29: 487-499.
Umeda M, Uchimiya H. 1994. Differential transcript levels of
genes associated with glycolysis and alcohol fermentation in
rice plant (Oryza sativa L.) under submergence stress, Plant
Physiol, 106: 1015-1022.
van Der Straeten D, Rodrigues R A, Goodman H M, van Montagu
M. 1991. Plant enolase: gene structure, expression, and
evolution. Plant Cell, 3: 719-735.
Verma M. 1989. Primary structure of Bacillus subtilis enolase
gene deduced from c-DNA sequence. Biochem Int, 18: 667-
672.
Weng M, Makaroff C A, Zalkin H. 1986. Nucleotide sequence of
Escherichia coli pyrG encoding CTP synthetase. J Biol Chem,
261: 5568-5574.
Zhang F, Lin J J, Fox T C, Kennedy R A. 1994. Effect of aerobic
priming on the response of Echinochloa crus-pavonis to
anaerobic stress, Plant Physiol, 105: 1149-1157.
(Managing editor: ZHAO Li-Hui)