全 文 ：*This work was supported by grants from Program for New Century
Excellent Talents (NCET-06-0256) and National Basic Research
Program of China (2006CB910600).
**Corresponding author.
Tel: 86-311-86269144, E-mail: cuisujuan@263.net
Received: November 15, 2008 Accepted: January 9, 2009
Site-directed Mutagenesis of Arabidopsis
Calmodulin Isoform 2 and Its Application
in Detecting Calcium-independent
Calmodulin-binding Proteins*
GAO Li, WANG Zhen-Jie, CUI Su-Juan**
(Institute of Molecular and Cell Biology, Hebei Key Laboratory of Molecular and Cellular Biology,
Hebei Normal University, Shijiazhuang 050016, China)
Abstract Not only calmodulin (CaM) with Ca2+ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca2+
bound) and Ca2+-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the
interaction between free-CaM and Ca2+-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase
chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM21, mCaM212, mCaM2123, mCaM2124 and
mCaM21234 were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more
EF-hand Ca2+-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently
separated on SDS-PAGE in the presence of Ca2+ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand
motifs. 45Ca2+ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca2+ in the mCaM2
proteins. The mCaM21234 mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca2+. Using yeast two-hybrid technique with
mCaM21234 as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding
protein. Site-directed mutation of AtCaM2 will aid the research of Ca2+, CaM and Ca2+-independent CaMBPs in plant biological
processes.
Key words site-directed mutagenesis, Arabidopsis, calmodulin, calcium-independent, calmodulin-binding protein
DOI: 10.3724/SP.J.1206.2008.00786
生物化学与生物物理进展
Progress in Biochemistry and Biophysics
2009, 36(7): 890~896
www.pibb.ac.cn
研究报告Research Papers
In plants, calcium ion (Ca2+) has important roles
in regulating cellular responses to extensive stimuli of
both biotic and abiotic stresses[1, 2]. Calmodulin (CaM),
which is a highly conserved and heat-stable protein
with four EF-hand motifs, is a vital Ca2+ sensor. The
CaM existence form is different between animal and
plant: the genomes of vertebrates contain multiple
CaM genes that only encode a single CaM isoform,
and the genomes of yeasts and filamentous fungi
contain single genes encoding one CaM isoform [3～5].
But in higher plants encode and express a variety of
CaM isoforms[6]. In the Arabidopsis genome, there are
11 CaM genes that encode at least seven isoforms [7].
This suggests that plants have a more-complex
regulatory mechanism for Ca2+ -signal via Ca2+ -CaM
than animals do.
In most cases, activated CaM (i.e. Ca2+-CaM)
mediates the activity of many CaM-binding proteins
(CaMBP), such as protein kinases [8], transcription
factors [6], nuclear proteins [9], metabolic enzymes [10],
cytoskeleton proteins [11], ion transporters, and
channels [12], and this kind of Ca2+-dependent CaMBPs
have been well characterized. But there is another
CaM existing form, Ca2+-free form (apo-CaM) [13, 14],
which can bind another kind of Ca2+-independent
CaMBPs. At first Ca2+-independent CaMBPs were
studied in animals, including structural proteins, and
signaling proteins involved in neurotransmitter
production and release, nerve growth, muscle
高丽等：拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用2009; 36 (7)
relaxation, and intracellular movement of organelles
along actin filaments [15～17], but in plants they have not
receive a great deal of attention. Most of the
Ca2+-independent CaMBPs are much less well
characterized and its functions are insufficient
understood. It was proposed that these
Ca2+-independent CaMBPs alter the Ca2+-binding
dynamics of free CaM and activated CaM [18]. The
Ca2+-independent CaMBPs influence the formation of
activated CaM by accelerating the rates of association
and dissociation of Ca2+ from the free CaM. However
the Ca2+-dependent CaMBPs depend on the activation
of activated CaM. In this way the Ca2+-independent
CaMBPs indirectly regulate the biological function of
the Ca2+-dependent CaMBPs[18～20]. But, until now, there
was no evidence of an in vivo interaction between
CaM and Ca2+-independent CaMBPs, which restrict us
to elucidate the activities of Ca2+-independent CaMBPs
regulated by free-CaM.
In our research we first isolated five mutated
Arabidopsis calmodulin isoform 2 (AtCaM2) genes. Of
these, mutant CaM21234 (mCaM21234) (E32Q; E68Q;
E105Q; E141Q) could not bind with Ca2+ in the
presence of 1×10-7 mol/L Ca2+, which is similar to the
cytosolic Ca2+ concentration in plant cells [7]. We use
mCaM21234 in yeast to test the interaction between
CaMBPs and CaM2 depending on Ca2+ or not. We also
can use it as bait in yeast two-hybird system to detect
the novel Ca2+-independent CaMBPs in Arabidopsis.
Using mCaM21234 as bait in yeast two-hybrid, we
provided in vivo evidence of interactions between
CaM and AtIQD26 (At3g16490), one Ca2+-independent
CaMBP in Arabidopsis[21, 22].
1 Materials and methods
1.1 Materials
Synthetic oligonucleotide primers for polymerase
chain reaction (PCR) and PCR-based site-directed
mutagenesis were obtained from Sangon (China,
http://www.sangon.com). Taq DNA polymerase,
restriction enzymes and T4-DNA ligase were obtained
from TaKaRa (Japan, http://www.takara.com.cn). The
Muta-directTM site-directed mutagenesis kit used in our
study was a product of Saibaisheng (SBS) Genetech
Company (China, http://www.sbsbio.com).
Escherichia coli BL21 and the plasmid pET28b
were from Novagen (Germany, http://www.novagen.
com). Plasmid vectors (pGADT7 and pGBKT7) and
AH109 yeast cells were bought from Clontech (Japan,
http://www.clontech.com). pET5a-AtCaM2 (GenBank
accession number NM_179766) was a gift from the
Zielinski laboratory (Ray Zielinski’s Laboratory at the
University of Illinois, http://www.life.uiuc.edu/zielinski).
1.2 Gene cloning and plasmid construction
Full-length cDNA for AtCaM2 was amplified by
PCR from the plasmid pET5a-AtCaM2 using the
forward primer CaM2f (5′ GGAATTCCATATGGCA-
GATCAGCTCACCG 3′ ) and reverse primer CaM2r
(5′ CGGAATTCTCACTTTGCCATCATAACTTTG 3′).
The PCR product, which was cut by the restriction
endonucleases enzymes NdeⅠ and EcoRⅠ, was ligated
into plasmids to form the plasmid pET28b-CaM2.
pET28b-CaM2 was then used to produce the CaM2
recombination protein and the yeast two-hybrid bait
vector pGBKT-CaM2.
IQD26 from RNA extracted from the pollen of
Arabidopsis thaliana (Col-0) seedling was amplified by
reverse transcriptase PCR (RT-PCR) using the IQD26f
(5′ GGAATTCATGGGAAGAGCTGCGAGATGGT-
TCA 3′ ) and IQD26r (5′ CGGGATCCCTAATTAT-
GAATCTAAATCAGTCT 3′ ) primers. The RT-PCR
product, cut with EcoRⅠ and BamHⅠ enzymes, was
ligated into plasmids to produce a pET28b-IQD26
plasmid. This was, in turn, used to produce the IQD26
recombination protein and pGADT7-IQD26, which
was used to test the pGBKT7-CaM2 interaction in
yeast.
1.3 Site-directed mutagenesis by PCR
The point mutations of AtCaM2 were made by
PCR using synthetic oligonucleotide primers (Table 1)
containing the desired mutation, with pET28b-CaM2
as amplified template. The method was performed
using Muta-DirectTM Enzyme DNA polymerase and a
temperature cycle (stepⅠ: 95℃ for 30 s for one cycle;
stepⅡ : 95℃ for 30 s, then 55℃ for 1 min; finally,
72℃ for 1 min for 15 cycles) according to the
Muta-directTM site-directed mutagenesis kit manual.
The PCR product was treated with MutazymeTM
Enzyme to digest the parental DNA template. The
mutation-containing synthesized DNA was then
transformed into DH5α supercompetent cells. DNA
sequencing was performed to determine the changes in
base pairing.
1.4 Recombinant protein expression and
purification
BL21 E. coli bacteria, with ahead construct
pET28b, were used to produce recombinant proteins
CaM2-His, mutant CaM2-His. The single-clone
891· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2009; 36 (7)
bacteria were grown overnight in 5 ml Lysogeny broth
containing 50 mg/L Kanamycin (LB-Kan50) at 37℃.
5 ml of the culture was inoculated with 250 ml of fresh
LB-Kan50 and grown at 37℃ until the cells reached a
density of A600= 0.6. Isoporopylthio-β-D-galactoside
(IPTG) was then added to a final concentration of
1 mmol/L and the culture was allowed to grow for a
further 3 h. Finally, the cells were collected and
centrifuged at 6 000 g at 4℃ for 10 min. The cell
pellet was suspended in bufferⅠ(50 mmol/L Tris
pH 7.5; 0.5 mmol/L DTT) on ice for 30 min, and
sonicated on ice for 10 min. The lysate was centrifuged
at 40 000 g at 4℃ for 45 min, and the resultant
supernatant was loaded onto a His-selected Nickel
Affinity Gel from Sigma (United States, http://www.
sigmaaldrich.com) according to their manuals, to
obtain the purified recombinant proteins CaM2-His
and mutant CaM2-His.
1.5 SDS-PAGE mobility shift
2 μl of BL21 harboring the expression plasmids
pET28b-CaM2, pET28b-CaM21, pET28b-CaM212,
pET28b-CaM2123, pET28b-CaM2124, pET28b-CaM21234
were dissolved in SDS sample buffer (0.1 mol/L
Tris-HCl, pH 6.8, 30% glycerol, and 2% SDS) in the
presence of either 5 mmol/L CaCl2 or 5 mmol/L EGTA,
then heated at 95～100℃ for 5 min. The samples were
centrifuged at 13 000 g at 4℃ for 5 min prior to
loading on gels. 12% SDS-PAGE was used to separate
the proteins (i.e. the recombinant CaM and the various
mutant CaM proteins). The mobility ratio was
calculated as the distance migrated by the molecule to
that migrated by bromophenol blue. The data
processed in GraphPad Prism 4 (http://www.graphpad.
com).
1.6 45Ca2+ overlay
2 μg of recombinant CaM2 and mutated-CaM2
purified by His-Select Nicked Affinity Gel were run on
a 12% SDS-PAGE gel and then transferred to a
Polyvinylidene-Fluoride (PVDF) membrane. The
PVDF membranes were incubated in 45Ca2+ buffer
(60 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L
imidazole-HCl pH 6.8) with 2.96 × 1012 μBq/L 45Ca2+
(7.4×1010 Bq/L, 95.3 mg/L, Amersham Biosciences
from Sweden, http:// www.amersham.com) at 23℃ for
30 min with shaking. After rinsing three times with
50% ethanol for 5 min, the membranes were dried at
room temperature for 3 h. Finally, they were exposed
to a storage phosphor screen for 12 h. Images were
scanned using a Typhoon 9210 imager (Amersham
Biosciences) and analyzed using Quantity One
software and the data processed in GraphPad Prism 4
(http://www.graphpad.com).
1.7 Yeast two-hybrid
The coding regions of CaM2, mutated-CaM2 or
IQD26 were constructed by PCR and confirmed by
DNA sequencing. The constructs were then introduced
into the NcoⅠ/BamHⅠ site of pGBKT7 and pGADT7,
respectively. Yeast cells (AH109 strain) were
co-transformed with different pGADT7 and pGBKT7
constructs. The transformants were streaked onto
media lacking Tryptophan and Leucine (media lacking
Trp and Leu), and cultured at 30℃ for 3 days. The
yeast cells were then streaked onto media lacking
Adenine, Histidine, Trp and Leu (media lacking Ade,
His, Trp and Leu), and incubated at 30℃ for 3 days.
pGADT7-RecT/pGBKT7-p53 and pGADT7-RecT/
pGBKT7-Lam served as positive and negative
controls. LacZ activity was detected in the yeast cells
using X-gal as a substrate.
2 Results and Discussion
2.1 Site-directed mutagenesis of AtCaM2
CaM has four Ca2+ binding EF-hand domains. The
Ca2+ binding ability of CaM depends on the conserved
amino acid in the EF-hand domain. The more
conserved amino acid in the EF-hand domain was
mutated, the weaker binding ability with Ca2+ [7]. Here
we wanted to alter the Ca2+ binding ability of CaM2 by
site-directed mutagenesis of the conserved amino acid
in the EF-hand domain. AtCaM2 was cloned into
pET28b vector to obtain pET28b-CaM2 expression
plasmid. According to the Muta-directTM site-directed
mutagenesis kit manual, we got pET28b-mCaM21 using
pET28b-CaM2 as the PCR template with the
mCaM21F, and mCaM21R primers (Table 1); we
generated pET28b-mCaM212 using the PCR template
pET28b-mCaM21 and the mCaM22F, and mCaM22R
primers (Table 1); then with the similar method we got
pET28b-mCaM2123, pET28b-mCaM2124 and pET28b-
mCaM21234 one by one based on the former well being
done. These expression plasmids were transformed
into E. Coli BL21 strain to produce recombinant
mCaM2 mutant proteins, with Glutamate (E) replaced
by Glutamine (Q) in the EF-hand motifs of CaM2
(Table 2).
892· ·
高丽等：拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用2009; 36 (7)
2.2 The electrophoresis mobility and Ca2+-binding
ability of mutant CaM
The same amount of induced BL21, harboring the
expression plasmids pET28b-CaM2, pET28b-mCaM21,
pET28b-mCaM212, pET28b-mCaM2123, pET28b-mCaM2124
or pET28b-mCaM21234 was directly dissolved in SDS
buffer, and analyzed by SDS-PAGE in the presence of
either 5 mmol/L CaCl2 or 5 mmol/L EGTA(Figure 1a).
All proteins except mCaM21234 exhibited the
characteristic Ca2+-dependent electrophoretic mobility
shift, which is dependent on the number and location
of the mutated EF-hand domain of CaM2. The
mobility-shift difference of wild-type CaM2 in the
presence of CaCl2 or EGTA was larger than that of
mutant CaM2 (Figure 1a). It is obviously that the
relative mobility ratio of mCaM21234 with Ca2+ to
EGTA was almost equal to 1 (Figure 1b). Thus, our
results show that the speed of Ca2+-dependent
electrophoretic mobility is relative to the number of
EF-hand motifs present. However, mCaM21234 is
different because its mobility shift was identical in the
presence of both CaCl2 and EGTA (Figure 1a and 1b).
To identify the calcium-binding ability of the
mutant CaM2 proteins, the more-sensitive method of
45Ca2+ overlay was adopted (Figure 2). From the protein
band stained by amino-black in the PVDF, all the lanes
were with the approximately same amount of protein,
but autoradiography film of 45Ca2+ overlay showed
different radiations of the protein bands, and the 45Ca2+
radiation in wild-type CaM2 band was the strongest
(Figure 2a). The mutation CaM2 binds Ca2+ ability is
obviously weakened. The more EF-hand motifs were
altered, the weaker binding with Ca2+ (Figure 2a).
Ca2+-binding ability was slightly reduced by the
alteration of the EF-hand 1, of EF-hand 1 and 2, or of
EF-hand 1, 2, and 3. However, it was markedly
weakened by the alteration of EF-hand 1, 2, and 4.
When all four EF-hand (1～4) were mutated, mCaM21234
displayed no Ca2+ binding at all (Figure 2a). We
defined the relative 45Ca2+-binding ability of the mutant
CaM2 proteins using Quantity One software based on
the results of 45Ca2+ overlay assay. The 45Ca2+-binding
ability of normal CaM2 was defined as 100% . Then
the relative 45Ca2+-binding ability of mutant CaM2 were
defined (Figure 2b). Bovine serum albumin (BSA)
Primer Primer sequence (5′～ 3′)1)
mCaM21F GGTTGCATCACAACGAAACAGCTAGGAACAGTGATGA
mCaM21R TCATCACTGTTCCTAGCTGTTTCGTTGTGATGCAACC
mCaM22F GGAACCATAGACTTCCCTCAGTTTCTGAACCTAATGG
mCaM22R CCATTAGGTTCAGAAACTGAGGGAAGTCTATGGTTCC
mCaM23F GGTTTCATCTCGGCAGCTCAGTTAAGACATGTAATGA
mCaM23R TCATTACATGTCTTAACTGAGCTGCCGAGATGAAACC
mCaM24F GGTCAGATCAATTATGAACAGTTTGTCAAAGTTATGATGGC
mCaM24R GCCATCATAACTTTGACAAACTGTTCATAATTGATCTGACC
Table 1 The oligonucleotides primers used in
site-directed mutagenesis of AtCaM2
1) The mutated residues are underlined.
Table 2 Summary of CaM2 mutations
Mutant protein Mutation amino acid Mutant site in
mCaM21 E32Q EF-hand 1
mCaM212 E32Q; E68Q EF-hand 1,2
mCaM2123 E32Q; E68Q; E105Q EF-hand 1,2 and 3
mCaM2124 E32Q; E68Q; E141Q EF-hand 1,2 and 4
mCaM21234 E32Q; E68Q; E105Q; E141Q EF-hand 1,2 and 3,4
E: Glutamate; Q: Glutamine.
893· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2009; 36 (7)
could not bind Ca2+, and its 45Ca2+-binding ability was
equal to background of PVDF. Obviously the relative
Ca2+-binding ability of mCaM21234 was similar to that of
BSA, that was to say, the Ca2+-binding ability of
mCaM21234 was completely lost in our experiment
conditions(Figure 2b). We used 2.96×1012 μBq/L 45Ca2+
in the expreriment system, the final concentration of
45Ca2+ was equal to 1×10-7 mol/L, which is equal to the
resting cytosolic Ca2+ concentration in plant cells [7]. So
the mCaM21234 couldn’t bind Ca2+ at the resting
cytosolic Ca2+ concentration in plant cells.
To understand the structure and function of CaM
in the activation of its target proteins, several mutant
CaMs have been generated in vitro in plant and animal
systems. To determine which domains are responsible
for differential activation of target NAD kinase, a
series of chimeric soybean CaMs were generated by
exchanging functional domains between sCaM4 and
sCaM1 [23]. And, to learn more about the roles of the
individual Ca2+-binding site in Drosophila melanogaster
CaM, Maune et al.[24] changed the conserved glutamate
into glutamine or lysine in the four EF-hand motifs. In
our present study, we first changed the amino acids
glutamate into glutamine in the EF-hand domains of
Arabidopsis CaM2, which resulted in altered
Ca2+-binding ability. Especially the mCaM21234
displayed no Ca2+-binding ability under the normal
resting cytosolic Ca2+ concentration of plant cells
(Figure 2).
2.3 Detecting Ca2+-independent CaM-binding
proteins in yeast using mCaM21234
CaM overlays in vitro have been used to
determine whether the interaction of CaMBP with
CaM was dependent on Ca2+ or not, but there is no
valid method in vivo to identify the CaMBPs bind
CaM depending on Ca2+ or not. Now we can use the
mCaM21234 as bait in yeast two-hybird system to detect
the novel Ca2+-independent CaMBPs in Arabidopsis.
To prove the feasibility we chose the AtIQD26 as a
control in vivo, which is a putative Ca2+-independent
CaMBP[21, 22]. We had previously confirmed that IQD26
interacted with AtCaM2 in a Ca2+-independent manner
in vitro by Bio-CaM or 35S-CaM [22]. In the present
study, we used the mutant CaM21234 as bait to further
identify the Ca2+-independent CaM-binding characteristic
of IQD26 in yeast two-hybrid system (Figure 3). Yeast
co-transformed with pGBKT7-mCaM21234 and
pGADT7-IQD26 showed blue on X-gal filter assay and
grew on media lacking Trp, Leu, His and Ade, while
the system positive and negative controls were normal.
Recently, several Ca2+-independent CaMBPs have
been found in plant and animal, and their
894· ·
高丽等：拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用2009; 36 (7)
Ca2+-independent CaM-target interactions are
extensive, involving transcription factor OsCBT from
rice [25], unconventional myosins [20], Ca2+ channels and
PEP-19[18], GAP-43[26, 27] which exist in neuronal tissues.
All of these suggest that Ca2+-independent CaMBPs
play important roles in growth and development of
plant and animal. But until now only few of
Ca2+-independent CaMBPs in the plant cells have been
found and their functions have not been well
charactered. To investigate the biological roles of
Ca2+-independent CaMBPs, the first step is to isolate
them. Using the mutant CaM21234 as bait in yeast
two-hybrid system, we will identify more
Ca2+-independent CaMBPs in plant cells. Depending
on this method we have proved that AtIQD26
interacted with CaM2 in a Ca2+-independent manner
(Figure 3); or mutant CaM21234 gene was transformed
into plant to compete with free-CaM, breaking the
balance of Ca2+, free-CaM and Ca2+-CaM, and then we
could observe the phenotype for exploring the
functions of free-CaM and Ca2+-independent CaMBPs
in plant development and responses to environmental
stimuli.
Acknowledgement We thank Ray Zielinski for
presenting pET5a-AtCaM2 plasmid.
References
1 Jabs T, Tsch觟pe M, Colling C, et al. Elicitor-stimulated ion fluxes
and O2 - from the oxidative burst are essential components in
triggering defense gene activation and phytoalexin synthesis in
parsley. Proc Natl Acad Sci USA, 1997, 94 (9): 4800～4805
2 Reddy V S, Ali G S, Reddy A S. Genes encoding calmodulin-
binding proteins in the Arabidopsis Genome. J Biol Chem, 2002,
277 (12): 9840～9852
3 Davis T N, Urdea M S, Masiarz F R, et al. Isolation of the yeast
calmodulin gene: calmodulin is an essential protein. Cell, 1986, 47
(3): 423～431
4 Takeda T, Yamamoto M. Analysis and in vivo disruption of the gene
encoding for calmodulin in Schizosaccharomyces pombe. Proc Natl
Acad Sci USA, 1987, 84 (11): 3580～3584
5 Toutenhoofd S L, Strehler E E. The calmodulin multigene family as
a unique case of genetic redundancy: multiple levels of regulation to
provide spatial and temporal control of calmodulin pools?. Cell
Calcium, 2000, 28 (2): 83～96
6 Zielinski R E. Calmodulin and calmodulin-binding proteins in
plants. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49 (1): 697～
725
7 Yang T B, Poovaiah B W. Calcium/calmodulin-mediated signal
network in plants. Trends Plant Sci, 2003, 8 (10): 505～512
8 Sathyanarayanan P V, Cremo C R, Poovaiah B W, et al. Plant
chimeric Ca2+/calmodulin-dependent protein kinase. Role of the
neural visinin-like domain in regulating autophosphorylation and
calmodulin affinity. J Biol Chem, 2000, 275 (39): 30417～30422
9 Reddy A S, Day I S, Narasimhulu S B, et al. Isolation and
characterization of a novel calmodulin-binding protein from potato.
J Biol Chem, 2002, 277 (6): 4206～4214
10 Yap K, Yuan T, Mal T K, et al. Structural basis for simultaneous
binding of two carboxy-terminal peptides of plant glutamate
decarboxylase to calmodulin. J Mol Biol, 2003, 328 (1): 193～204
11 Reddy A S. Calcium: silver bullet in signaling. Plant Sci, 2001, 160
(3): 381～404
12 Chung W S, Lee S H, Kim J C, et al. Identification of a
calmodulin-regulated soybean Ca2+-ATPase (SCA1) that is located
in the plasmamembrane. Plant Cell, 2000, 12 (8): 1393～1407
13 Komeiji Y, Ueno Y, Uebayasi M. Molecular dynamics simulations
revealed Ca2+-dependent conformational change of Calmodulin.
FEBS Lett, 2002, 521 (19): 133～139
14 Komeiji Y, Uebayasi M. Peach-Grape system - a high performance
simulator for biomolecules. Chem-Bio Informatics J, 2002, 2 (4):
102～118
15 Akyol Z, Bartos J A, Merrill M A, et al. Apo-calmodulin binds with
its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0
region. J Biol Chem, 2004, 279 (3): 2166～2175
16 Serysheva I I, Ludtke S J, Baker M R, et al. Structure of the
voltage-gated L-type Ca2+ channel by electron cryomicroscopy. Proc
Natl Acad Sci USA, 2002, 99 (16): 10370～10375
17 Zhang M, Tanaka T, Ikura M. Calcium-induced conformational
transition revealed by the solution structure of apo calmodulin. Nat
Struct Biol, 1995, 2 (9): 758～767
18 Putkey J A, Kleerekoper Q, Gaertner T R, et al. A new role for IQ
motif proteins in regulating calmodulin function. J Biol Chem,
2003, 278 (50): 49667～49670
19 Shah V N, Wingo T L, Weiss K L, et al. Calcium-dependent
regulation of the voltage-gated sodium channel hH1: Intrinsic and
extrinsic sensors use a common molecular swich. Proc Natl Acad
Sci USA, 2006, 103 (10): 3592～3597
20 Houdusse A, Gaucher J F, Krementsova E, et al. Crystal structure of
apo-calmodulin bound to the first two IQ motifs of myosin V reveals
essential recognition feature. Proc Natl Acad Sci USA, 2006, 103
(51): 19326～19331
21 Abel S, Savchenko T, Levy M. Genome-wide comparative analysis
of the IQD gene families in Arabidopsis thaliana and Oryza sativa.
BMC Evol Biol, 2005, 5 (1): 72～96
22 韦慧彦,郭振清,王振杰,等.拟南芥钙调素结合蛋白 AtIOD26的
分离鉴定.生物化学与生物物理进展, 2008, 35 (6): 703～711
Wei H Y, Guo Z Q, Wang Zhi J, et al. Prog Biochem Biophys, 2008,
35 (6): 703～711
23 Sang H L, Hae Y S, Jong C K, et al. Differential activation of NAD
kinase by plant calmodulin isoforms. J Biol Chem, 1997, 272 (14):
9252～9259
24 Maune J F, Klee C B, Beckingham K. Ca2+ binding and
895· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2009; 36 (7)
拟南芥钙调素定点突变基因分离及其在
钙不依赖钙调素结合蛋白检测中的应用 *
高 丽 王振杰 崔素娟 **
(河北师范大学分子细胞生物学研究室，河北省分子细胞生物学重点实验室，石家庄 050016)
摘要 动植物系统研究表明，钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性，而且没有钙离子结合时，还可以通
过结合钙不依赖的钙调素结合蛋白，发挥多种生物学作用．然而，目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相
互作用的方法．首先，采用定点突变的方式，得到了拟南芥钙调素亚型 2的多个突变基因 mCaM2，随后，大肠杆菌重组表
达突变蛋白的电泳迁移率及 45Ca2+覆盖分析表明，得到了编码失去钙结合能力的钙调素的突变基因 mCaM21234, mCaM21234突变
钙调素中所有 4个钙结合 EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中，作为诱饵蛋白的
突变钙调素 mCaM21234与我们前期体外方法报道的钙不依赖性钙调素结合蛋白 AtIQD26存在相互作用．这将为钙不依赖性钙
调素结合蛋白提供有用的体内研究工具，有利于我们全面认识钙 -钙调素 -钙调素结合蛋白信号途径．
关键词 定点突变，拟南芥，钙调素，钙不依赖，钙调素结合蛋白
学科分类号 Q291 DOI: 10.3724/SP.J.1206.2008.00786
* 教育部新世纪优秀人才支持计划(NCET-06-0256)和国家重点基础研究发展计划(973)(2006CB910600)部分资助项目.
**通讯联系人.
Tel: 0311-86269144, E-mail: cuisujuan@263.net
收稿日期：2008-11-15，接受日期：2009-01-09
conformational change in two series of point mutations to the
individual Ca2+-binding sites of calmodulin. J Biol Chem, 1992, 267
(8): 5286～5295
25 Choi M S, Kim M C, Yoo J H, et al. Isolation of a calmodulin-
bindng transcription factor from rice. J Biol Chem, 2005, 280 (49):
40820～40831
26 Esdar C, Oehrlein S A, Reinhardt S, et al. The protein kinase C
(PKC) substrate GAP-43 is already expressed in neural precursor
cells, colocalizes with PKCeta and binds calmodulin. Eur
J Neurosci, 1999, 11 (2): 503～516
27 Liu Y C, Storm D R. Regulation of free calmodulin levels by
neuromodulin: neuron growth and regeneration. Trends Pharmacol
Sci, 1990, 11 (3): 107～111
896· ·