CuZnSOD - ResearchGate

Transcription

A r t i c l e
MOLECULAR CHARACTERIZATION
AND OXIDATIVE STRESS
RESPONSE OF AN
INTRACELLULAR Cu/Zn
SUPEROXIDE DISMUTASE
(CuZnSOD) OF THE WHITEFLY,
Bemisia tabaci
Jun-Min Li, Yun-Lin Su, Xian-Long Gao, Jiao He,
Shu-Sheng Liu, and Xiao-Wei Wang
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop
Pathogens and Insects, Institute of Insect Sciences, Zhejiang University,
Hangzhou, China
Superoxide dismutases (SODs) are important for the survival of insects
under environmental and biological stresses; however, little attention has
been devoted to the functional characterization of SODs in whitefly. In
this study, an intracellular copper/zinc superoxide dismutase of whitefly
(Bemisia tabaci) (Bt-CuZnSOD) was cloned. Sequence analysis
indicated that the full length cDNA of Bt-CuZnSOD is of 907 bp with a
471 bp open reading frame encoding 157 amino acids. The deduced
amino acid sequence shares common consensus patterns with the
CuZnSODs of various vertebrate and invertebrate animals. Phylogenetic
analysis revealed that Bt-CuZnSOD is grouped together with intracellular CuZnSODs. Bt-CuZnSOD was then over-expressed in E. coli and
purified using GST purification system. The enzymatic activity of
purified Bt-CuZnSOD was assayed under various temperatures. When
whiteflies were exposed to low (41C) and high (401C) temperatures, the in
vivo activity of Bt-CuZnSOD was significantly increased. Furthermore,
Grant sponsor: National Natural Science Foundation of China; Grant number: 30730061; Grant sponsor:
National Basic Research Program of China; Grant number: 2009CB119203; Grant sponsor: Earmarked fund for
Modern Agro-industry Technology Research System, Fundamental Research Funds for the Central Universities.
Correspondence to: Xiao-Wei Wang, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop
Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou
310029, China. E-mail: xwwang@zju.edu.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20428
2
Archives of Insect Biochemistry and Physiology, 2011
we measured the activities of several antioxidant enzymes, including
SOD, catalase and peroxidase, in the whitefly after transferring the
whitefly from cotton to tobacco (an unfavorable host plant). We found
that the activity of SOD increased rapidly on tobacco plant. Taken
together, these results suggest that the Bt-CuZnSOD plays a major role in
C 2011 Wiley
protecting the whitefly against various stress conditions. Periodicals, Inc.
Keywords: Bemisia tabaci; Cu/Zn superoxide dismutase (CuZnSOD);
oxidative stress; whitefly
INTRODUCTION
Reactive oxygen species (ROS) are constantly generated in all aerobic biological
systems as natural products of oxidative metabolism. These include superoxide,
hydrogen peroxide (H2O2), hydroxyl radicals, nitric oxide (NO), and singlet oxygen.
ROS cause oxidative stress and can be toxic to many cell components such as lipids,
proteins, and nucleic acids (Fridovich, 1986; Cadenas, 1989; Halliwell and Gutteridge,
1999). Recent studies indicated that ROS, such as H2O2 and NO, are not only just
acting as destructive molecules but also involved in various intracellular signaling
pathways as important second messengers (Neill et al., 2002; Rhee, 2006; Groeger
et al., 2009). To minimize the damaging effects of ROS, organisms have well-developed
defense systems, including nonenzymatic and enzymatic antioxidants, against
oxidative injury by limiting the formation of ROS as well as instituting its removal
(Fattman et al., 2003). The enzymatic antioxidants include superoxide dismutases
(SODs; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7) and
so on. SODs are one of the first lines of enzymatic defenses against ROS by catalyzing
the dismutation of superoxide radicals into molecular oxygen and H2O2 (Fridovich,
1975; Holmblad and Soderhall, 1999). SODs are a group of metalloenzymes which are
classified into three basic distinct groups according to their metal cofactor: copper/zinc
SOD (CuZnSOD), manganese SOD (MnSOD), and iron SOD (FeSOD) (Zelko et al.,
2002). CuZnSOD is primarily found in the cytosol of eukaryotes and rarely in bacteria
(Crapo et al., 1992). In contrast, MnSOD is found in prokaryotes and in the
mitochondria of eukaryotes, whereas FeSOD is found primarily in bacteria as well as in
the chloroplasts of some green plants (Ken et al., 2005).
CuZnSOD is an important type of SOD because of its physiological function and
therapeutic potential. It is a homodimeric enzyme that requires zinc and copper for its
structural integrity and enzymatic activity (Xu et al., 2009). The loss of copper results
in its complete inactivation and induces many diseases in organism (Mizuno, 1984;
Hough and Hasnain, 1999; Lindberg et al., 2004). Two types of CuZnSOD are found
in eukaryotes: extracellular CuZnSOD (ecCuZnSOD) with an N-terminal signal
cleavage peptide for secretion and intracellular CuZnSOD (icCuZnSOD) without
signal peptide. Previous studies indicated that ec-SOD can be secreted in two forms,
intact and cleaved, due to intracellular proteolytic processing of the carboxyl terminus
containing the polybasic residues. The proteolytic removal of the carboxyl terminus
could serve as a regulatory step by affecting both the affinity and distribution of
ec-SOD to the extracellular matrix (Enghild et al., 1999; Bowler et al., 2002).
icCuZnSOD is present in the cytoplasm and nucleus, whereas ecCuZnSOD is found in
Archives of Insect Biochemistry and Physiology
Intracellular CuZnSOD of the Whitefly
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the extracellular matrix of tissues such as lympha and plasma (Fattman et al., 2003).
Molecular characterization of CuZnSOD has been investigated in various species
(Ni et al., 2007). Previous studies showed that CuZnSOD is a main proximate cause of
aging. Over expression of CuZnSOD can extend the life span of adult fruit fly
Drosophila melanogaster (Sun and Tower, 1999) and yeast (Fabrizio et al., 2003). In
contrast, loss of CuZnSOD activity by mutation can dramatically reduce the viability and
lifespan of Drosophila (Phillips et al., 1989). Furthermore, CuZnSOD is also a major
defense mechanism to many biological stimulators such as heat shock (Yoo et al.,
1999a), heavy metals (Yoo et al., 1999b) and infection (Neves et al., 2000). For example,
the level of CuZnSOD from fat body cells of mole cricket Gryllotalpa orientalis was
significantly increased during the exposure to low (41C) and high (371C) temperature
compared with control (251C), suggesting that CuZnSOD might have an important role
in the protection of cricket against oxidative damage caused by temperature stress (Kim
et al., 2005b). In addition, the activity of SOD is related to pesticide resistance in insects.
When the granary weevils Sitophilus granarius were treated with fumigant insecticide
phosphine (PH3), elevated SOD activity occurred probably in response to an increase in
O
2 generation during the treatment (Bolter and Chefurka, 1990).
The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidea) is one of the
most damaging agricultural pests of vegetable, ornamental, and fiber crops (Brown
et al., 1995; De Barro et al., 2011). Recent phylogenetic analyses and crossing
experiments indicate that the whitefly complex contains at least 28 cryptic species
(Dinsdale et al., 2010; Xu et al., 2010; Wang et al., 2010b, 2011; De Barro et al., 2011;
Hu et al., 2011). Among the 24 putative species within B. tabaci delineated by Dinsdale
et al. (2010), one of them, namely the ‘‘Middle East-Asia Minor 1’’ (commonly referred
to as B biotype, hereafter MEAM1), has risen to international prominence since the
1980s due to its worldwide distribution (Brown et al., 1995; Boykin et al., 2007; Liu
et al., 2007). The global distribution of whitefly MEAM1 is partially due to its high
fitness parameters such as a wide range of host plants, high fecundity and high survival
rate under various environmental stresses (Lu and Wan, 2008). Preliminary studies on
the correlation between stress and the SOD activity of whitefly showed that SOD plays
a vital role for the survival of the whitefly in areas with extremely high temperature
(Rosell et al., 2008). Those findings indicate that the activity of SOD is probably
important for the survival of the whitefly under detrimental conditions. However, little
attention has been devoted to the functional characterization of SOD in whitefly. With
the aim at furthering understanding the potential role of SOD in the defense system
against various environmental stresses in MEAM1, we cloned and characterized
CuZnSOD gene of B. tabaci MEAM1 (hereafter Bt-CuZnSOD). To gain an insight into
the physiological roles of Bt-CuZnSOD, the activity of SOD and relative antioxidant
enzymes were further explored in vivo under extreme temperature and host shift
(from favorable to unfavorable host plant) stresses.
MATERIALS AND METHODS
Whitefly Cultures
The invasive whitefly MEAM1 (mtCO1 sequence GenBank accession no. GQ332577),
collected from Zhejiang, China, were used in this study. Whitefly cultures were
maintained on cotton plants (Gossypium hirsutum L. cv. Zhemian 1793). The details of
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the methods for maintaining the stock cultures were described in Liu et al. (2007)
and Jiu et al. (2007). The purity of the cultures was monitored every 3–5 generations
using the random amplified polymorphic DNA-polymerase chain reaction technique
with the primer H16 (50 -TCTCAGCTGG-30 ) (De Barro and Driver, 1997; Luo
et al., 2002).
RNA Extraction
Total RNA was extracted from 50 mg of adult whiteflies using SV total RNA isolation
system (Promega, Madison, WI) according to the manufacturer’s protocol (Wang et al.,
2010c). The quality of the total RNA samples was assessed by the ratio of absorbance
at 260 and 280 nm wavelength and further checked by electrophoresis in 1.2%
formaldehyde agarose gel.
Cloning and Sequencing of Full-length Bt-CuZnSOD cDNA
An EST homologous of copper/zinc SOD was found from the whitefly cDNA library
(GenBank accession no. EE598784) (Leshkowitz et al., 2006). Based on this fragment
sequence, 50 and 30 RACE were performed using the SMART RACE cDNA
Amplification Kit (Clontech, Palo Alto, CA) to obtain its 50 and 30 ends. Gene-specific
primers for the 50 (50 -ACGACCATGATATTCAGAGGGC-30 ) and 30 (50 -TTCAAGGATTAGCTCCAGGGC-30 ) RACE were used following the manufacturer’s instructions.
The PCR products were cloned into the pMD18-T vector (Takara, Dalian, China) and
sequenced in both directions using M13 primers.
Homology Analysis
The sequence was analyzed for similarity with known genes using the Basic Local
Alignment Search Tool (BLASTx) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide and deduced amino acid sequence of Bt-CuZnSOD cDNA were analyzed using
software DNAMAN version 6 (http://www.lynnon.com). The signal peptide of
Bt-CuZnSOD was predicted with the SignalP program version 3.0 (http://www.
cbs.dtu.dk/services/SignalP). The molecular mass and theoretical isoelectric point (pI)
of Bt-CuZnSOD were calculated using ProtParam tool (http://kr.expasy.org/tools/
protparam.html). The potential N-Glycosylation site was predicted by NetNGlyc 1.0
Server (http://www.cbs.dtu.dk/services/NetNGlyc/).
Phylogenetic Analysis
The deduced amino acid sequence of Bt-CuZnSOD was compared with ecCuZnSODs
and icCuZnSODs of other species available in GenBank (Table 1). Alignment of
multiple sequences was performed with the CLUSTAL W program version 2.0.12 at
the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw). A phylogenetic
tree was constructed based on the amino acid sequences (Table 1) using the Neighborjoining (NJ) algorithm in MEGA4 program (http://www.megasoftware.net/). The
reliability of the branching was tested using bootstrap method (1,000 replications).
Construction of Bt-CuZnSOD Expression Vector
The open reading frame (ORF) of Bt-CuZnSOD was PCR amplified using the forward
primer Bt-CuZnSOD-F 50 -GGATCCATGGCTGGCAAAACCAAAGC-30 (BamH1 restriction
site is underlined and the start codon is shown in bold, italic) and reverse primer
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Table 1. Sequences of CuZnSOD Used for the Alignment and Phylogenetic Analysis
Species
Invertebrates
Platyhelminths
Schistosoma mansoni
Taenia solium
Nematodes
Caenorhabditis elegans
Trichinella pseudospiralis
Arthropods
Insects
Apis mellifera ligustica
Bemisia tabaci
Drosophila melanogaster
Drosophila melanogaster
Lasius niger
Musca domestica
Plutella xylostella
Crustaceans
Callinectes sapidus
Macrobrachium rosenbergii
Mollusks
Aplysia californica
Crassostrea gigas
Vertebrates
Fish
Danio rerio
Oncorhynchus mykiss
Amphibia
Bufo gargarizans
Xenopus laevis
Reptilia
Caretta caretta
Birds
Gallus gallus
Melopsittacus undulates
Mammals
Bos taurus
Canis familiaris
Homo sapiens
Homo sapiens
Mus musculus
Abbreviation
GenBank no.
Type of CuZnSOD
S. mansoni-ic
T. solium-ic
AAA29936.1
AAL66230.1
Intracellular CuZnSOD
C. elegans-ec
T. pseudospiralis-ic
NP_499091.1
AAM76075.1
Extracellular CuZnSOD
A. mellifera-ic
B. tabaci-ic
D. melanogaster-ec
D. melanogaster-ic
L. niger-ec
M. domestica-ic
P. xylostella-ic
AY329355
HQ230310
AAL25378.1
P61851
AAV85459.1
AAR23787.1
BAD52256.1
C. sapidus-ec
M. rosenbergii-ec
AAF74772.1
AAZ29240.1
A. californica-ic
C. gigas-ic
AAM44291.1
CAD42722.1
D. rerio-ic
O. mykiss-ic
AAH55516.1
AAL79162.1
B. gargarizans-ic
X. laevis-ic
ABD75370.1
P15107.3
C. caretta-ic
P80174.2
G. gallus-ic
M. undulates-ic
NP_990395.1
AAO72711.1
B. taurus-ic
C. familiaris-ic
H. sapiens-ec
H. sapiens-ic
M. musculus-ec
NP_777040.1
NP_001003035
AAA66000.1
NP_000445.1
AAB51106.1
-ic indicates intracellular CuZnSOD; -ec indicates extracellular CuZnSOD.
Bt-CuZnSOD-R 50 -GTCGACTCAATACTTGGTGATTCCAAT-30 (Sal1 restriction site
is underlined). The PCR product was first ligated with pMD18-T vector (Takara,
Dalian, China) and transformed into E. coli DH5a competent cell. For expression of
GST fusion proteins, the pMD18-T-SOD vector was digested with BamH1 and Sal1, gel
purified, and ligated into the pGEX-4T-3 plasmid (GE Healthcare, Piscataway, NJ)
digested with the same enzymes. The recombinant plasmid was transformed into E.
coli BL21 (DE3) pLysS cells (Novagen, Madison, WI) and positive clones were further
confirmed by DNA sequencing.
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Expression and Purification of Bt-CuZnSOD
The Bt-CuZnSOD was over-expressed in E. coli BL21 (DE3) pLysS cells by the addition
of 0.5 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) to cells in logarithmic phase
(OD600 approached 0.4–0.6). The growth conditions for expression were 301C and
200 rpm. After 3 h of induction, the cells were harvested by centrifugation at 5,000g for
15 min at 41C and resuspended in lysis buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 5 mM
DTT). The cells were sonicated at 400 W for 30 times with a 10 sec pause between
sonication intervals on ice. The resulting suspension was centrifuged at 14,000g for
15 min at 41C to remove cell debris. The supernatant containing soluble proteins was
ready for purification. The purification of fusion protein was performed using the GST
gene fusion system as described (Guan and Dixon, 1991). In brief, the supernatant
containing the soluble GST-SOD recombinant protein was incubated with Glutathione
Sepharose 4B (GE Healthcare, Piscataway, NJ) equilibrated with 1 PBS for 30 min at
room temperature. Bound fusion protein was then eluted and collected using elution
buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The GST-tag was cleaved
from fusion protein at 41C for 5 h using twenty units of thrombin solution which
specifically recognizes and cleaves a sequence located upstream of the multiple cloning
sites. The purity of the protein from various steps was evaluated by 12% sodium
denaturing dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Enzyme Assays and Thermal Stability of Bt-CuZnSOD
The enzyme activity of Bt-CuZnSOD was determined by the pyrogallol auto-oxidation
method (Marklund and Marklund, 1974). One unit of enzyme activity was defined as
the amount of enzyme that results in 50% inhibition of the auto-oxidation rate of
pyrogallol under the assay condition. In order to determine the thermal stability of the
purified Bt-CuZnSOD, the enzyme was incubated at various temperatures of 37, 50,
60, 70, 801C for 0 (control), 5, 10, 30, 60, 120, 240 min and the residual enzyme
activity was measured. Three replicates of each treatment were conducted. The data
obtained from treatments were analyzed using one-way analysis of variance. When a
significant effect was detected at Po0.01 level, the mean values among the treatments
were compared using an LSD test. All statistical analyses were conducted using the
DPS data processing system software 8.50 for Windows (Tang and Feng, 2007).
The in vivo Activity of Bt-CuZnSOD under High- and Low-Temperature Stress
Newly emerged whiteflies were obtained from the culture and sexed under a stereomicroscope. For each treatment, 50 female whiteflies were collected in a 5 ml tube and exposed to
4, 25, 401C for 0 (control), 10, 30, and 60 min in a climatic chamber, respectively. After each
treatment, the samples were frozen immediately with liquid nitrogen and stored at 801C.
Bt-CuZnSOD activity of those samples was determined as described above. In all treatments,
each assay was replicated three times. Statistical analyses for the differences among various
sampling times of each temperature and comparison among the three temperature regimes
within each sampling time were carried out. All statistical analyses were performed as
described in ‘‘enzyme assays and thermal stability of Bt-CuZnSOD.’’
The in vivo Activity of SOD, CAT, POD in Response to Host Shift
Whitefly adults obtained from the culture on cotton were transferred to tobacco,
Nicotiana tabacum cv. NC89—an unfavorable host plant of the whitefly (Xu et al., 2011).
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After 0 (control), 1, 3, and 5 days of feeding on the tobacco, whiteflies were collected
for the measurement of the SOD, CAT, and POD activity, respectively. The collection
method for the whiteflies and the determination of the total SOD activity were
performed as described above. CAT activity was measured according to Goth (1991)
and POD activity was performed with the method provided by Simon et al. (1974). All
statistical analyses were performed as described in ‘‘enzyme assays and thermal
stability of Bt-CuZnSOD.’’
RESULTS
Cloning and Characterization Analyses of Bt-CuZnSOD
The full-length Bt-CuZnSOD cDNA, obtained by 50 and 30 RACE PCR, consists of
907 bp, with a 471 bp ORF encoding for 157 amino acids residues. Bt-CuZnSOD cDNA
sequence contains a 50 -untranslated region (UTR) of 111 nucleotides, a long 30 -UTR of
325 nucleotides including a stop codon (TGA), a putative polyadenylation consensus
signal (AATAAA) and a poly (A) tail (Fig. 1A and B). The calculated molecular mass of
the Bt-CuZnSOD is 16.08 kDa with an estimated pI of 5.85. SignalP program analysis
revealed that no signal peptide is present in Bt-CuZnSOD. One putative N-glycosylation
site was found, suggesting that Bt-CuZnSOD might be a glycoprotein (Fig. 1B).
Bt-CuZnSOD cDNA sequence and its deduced amino acid sequence were submitted to
the NCBI GenBank under accession number HQ230310.
Homology Analyses of Bt-CuZnSOD
Multiple sequences alignment revealed that Bt-CuZnSOD shares a common consensus
pattern with the CuZnSODs of various species (Fig. 2). The residues required for
copper (His-48, -50, -65, and -122) and zinc (His-65, -73, -82, and Asp 85) binding and
the two cysteines (Cys-59 and Cys-148) involved in disulphide bridge formation are
well conserved in different species. The disulfide bond Cys59-Cys148 stabilizes the
loop region containing the metal ligands, whereas the Arg145 might guide the
superoxide radical to the active site (Djinovic et al., 1992; Malinowski and Fridovich,
1979). Furthermore, two Cu/Zn signatures from 46 to 56 (GFHVHEFGDNT), and
from 140 to 151 (GNAGARLSCGVI) are highly conserved as well (Fig. 2).
Phylogenetic Analyses of Bt-CuZnSOD
Using the NJ method, a phylogenetic tree was constructed based on the amino acid
sequences of selected CuZnSODs. All the icCuZnSODs clustered together as a
subgroup and ecCuZnSODs clustered to another group (Fig. 3). Bt-CuZnSOD was in
the group of icCuZnSODs, suggesting that it is an intracellular CuZnSOD. In addition,
Bt-CuZnSOD is clustered with A. mellifera and then formed a sister group to
D. melanogaster and M. domestica. The topology approximately reflected the taxonomic
classification of the corresponding species (Fig. 3).
Expression and Purification of Recombinant Bt-CuZnSOD
The recombinant Bt-CuZnSOD was successfully over-expressed in E. coli BL21 (DE3)
when induced with 0.5 mM IPTG for 3 h at 301C as shown in Figure 4 (lane 1).
Moreover, the over-expressed Bt-CuZnSOD was proved to be in soluble form after the
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Figure 1. Domain structure and amino acid sequence of Bt-CuZnSOD. (A) Bt-CuZnSOD is composed of
two Cu/Zn SOD signatures (red), an N-glycosylation site (green) and disulfide bonds (red solid line). (B) The
nucleotide and deduced amino acid sequences of Bt-CuZnSOD cDNA. The letters in black box indicate the
start codon (ATG) and the stop codon (TGA). The polyadenylation signal sequence (AATAAA) is double
underlined. Potential N-glycosylation site is shown in bold green italic letters (NITD). The two conserved
Bt-CuZnSOD signatures are shown in bold red letters. Highly conserved amino acids critical for Cu (His-48,
-50, -65, and -122) and Zn (His-65, -73, -82, and Asp 85) binding are circled. Two cysteines (Cys-59 and
Cys-148) predicted to be engaged in the disulfide bond formation are in blue box.
analysis of the supernatant (lane 2). The expressed protein was purified with GST
purification system. The purified GST-fusion protein migrated as a 42 kDa band which
is consistent with the estimated molecular weight of the recombinant protein
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Figure 2. Multiple sequences alignment of deduced protein sequence of the whitefly intracellular
CuZnSOD (icCuZnSOD) with the extracellular CuZnSOD (ecCuZnSODs) and icCuZnSODs of other species.
The conserved amino acid residues are shaded with different color. Four residues required for binding
copper (Cu), four residues required for binding zinc (Zn), and two cysteines residues that form a disulfide
bridge (SS) are indicated by arrows. The two conserved CuZnSOD signatures (Signature 1 and Signature 2)
are boxed in blue. The consensus head and consensus tail for icCuZnSODs are boxed in black. See Table 1
for the abbreviation of species and GenBank accession numbers.
(Bt-CuZnSOD: 16 kDa and GST tag: 26 kDa) (lane 5). When the fusion protein
was further cleaved by thrombin, Bt-CuZnSOD migrated as a 16 kDa band during
SDS-PAGE analysis (lane 6).
Thermal Stability of Bt-CuZnSOD
To study the temperature stability of Bt-CuZnSOD, we treated the purified BtCuZnSOD under different temperatures. As shown in Figure 5, Bt-CuZnSOD
maintained more than 80% activity after incubated at 371C for 240 min. With the
temperature of 50 and 601C, the enzymatic activity of Bt-CuZnSOD decreased
significantly after 5 min (601C) and 10 min (501C) (Po0.01) but still retained up to 50%
after 240 min incubation. When the temperature increased up to 70 and 801C, the
enzyme activity decreased rapidly in the initial 30 min (Po0.01) and was completely
inactivated after 120 min (801C) and 240 min (701C) incubation, respectively (Fig. 5).
In vivo Activity of Bt-CuZnSOD under High- and Low-Temperature Stress
Extreme temperature is one of the major stress factors faced by all living organisms.
To characterize the effect of external temperature stimulus on the in vivo activity of
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Figure 3. A phylogenetic tree of ecCuZnSODs and icCuZnSODs amino acid sequences from 27 different
species was constructed. Numbers at each branch node represent the values given by bootstrap analysis. The
abbreviation of CuZnSODs and the GenBank accession numbers used to construct phylogenetic tree are
given in Table 1. GenBank accession numbers are in brackets.
Bt-CuZnSOD, female adult whiteflies were exposed to 4, 25, 401C for various time
points (0, 10, 30, and 60 min). The differences of Bt-CuZnSOD activity between
various sampling times are shown in Figure 6. No significant differences were detected
at 251C from 0 to 60 min. When whiteflies were exposed at low temperature (41C) and
high temperature (401C), still, no significant differences of the activity were observed
after 10 and 30 min exposure compared with 0 min. After 60 min of incubation, both
activities at 40 and 41C increased significantly compared with 0 min (marked with two
asterisks as shown in Fig. 6) (Po0.01). Furthermore, we compared the activities of
Bt-CuZnSOD among the three temperature regimes of each sampling time. No
significant differences were found at 0, 10, and 30 min. However, after 60 min of
exposure, significant differences were recorded among each of the three temperatures
(marked with majuscules as shown in Fig. 6) (Po0.01). Interestingly, the activity of
CuZnSOD increased significantly higher under 401C than that of 41C (Po0.01),
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Figure 4. Expression and purification of the Bt-CuZnSOD. Lane M, molecular weight marker; lane 1, cell
lysate after induced with 0.5 mM IPTG and grown at 301C for 3 h; lane 2, soluble portion of cell lysate;
lane 3, insoluble portion of cell lysate; lane 4, un-induced cell lysate; lane 5, purified GST-Bt-CuZnSOD
fusion protein; lane 6, GST-Bt-CuZnSOD fusion protein after treatment with thrombin.
Figure 5. Thermal stability of Bt-CuZnSOD. The enzyme was incubated at various temperatures for
different time intervals and the residual enzyme activities were measured. Maximal activity is shown as 100%.
Bars represent means7SD (N 5 3). Significant differences among various time points of each temperature
compared with control (0 min) are indicated with different letters (LSD test, Po0.01).
indicating that the in vivo CuZnSOD activity of whitefly might be more sensitive to
high temperature treatment.
The in vivo Activity of SOD, CAT, POD of Whitefly in Response to Host Shift
Furthermore, we measured the in vivo activity of SOD, CAT, and POD in the whitefly
after transferring from cotton to tobacco for various days as shown in Figure 7. After
transferring and feeding on new host for 1, 3, and 5 days, we found that the activity of
SOD significantly increased compared with 0 day (Po0.01). However, no significant
differences were found for the activity of CAT and in contrast, the activity of POD
significantly decreased with the lapse of the days (Po0.01).
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Figure 6. In vivo Bt-CuZnSOD activity analysis of whitefly exposed to different temperatures for different
time intervals. Bars represent means7SD (N 5 3). Significant differences between various sampling times of
each temperature compared with control (0 min) are indicated with two asterisks (LSD test, Po0.01).
Significant differences among the three temperature regimes within each sampling time are indicated with
majuscules (LSD test, Po0.01).
Figure 7. In vivo activity of SOD, CAT, POD in the whitefly in response to host shift. Bars represent
means7SD (N 5 3). Significant differences of each treatment compared with control (0 day) are indicated
with two asterisks (LSD test, Po0.01).
DISCUSSION
B. tabaci MEAM1 causes billions of dollars worth of crop losses each year worldwide
through phloem feeding, transmission of plant viruses, induction of phytotoxic
disorders, and excretion of honeydew (Brown et al., 1995). MEAM1 invaded China
in the mid-1990s and has become one of the most important agricultural pests because
of its wide range of host plants, high survival rate under stressful conditions and ability
of transmitting plant viruses. Many of these parameters of MEAM1 are critical to
its global invasion and displacement of the indigenous species (Luo et al., 2002). Shah
et al. (2007) indicated that the ability of the whitefly to survive in extreme subtropical
climates is related to the regulation of SOD. Thus, we speculate that the prominent
antioxidant—SOD plays important roles in the successful invasion of MEAM1
worldwide. To investigate its potential function, one type of SODs—Bt-CuZnSOD
gene was successfully cloned, expressed, and purified from whitefly MEAM1 in
our study.
13
The ORF of Bt-CuZnSOD includes 471 nucleotides encoding a deduced peptide
of 157 amino acids residues (Fig. 1B). No signal peptide in Bt-CuZnSOD was predicted
by Signal P, indicating that it should be a member of the intracellular CuZnSOD family
(Folz et al., 1997). Multiple sequences alignment of deduced amino acid of icCuZnSODs
and ecCuZnSODs from various species shows that all ecCuZnSODs have a signal
peptide extension in front of the N-terminus, whereas such a signal peptide is absent in
icCuZnSODs. We noticed that all icCuZnSODs have a consensus head sequence
(KAVCVL) in the N-terminus and a consensus tail sequence (GVIGI) in the C-terminus
(Fig. 2). Furthermore, as shown in Figure 2, the eight metal-coordination residues are
highly conserved in all icCuZnSODs and ecCuZnSODs selected for alignment,
indicating that these sites are essential for its structure and function.
CuZnSOD is considered as a general stress responsive factor influenced by a variety of
environmental stresses at transcriptional and/or translational levels (Zelko et al., 2002).
Temperature stress was reported as one of the key mediators of the formation of ROS
(Harari et al., 1989; Rauen et al., 1999). In our experiment, whitefly MEAM1 was exposed
to high (401C), normal (251C), and low (41C) temperatures. As a result, the activity of
Bt-CuZnSOD increased significantly after 60 min under 40 and 41C. In addition, the in
vivo CuZnSOD activity of the whitefly is more sensitive to high-temperature stress. Our
observation is consistent with previous studies in G. orientalis CuZnSOD (Kim et al., 2005a),
Bombus ignitus CuZnSOD (Choi et al., 2006) and Phascolosoma esculenta MnSOD (Wang
et al., 2010a). The ability of the MEAM1 to survive in extreme subtropical climates might
be related to CuZnSOD which acts as an antioxidant protein by reducing the high level of
intracellular superoxide radical induced by high temperature.
Adaptation to new host plants may assist in the successful invasion of the whitefly to
new environments. However, host shift from a favorable plant to an unfavorable plant is
always a big challenge for insects. Jankovic-Hladni et al. (1997) reported that a
nutritionally deficient food and the presence of secondary metabolites in an unfavorable
new host plant induced detoxification and antioxidant responses in Lymantria dispar, and
feeding the moth larvae on an unfavorable host plant led to increases in GST and SOD
activities. Interestingly, in our study, an increase of SOD activity was also found when
whiteflies were transferred from cotton to tobacco—an unfavorable host. We speculate
that adaptation to an unfavorably host plant might activate the antioxidant responses in
the whitefly and SOD probably plays an important role during this process.
In conclusion, the full length of CuZnSOD was successfully cloned, and
characterized from whitefly. Its potential functions in antioxidant defense, which
might contribute to the successful invasion of the whitefly, were elucidated. Our
findings may provide clues for further study on the defense mechanisms of the
whitefly, especially invasive cryptic species such as MEAM1, against oxidative stress
under various unfavorable environmental conditions. More studies are required to
achieve a better understanding of the expression pattern with different types of
whitefly SODs and other antioxidant enzymes in response to various stresses such as
begomovirus infection and pesticide treatments.
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