Curr Microbiol (2009) 58:52–57
DOI 10.1007/s00284-008-9265-y
Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene
from Bacillus thuringiensis with the Neurotoxin Gene hwtx-I
LiQiu Xia Æ XiaoShan Long Æ XueZhi Ding Æ
YouMing Zhang
Received: 19 April 2008 / Accepted: 25 August 2008 / Published online: 25 October 2008
Ó Springer Science+Business Media, LLC 2008
Abstract A fusion gene was constructed by combining the
cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a
neurotoxin gene, hwtx-1, which was synthesized chemi-
cally. In this process, an enterokinase recognition site
sequence was inserted in frame between two genes, and the
fusion gene, including the promoter and the terminator of
the cry1Ac gene, was cloned into the shuttle vector pHT304
to obtain a new expression vector, pXL43. A 138-kDa
fusion protein was mass-expressed in the recombinant strain
XL002, which was generated by transforming pXL43 into
B. thuringiensis acrystalliferous strain XBU001. Quantita-
tive analysis indicated that the expressed protein accounted
for 61.38% of total cellular proteins. Under atomic force
microscopy, there were some bipyramidal crystals with a
size of 1.0 9 2.0 lm. Bioassay showed that the fusion
crystals from recombinant strain XL002 had a higher tox-
icity than the original Cry1Ac crystal protein against third-
instar larvae of Plutella xylostella, with an LC50 (after 48 h)
value of 5.12 lg/mL. The study will enhance the toxicity of
B. thuringiensis Cry toxins and set the groundwork for
constructing fusion genes of the B. thuringiensis cry gene
and other foreign toxin genes and recombinant strains with
high toxicity.
LiQiu Xia and XiaoShan Long contributed equally to this work.
L. Xia (&)
X. Long
X. Ding
Y. Zhang
College of Life Sciences of Hunan Normal University, Key
Laboratory of Microbial Molecular Biology, Changsha 410081,
Hunan Province, China
e-mail: xialq@hunnu.edu.cn
123
Introduction
Bacillus thuringiensis is a gram-positive bacterium that
produces regular parasporal crystals during the sporulation
phase. The crystal proteins are specific and lethal only to
larvae in the orders Lepidoptera, Diptera, Coleoptera, and
Orthoptera [1, 6]. In addition, showing no toxicity to
humans, animals, and nontarget insects, the crystal protein
has great ecological value in agriculture and has become
the most widely used microbiological insecticide in the
world. However, biopesticides based on B. thuringiensis
have some shortcomings, such as the narrower activity
spectrum, inferior stability, and shorter persistence, which
restricts adoption more widely. At present, many research
institutes have constructed recombinant crystal proteins
and B. thuringiensis strains which have a high toxicity and
broad activity spectrum by genetic engineering technology,
such as site-directed mutations, transgenic plants, and
fusion genes [5, 13, 15]. In studies of fusion including
different B. thuringiensis toxin genes and their foreign
toxin gene, researchers have gained some important
advantages in the application of B. thuringiensis [9].
Huwentoxin-I (HWTX-I) is a peptide neurotoxin iso-
lated from the venom of the Chinese bird spider
(Selenocosmia huwena), which can block neuromuscular
transmission in an isolated mouse phrenic nerve-diaphragm
preparation [12]. It is of interest for research on neurobi-
ology and for potential applications as insecticides and
pharmaceuticals. It has been shown that HWTX-I acts as a
presynaptic toxin and can block high-voltage activated
calcium channels. It functions as an inhibitor of the N-type
acetylcholine receptor [11]. The mature peptide of this
toxin is composed of 33 amino acids and the relative
molecular weight is 3.75 kDa. Two-dimensional (2D)-
NMR analysis indicated that it has three intramolecular
L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene
disulfite bonds, and the 3D structure appears to be the
pattern of ‘‘three sheets, three bridges’’ [17]. Previously,
we isolated a B. thuringiensis strain 4.0718 expressing
Cry1Ac with a high toxicity to insects and combined the
cry1Ac with the hwtx-I gene to form a new fusion gene
under the native promoter of the cry1Ac gene, to enhance
the toxicity and broaden the activity spectrum as a bio-
pesticide. After transforming the expression plasmid into
B. thuringiensis acrystalliferous strain (XBU001), the
fusion protein was mass-expressed. Serial examinations of
the protein were done and the toxicity to the larvae of
Plutella xylostella was tested.
Materials and Methods
Bacterial Strains, Plasmids and Medium
Bacterial strains and plasmids are reported in Table 1. B.
thuringiensis (Bt) strain 4.0718 (CCTCC No. M200016)
was from our laboratory collection. Bt acrystalliferous
strain XBU001 was used for expression of the fusion gene
and E. coli DH5a for the cloning. pUC19 was used for
DNA subcloning and sequencing and the shuttle vector
pHT304 was used for expression in XBU001. Recombinant
strain XL002 was grown at 28°C in fermentation medium,
pH 7.3. For solid media, 2% agar was added to the liquid
medium.
Table 1 Strains and plasmids
Strains
DH5a
B. thuringiensis 4.0718 B. thuringiensis strain wild type (CCTCC No. M200016)
XBU001
HC42
UAc19
UAcH19
HAcH43
XBU304
HTX42
XL002
Plasmids
pUC19
pHT304
pHC42
pUAc19
pUAcH19
pXL43
53
Construction of Recombinant Plasmids
A fragment of the cry1Ac gene including the upstream
promoter region was obtained by PCR using the extracted
plasmids as a template. Primer sequences were as follows:
Ac-F SalI, 50 ACGCGTCGACTTGCA GGTAAATGGT
TC 30 ; and Ac-R BglII, 50 GCGC AGATCT AGATT
CCTCCATAAGAGTAA30 . The cDNA sequences (No.
AF157504) of the hwtx-I and Enterokinase sites described
by Ausubel et al. [2] and Liang et al. [12] were designed
according to the preference codon usage of B. thuringien-
sis. The designed sequences were then synthesized
chemically with a BglII restriction site and Enterokinase
recognition site at 5’. After BglII digestion of cry1Ac, PCR
fragments and synthesized hwtx-I fragments were ligated
with T4 DNA ligase to form the fusion gene.
The construction process of expression vector pXL43 is
shown in Fig. 1a. The digestion and transformation were
carried out as elaborated by Sambrook [20]. The fusion
gene fragment carrying the upstream promoter region and
the downstream terminator region of the cry1Ac gene was
cloned into the shuttle vector pHT304, and the resulting
plasmid was designated pXL43. Final expression construct
pXL43 was verified by sequencing.
Electroporation
Electroporation was performed as described by Bauer [19],
and in a Gene Plusher Xcell (Bio-rad), pXL43 was
Characterization Resource
E. coli strain Dr. Jian Zhang
Lab store
B. thuringiensis acrystalliferous strain Lab store
DH5a (pHC42) Lab store
DH5a (pUAc19) This work
DH5a (pUAcH19) This work
DH5a (pXL43) This work
XBU001 (pHT304) Lab store
XBU001 (pTX42Ac) (cry1Ac) Lab store
XBU001 (pXL43) (cry1Ac ? hwtx-1) This work
Ampr TaKaRa Co.
Ampr and Ermr, shuttle vector of E. coli and B. thuringiensis Dr. Sun Ming
pHT304 carried 4.2-kb fragment from PCR Lab store
pUC19 carried 4.2-kb fragment from PCR This work
pUC19 carried 4.3-kb fragment of fusion gene This work
pHT304 carried 4.3-kb fragment of fusion gene This work
123
54
Fig. 1 (a) Diagram of construction of fusion gene expression. (b) Identification of recombinants by PCR of a molecular weight marker (kDNA/
HindIII; lane M), PCR of B. thuringiensis acrystalliferous strain XBU001 (lane 1), and PCR of different recombinant strains (lanes 2–4)
transformed into XBU001 and called XL002, under the
following conditions: 2.0 kV, 200 X, and 25 lF. Positive
recombinants were selected on BHI medium (3.7% brain
heart infusion, 17.1% sucrose, 2.0% agar).
Purification of Fusion Inclusions and SDS-PAGE
The crystal-spore mixture was transferred into a tundish
and mixed with Na2SO4 and CCl4 at the following pro-
portions: crystal-spore suspension:1% Na2SO4:CCl4, 7:6:7
(v/v). The tundish was shaken rigorously for 10 min. The
aqueous phase was kept and centrifuged at 10,000 g and
4°C for 15 min, and the pellets were washed once with
distilled water, twice with 5% acetone, and twice with
distilled water. Finally, the pellets were collected, freeze-
dried, and stored at -80 °C.
Purified fusion inclusions were boiled with loading
buffer and loaded onto sodium dodecyl sulfate (SDS)/8%
polyacrylamide gels and subjected to electrophoresis. They
were then stained with Coomassie brilliant blue and
123
L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene
quantitative analysis was performed with the Gel-pro
Analyzer software (American).
Scanning of Atomic Force Microscope and Insecticidal
Activity Assays
Strain XL002 was grown in fermentation medium with
shaking at 28°C for 72 h. The collection was washed with
distilled water repeatedly and diluted ton a certain optimum
concentration. A trace of solution was dripped on the mica
disk. After air-drying by blowing, the sample was scanned
under the atomic force microscope.
Plutella xylostella eggs were hatched and reared to the
third instar using standard protocols [14]. Purified fusion
inclusions were prepared at five different concentrations.
Groups of 12 larvae were allowed to eat cabbage leaf disks
dipped in the crystal protein suspension of each concen-
tration, and each bioassay was preformed three times.
Bioassay conditions were as follows: 14 h of light and 10 h
of darkness, alternately, a light intensity of 3000 lux, and a
L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene
temperature of 25°C. Larval mortality was recorded at 48 h
after inoculation, each experiment was replicated three
times, and 50% lethal concentration (LC50) values were
calculated as disintegrations per second (dps).
Results
Construction of Fusion Genes and Expression Vector
The plasmid template from B. thuringiensis strain 4.0718
was used for PCR with primer Ac-F and primer Ac-R. A
3.9-kbp fragment was amplified, which was consistent with
the predicted result. A BglII fragment of 3.9 kb was ligated
with the synthetic fragment of hwtx-I and Enterokinase
recognition site. Using this ligated fragment as a template
for amplification of the fusion gene, a 4.0-kb fragment was
amplified with a size as predicted.
Through a series of restriction enzyme digestions and
ligations (Fig. 1a), the fusion gene fragment of cry1Ac
gene was cloned into the shuttle vector pHT304, and the
recombinant plasmid was named pXL43. The fragment in
pXL43 was verified by sequencing and Blast analysis of
the sequencing result shows that the orf of cry1Ac from
strain 4.0718 is identical to the cry1Ac5 gene (accession
no. M73248), and the sequence of the hwtx-I gene and
Enterokinase site corresponds with the designed sequence.
Expression of Fusion Gene Analysis
Identification of recombinants by PCR indicated that the
expression vector was transformed in strain XBU001
(Fig. 1b). The fusion protein was mass-expressed in strain
XBU001 (Fig. 2a). The cry1Ac gene encodes 1178 amino
acids, while the hwtx-1 gene encodes 33 amino acids and
the Enterokinase recognition site encodes 5 amino acids.
The theoretical molecular weight of the fusion protein
approximates 138 kDa, and the experimental data were
approximately equivalent to the theoretical value. Quanti-
tative analysis indicates that the expressed fusion protein
accounted for 61.38% of the total cellular proteins.
Fig. 2 (a) SDS-PAGE analysis
of expressed products of the
fusion gene, protein markers
(lane M), XBU001 strain (lane
1), XBU304 strain (lane 2),
XL002 strain (lane 3), and
purified recombinant crystals
(lane 4). (b) Atomic force
microscopy of recombinant
crystals of the engineering
XL002 strain with different
scanning sizes
55
Scanning and Bioassay Analysis
Cry1A-type protoxins can accumulate in big rhombus
crystal inclusions. The fusion gene cloned also expressed a
large amount of protoxins and big rhombus crystal inclu-
sions were formed in the XL002 strain during sporulation.
Maps of atomic force microscope scans are shown in
Fig. 2b. There were some bipyramidal crystals, 1 9 2 lm.
Bioassays showed that the fusion protein crystals from the
XL002 strain had a high toxicity against larvae of Plutella
xylostella, with an LC50 value of 5.12 lg/mL, and for
HTX42 tthis value was 70.78 lg/mL at 48 h. The toxicity of
XL002 was 13.8 times higher than that of HTX42 at 48 h.
Discussion
We used B. thuringiensis strain 4.0718 as the original
strain, and its genome was analyzed by PCR-RFLP. There
were some cry genes existing on plasmids and 20-kb DNA
in bipyramidal crystals, such as cry1Ac, cry1Aa, cry2Aa,
and cry2Ab [7]. The toxicity experiment demonstrated that
strain 4.0718 was highly toxic to larvae of P. xylostella [8].
Palidam [18] reported that the Cry1Ac crystal of the Cry1
family crystal protein was most toxic to larvae of Heli-
courpa armigori and P. xylostella. Therefore our research
on the fusion gene was based on the cry1Ac gene, and we
tried to construct a new fusion gene with an effective
encoded insecticidal fusion protein.
We aligned the sequence of some cry1Ac genes and
cry1Aa genes published in GenBank and found that both
the 50 -end upstream promoter and the 30 -end orf of the two
genes were highly homologous. Because the cry1Ac gene
and cry1Aa gene coexist on the plasmid from B. thurin-
giensis strain 4.0718, the 3.9-kb PCR fragment probably
contains both genes. In order to obtain the correct recom-
binant plasmid including the cry1Ac gene fragment, we
identified and selected the recombinant plasmid pUAcH19
with PstI digestion. The result showed that both the cry1Aa
gene and cry1Ac gene existed in different recombinant
plasmids.
123
56 L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene
The hwtx-I gene was from venom of the Chinese bird
spider, which was also a foreign gene to B. thuringiensis.
So we designed the DNA sequence of the hwtx-I gene with
the partial codon usage of the B. thuringiensis strain. As for
construction of the expression vector of the fusion gene, we
used the shuttle vector pHT304, which had been used most
widely and successfully as the expression vector, pHT304,
which has two replicons, which can multiply in two kinds
of bacteria (E. coli and B. thuringiensis). However, its lacZ
promoter cannot be recognized by RNA polymerase of B.
thuringiensis. We retained the 50 -end promoting-region
376 bp and the 30 -end terminator-region 248 bp of cry1Ac
gene at the two ends of the fusion gene, respectively.
Within the promoter region, Wong et al. [21] discovered
two adjacent transcript start sites (Bt I and Bt II) by S1
nuclease mapping which belong to the sporulation-depen-
dent-type promoter. Sequencing analysis of terminator
region reveals that there are two stem-loop structures,
which are composed of four reverse repeats and one
poly(T) structure. The two kinds of structures had two
functions of stopping transcription and promoting the sta-
bility of mRNA [16]. From Fig. 2, we can see that the
fusion protein was mass-expressed in XBU001. The causes
are as follows: the two promoters were very suitable for
expression in XBU001; the two stem-loop structures and
poly(T) structure would stop further transcription and
promote the stability of mRNA efficiently; and the
designed DNA sequence of the hwtx-I gene and Enteroki-
nase recognition site with codon usage of the B.
thuringiensis strain was very suitable for expression in
strain XBU001. Formation of the crystals prevented the
protein from being degraded efficiently by the exogenous
proteases.
The fusion protein could still form crystals as cry1
family proteins do. Some reports have shown that the Cry1
family crystals were sustained by intramolecular disulfate
bonds in the C-end conservative region [4], and this was
the key basis for sustaining the stability of the fusion
protein constructed in this study. In addition, the mature
HWTX-I peptide contains three intramolecular disulfate
bonds, which can help fusion proteins form crystals.
Constructed of fusion insecticidal proteins has been
reported previously. Bosch et al. [3] constructed a fusion
gene with a toxic domain of the cry1C gene combined with
the cry1E gene. Compared with the Cry1C protein, the
fusion protein has different acceptors, a higher toxicity, and
a combination of the cry1A gene with the proteinase
inhibitor gene (CpTi). The fusion gene was introduced into
the elite cotton, which resulted in double-effect-resistant
insects in transgenic cotton [10]. We combined the cry1Ac
gene with a neurotoxin gene (hwtx-I) to form a new fusion
gene. The expression plasmid of the fusion gene was
transformed into XBU001 and the fusion protein was mass-
123
expressed successfully. We make the following specula-
tions on the active mechanism of the fusion protein: (1)
when the fusion crystal proteins were ingested by suscep-
tible insects, they were dissolved in the alkaline midgut,
followed by proteolytic processing by midgut protease; and
(2) the fusion proteins were divided into two parts (Cry1Ac
and HWTX-I) by Enterokinase. The stable 60-kDa Cry1Ac
toxin bound to midgut receptors and inserted into the apical
membrane of brush border epithelial cells to form pores,
which disrupted the functional membrane process. The
HWTX-I peptides entered into the lymphokinesis through
these pores and inhibited the nervous system of the insect.
The two toxins may have synergized the toxicity to the
target insects. From the bioassay results, we observed that
the toxicity of the fusion protein increased compared to the
Cry1Ac protoxin. In addition, the fusion protein has the
characteristic of safety: the fusion crystal protein cannot be
dissolved in the acidic midgut of human or animal, hence
the active 60-kDa toxins should not be released. The
HWTX-I peptide can inhibit nerve conduction in the cir-
culatory system but not in the digestive tubes. Therefore,
the fusion protein should be safe to humans and animals.
This study has laid the groundwork for constructing fusion
genes of B. thuringiensis cry genes and other foreign toxin
genes with a higher toxicity.
Acknowledgments This research was supported by the National
Natural Science Foundation of China (No. 30670052 and 30870064),
the National 863 Project of China (Nos. 2006AA02Z187 and
2006AA10A212), the Research Fund for the Doctoral Program of
Higher Education (No. 20060542006), and the Provincial Natural
Science Foundation of Hunan (No. 06JJ2009).
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