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Increase in Insecticidal Toxicity by Fusion of the cry1Ac Genefrom Bacillus thuringiensis with the Neurotoxin Gene hwtx-I

LiQiu Xia XiaoShan Long XueZhi Ding YouMing Zhang

Received: 19 April 2008 / Accepted: 25 August 2008 / Published online: 25 October 2008

Springer Science+Business Media, LLC 2008

Abstract A fusion gene was constructed by combining the

cry1Ac gene of Bacillus thuringiensis strain 4.0718 with aneurotoxin 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 ofPlutella 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.

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

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: [email protected]

123

Curr Microbiol (2009) 58:5257

DOI 10.1007/s00284-008-9265-y


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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 28C in fermentation medium,

pH 7.3. For solid media, 2% agar was added to the liquid

medium.

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

CCTCCATAAGAGTAA3 0. 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 ofcry1Ac, 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 andthe 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

Table 1 Strains and plasmidsCharacterization Resource

Strains

DH5a E. coli strain Dr. Jian Zhang

B. thuringiensis 4.0718 B. thuringiensis strain wild type (CCTCC No. M200016) Lab store

XBU001 B. thuringiensis acrystalliferous strain Lab store

HC42 DH5a (pHC42) Lab store

UAc19 DH5a (pUAc19) This work

UAcH19 DH5a (pUAcH19) This work

HAcH43 DH5a (pXL43) This work

XBU304 XBU001 (pHT304) Lab storeHTX42 XBU001 (pTX42Ac) (cry1Ac) Lab store

XL002 XBU001 (pXL43) (cry1Ac ? hwtx-1) This work

Plasmids

pUC19 Ampr TaKaRa Co.

pHT304 Ampr

and Ermr, shuttle vector of E. coli and B. thuringiensis Dr. Sun Ming

pHC42 pHT304 carried 4.2-kb fragment from PCR Lab store

pUAc19 pUC19 carried 4.2-kb fragment from PCR This work

pUAcH19 pUC19 carried 4.3-kb fragment of fusion gene This work

pXL43 pHT304 carried 4.3-kb fragment of fusion gene This work

L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene 53

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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. Theaqueous phase was kept and centrifuged at 10,000 g and

4C 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

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 28C 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 scannedunder 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

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 24)

54 L. Xia et al.: Increase in Insecticidal Toxicity by Fusion of the cry1Ac Gene

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temperature of 25C. 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 cry1Acgene 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.

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 DNAin bipyramidal crystals, such as cry1Ac, cry1Aa, cry2Aa,

and cry2Ab [7]. The toxicity experiment demonstrated that

strain 4.0718 was highly toxic to larvae ofP. 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.

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


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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 kindsof 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 twofunctions 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 cry1Cgene 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-

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 apicalmembrane 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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