A Novel Bacillus thuringiensis Cry57 Protein Domain Swap In fluence on Insecticidal Activity

Shi Chen-chen, Zhou Guo-wang, Liu Zhi-yang, Gao Ji-guo, and Li Hai-tao*

1 College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

2 Laboratory of Biochemistry and Molecular Biology, Harbin Academy of Agricultural Sciences, Harbin 150070, China

Abstract: Bacillus thuringiensis (Bt) is widely used in insecticides. Bt is a gram positive sporulation bacterium belonging to Bacillaceae family. It produces different insecticidal proteins like Cry toxin, Vip toxin, β-exotoxin and chitinase, etc. Bt insecticidal crystal proteins (ICPs) are not homologous to other known Vip protein and then act against lepidopteran, dipteran, coleopteran and nematodeslarvae via a unique process. In this experiment, modern high-throughput sequencing technique and sequencing were used and the whole genome sequence of BtLTS290 was obtained. The results compared to the database of GenBank showed that there was a cry57 gene in the genome sequence of BtLTS290. A novel cry57 gene was cloned and named cry57Ab1 (accession number is KF638650) by International Nomenclature Committee of Bt Endotoxin. cry57Ab1 gene could be expressed with the molecular weight of 90 ku. Cry57Ab1 protein had no obvious activity against Spodoptera exigua and Helicoverpa armigera. And Cry57Ab1 protein had a slight insecticidal activity against Ostrinia furnacalis and Plutella xylostella. Furthermore, the domain Ⅱof Cry57Ab1 and Cry1Bb were exchanged by overlapping extension PCR. SDS-PAGE showed that the molecular weight of Cry57Ab/1Bb/57Ab was about 90 ku. The insecticidal activity of Cry57Ab1 protein and Cry57Ab/1Bb/57Ab recombinant protein were determined. The results showed that the insecticidal activity of the recombinant protein to Spodoptera exigua and Helicoverpa armigera was very low, and the corrected mortality was less than 10%. The insecticidal activities against Ostrinia furnacalis and Plutella xylostella were reduced. The corrected mortality of Ostrinia furnacalis was 4.4%, and the corrected mortality rate for Plutella xylostella was 6.7%. Domain Ⅱof cry toxin played a key role on affecting host specificity.

Key words: Bacillus thuringiensis, cry57Ab1 gene, domain exchange, bioassay

Introduction

Bacillus thuringiensis (Bt) is a gram positive sporulating bacterium belonging to Bacillaceae family. Bt produces insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are predominantly comprised of one or more proteins Crystal (Cry) and Cytolitic (Cyt) toxins, also called d-endotoxins (Bravoa and Gill, 2007). Cry proteins are parasporal inclusion (Cry) proteins from Bt that exhibits experimentally verifiable toxic effect to a target organism or has significant sequence similarity to a known Cry proteins. These toxins are highly specific to their target insects, are innocuous to humans, vertebrates and plants, and are completely biodegradable. Therefore, Bt is a viable alternative for the control of insect pests in agriculture and of important human disease vectors (Bravo et al.,2011).

Cry proteins are specifically toxic to the insect orders Lepidoptera, Coleoptera, Hymenoptera and Diptera, and also to nematodes. Because cry protein toxin has three conserved domains (Bravo et al.,2011) and has a similar mechanism of action, it is entirely possible to transform the cryotoxin by the method of toxin protein molecular design to obtain a new cryotoxin for the expansion and virulence of insecticides. In all the domains of cry gene, the domain Ⅱ is the most divergent domain among the Cry toxins and its replacement or switching with domainsⅡand Ⅲof other toxins can affect host specificity (Gomez et al., 2002). There are four ring regions (loop α8, loop1, loop2 and loop3) in the toxin protein domainⅡ, and there is a large gap between the sequence and structure of each cyclic region. The toxin protein domainⅡ is responsible for the receptor so that the amino acids on the four annular regions can be individually mutated to demonstrate the function of each annulus (Lee et al.,1996). Studies have found that the mutants S283E and G2828A of Cry1Ab2 were up to 10 times more biologically active in the gypsy moth (Lee et al.,1996). Related reports indicated that the insecticidal activity of the target pests can be improved by the domain swap of Cry protein, not only can be directly applied to the prevention and control of agricultural pests, but also to understand the relationship between Cry protein structure and function and the role of Cry protein mechanism. Because of the need of novel toxin with improved toxicity against Lepidopteran insect pest, present study was undertaken to construct a novel hybrid Bt toxin of Cry57Ab1 and Cry1Bb by domain swapping between domainⅡof Cry57Ab1 and domainⅡof Cry1Bb by overlapping extension PCR to enhance toxicity against Lepidopteran insect pests Helicoverpa armigera, Spodoptera exigua, Ostrinia furnacalis and Plutella xylostella.

Materials and Methods

Bacterial strains, growth conditions and plasmids

Escherichia coli JM109 cells [recA1, endA1, gyrA96,thi-1, hsdR17 (), e14-(mcrA-), supE44, relA1,Δ(lac-proAB)/F' [traD36, proAB+, lacIq, lacZΔM15]and Rosetta (DE3)[F-ompT hsdSB() gal dcm λ(DE3) pRARE (argU, argW, ilex, glyT, leuW, proL)(Camr)] were used for standard transformations. E. coli was incubated at 37℃ in LB medium (1% tryptone, 1%NaCl and 0.5% yeast extract). Ampicillin (100 mg · L-1)was added to the culture medium to select for antibiotic-resistant E. coli. All the Cry protein expression vectors used in this study were based on pEB, which was a derivative of pET-21b. Next, the products were inserted into Ecl136II site of pEB vector. pMD19-T vector, Markers DNA, protein Markers which were purchased from TaKaRa Company; KOD high fidelity DNA polymerase was purchased from TOYOBO Company; DNA recovery kit and Escherichia coli plasmid extraction kit were purchased from Invitrogen Company. All the PCR primers were synthesized by Beijing Jin Weizhi Company.

PCR amplification primers

Cry57Ab and Cry1Bb domain swap primer sequences were shown as the followings. 57Ab-1F: 5'-CGGGAT CCGATGGGGACATGGTGGCC-3'; 57Ab-1R: 5'-GG CTACTAATACATTAATAGTCATAGTAAGACG-3';1Bb-2F: 5'-GTATTAGATTTAGTAGCCCTATTCCC AAG-3'; 1Bb-2R: 5'-TGGAAATATTGTAATTCTAT TTGGTCCAATCGTAT-3'; 57Ab-3F: 5'-AGAATTA CAATATTTCCAGCTATAAAAAG-3'; 57Ab-3R:5'-CCCAAGCTTATTTGATAAATAATTAAATAA AGTATCAGG-3'.

PCR reaction

PCR reaction system was carried on 5 μL 10×PCR Buffer, 1 μL dNTP, 0.5 μL Taq enzyme, 1 μL upstream primer, 1 μL downstream primer, 1 μL tem-plate DNA and 40.5 μL ddH2O. PCR reaction conditions were pre-denaturation 94℃ for 5 min, denaturation 94℃ for 1 min, annealing 52-56℃ for 1 min,extension 72℃ for 2-4 min and posterior extension 72℃ for 10 min, in addition, the process from denaturation to extension took 30 cycles. The detection of amplified product: after the reaction, took 5 µL of the reaction liquid, and 1% agarose gel electrophoresis was conducted. The results were observed under UV light and then glue recovery.

The target DNA fragment was ligated with the vector, which was selected as pEB. The vector was ligated according to the following connection system:target fragment DNA 4 µL, vector DNA 1 µL, ligation kit solution I 5 µL. The three liquids were well mixed and allowed to stand at 4℃ overnight. According to the concentration of the recovered product, the amount of the product was adjusted. In general, the fragment DNA to vector DNA was 3 : 1 and then transformed E. coli by heat shock.

Completed the full-length primer according to the sequencing results 57Ab-1F and 57Ab-1R, primers were added to the 5' end of the digestionytm sites BamHⅠand Hind Ⅲ, respectively (underlined place).PCR was carried out using KOD high fidelity DNA polymerase as template for the strain BtLTS290.PCR product was then recovered and the product was ligated with the digested pEB vector. The recombinant plasmid was transformed into competent E. coli JM109, and finally the correct positive transformants were screened for sequencing. DNA sequencing results were compared by DNAMAN software and NCBI database.

Construction of recombinant plasmid

The purified Cry57Ab/1Bb/57Ab and vector pEB were digested with restriction endonucleases BamHⅠand Hind Ⅲ, and then the two fragments were ligated with DNA ligase. The recombinant plasmid was transferred into Escherichia coli Rosetta (DE3) and then extracted E. coli plasmid DNA. The expression recombinant plasmids was induced by IPTG in E.coli and protein electrophoresis was detected by He(2011).

Test insects

Those standardized test insects Spodoptera exigua,Helicoverpa armigera, Ostrinia furnacalis and Plutella xylostella were provided by our laboratory.

The expressed protein was dissolved in 10 mmol · L-1Tris-Cl (pH 8.0), and the protein was quantified and mixed with the feed. The mixture was mixed in a sterile 24-well plate (Wu et al., 2010). The biological activity was measured, each of which was repeated.The treatments of Plutella xylostella feed were different. Fresh cabbage leaves were selected and immersed in the crude protein for 10 min, and then the excess proteins on the surface were dried. A 10 mmol · L-1Tris-Cl (pH 8.0) solution was used as the control.The larvae were planted for 2 days. The numbers of dead insects and the numbers of live insects were investigated after 6 days of other larvae feeding, and the larvae of the treatment group were calculated. The mortality and corrected mortality of treatment group were calculated.

Results

Cloning and sequence analysis of cry57Ab gene

After obtaining the correct size of the target band by PCR amplification of 57F and 57R, the target band was purified, and connected with the carrier pEB, and then transferred it into JM109 strain. The positive clones were selected and plasmid was extracted and sequenced. Sequence analysis showed that the size of cry57 gene from BtLTS290 strain was 2 064 bp,encoding 678 amino acid residues. There was 90.2%similarity with Cry57Aa1. This was registered in the international GenBank with accession number KF638650 and received an official name cry57Ab1 from Bt δ-endotoxin Gene International Nomenclature Committee.

By using the European Molecular Biology Open Software Package EMBOSS online, Cry57Ab1 protein amino acid sequence was input, its primary structure and a few basic parameters were gotten. Cry57Ab1 protein consisted of 687 amino acids (Table 1), with a molecular weight of 79.38 ku and the protein's isoelectric point of 7.42.

Table 1 Amino acid composition of Cry57Ab1 protein

The analysis result of NCBI Conserved Domain Summary showed that the protein domainⅠconsisted of 68-279 bits, totally 212 amino acids at the amino terminal (Fig. 1). Domain Ⅱ consisted of 532-678 bits,totally 147 amino acids, and the comparison of domain Ⅱcame to nothing.

Fig. 1 Domains of Cry57Ab1

Exchange between domain II of Cry1Bb and domain II of Cry57Ab

Construction of chimeric gene cry57Ab/1Bb/57Ab By using overlapping PCR method to perform PCR for 6 times totally, base sequences corresponding to Cry1Bb domainⅡwas embedded into the middle of Cry57Ab successfully. Firstly, obtained the base sequences corresponding to domain I of Cry57Ab by PCR (Fig. 2, Lane 1), domainⅡof Cry1Bb (Fig. 2,Lane 2) and domain Ⅲ of Cry57Ab (Fig. 2, Lane 3),respectively. Secondly, by using overlapping PCR with upstream primer 57Ab-1F and downstream primer 1Bb-2R, the base sequences corresponding to the combination of Cry57Ab's domainⅠand Cry1Bb's domainⅡwere obtained (Fig. 2, Lane 4). Similarly,obtained the base sequences corresponding to the combination of Cry1Bb's domainⅡand Cry57Ab's domainⅢ (Fig. 2, Lane 5) by PCR. Thirdly, used PCR that carried out with the upstream primer 57Ab-1F and the downstream primer 57Ab-3R to obtain the nucleotide sequence corresponding to the combination of Cry57Ab's domainⅠ, Cry1Bb's domainⅡand Cry57Ab's domainⅢ, and then chimeric gene of cry57Ab/1Bb/57Ab was got (Fig. 2, Lane 6).

Domain analysis after swapping

The domainⅡof Cry1Bb which situated from 237 to 483 sequence of Cry1Bb protein, had 247 amino acids residues in total. After replacement, the structure of Cry57Ab contained the domainⅡof Cry1Bb. After the domain replacement, domainⅡof Cry1Bb was exactly located in the middle of Cry57Ab domainⅠand domain Ⅲ (Fig. 3). And Cry57Ab/1Bb/57Ab was made up of 648 amino acids totally.

Construction of Cry57Ab/1Bb/57Ab heterologous expression vector

DNA rubber of target band (cry57Ab/1Bb/57Ab) after PCR amplification was recovered, purified, and double digested by the restriction enzymes BamHⅠand SalⅠ, and vector PEB was double digested too. Then,DNA ligase was used to connect target fragment with vector DNA fragment. After that, transformed it into E. coli Rosetta (DE3) strain and did PCR validation and enzyme digested verification.

Fig. 2 Results of overlapping PCR

Fig. 3 Domains of Cry57Ab/1Bb/57Ab

Protein expression of Cry57Ab and Cry57Ab/1Bb/57Ab

The results of SDS-PAGE electrophoresis showed that by using Rosetta (DE3) strain with empty plasmid pEB transferred into as a negative control, the recombinant plasmid was transferred into Rosetta (DE3) strain, and the expression vector pEB in E. coli could produce proteins efficiently.

The recombinant plasmids pEB57Ab and pEB57Ab/1Bb/57Ab were transformed into E. coli Rosetta (DE3).And after IPTG inducting, SDS-PAGE (10%) gel electrophoresis of the extracted protein was carried out. The results showed that the empty vector pEB inducted into Rosetta (DE3) via IPTG produced no specific target band (Fig. 4, Lane 1), while gene cry57Ab was highly expressed in E. coli with approximately 90 ku protein by expression vector pEB( Fig. 4, Lane 2). And Cry57Ab/1Bb/57Ab were highly expressed in E. coli with the molecular weight of 85 ku ( Fig. 4, Lane 3).

Fig. 4 Expression of new genes in Rosetta (DE3)

Insecticidal activity assay of Cry57Ab and Cry57Ab/1Bb/57Ab

In this study, the preliminary insecticidal activity of protein Cry57Ab was conducted. The protein concentration in screening test was set at 100 μg · mL-1(Luo et al., 1999; Wang et al., 2007; Wang et al.,2008), and each treatment was repeated three times with 10 mmol · L-1Tris-Cl used as a negative control.Results of screening are shown in Table 2. The death rates of Helicoverpa armigera and Spodoptera exiguacaused by protein Cry57Ab were less than 10%both after treated, indicating that the protein Cry57Ab had few insecticidal activity against Helicoverpa armigera and Spodoptera exigua. As for Ostrinia furnacalis, the death rate was 10.0%, and Plutella xylostella was 13.4%, indicating that the protein Cry57Ab had slight insecticidal activity against them.

To test whether the activity had changed after the replacement of Cry57Ab domain, the preliminary insecticidal activity of protein Cry57Ab/1Bb/57Ab was conducted too. And the protein concentration in screening test was set at 100 μg · mL-1(Luo et al.,1999) and each treatment was repeated three times with protein concentration screening when set at 100 μg · mL-1, each treatment was repeated three times with 10 mmol · L-1Tris-Cl used as a negative control.Screening results shown in Table 3 indicated that the death rate of Helicoverpa armigera and Spodoptera exigua caused by protein Cry57Ab/1Bb/57Ab was less than 10% after treated, indicating that it had few insecticidal activity against Helicoverpa armigera and Spodoptera exigua. And the death rates of Ostrinia furnacalis and Plutella xylostella were 4.4% and 6.7%,respectively, which indicated that insecticidal activity against them was declining compared with protein Cry57Ab.

Table 2 Bioassay results of Cry57Ab protein against certain insect larvae

Table 3 Bioassay results of Cry57Ab/1Bb/57Ab protein against certain insect larvae

Discussion

Pests cause major limitation in high production of food crops (Karthikeyanet al., 2012). Advantages of Bt bioinsecticides include target specific killing,lacking of toxicity to humans and ease of commercial production (Deist et al., 2014; Bravo et al., 2011).Though Spodoptera exigua, Helicoverpa armigera,Ostrinia furnacalis and Plutella xylostella belonged to Lepidoptera, they showed variation in susceptibility to Cry toxins (Gujar et al., 2007). Thus, it required the use of novel toxins with improved toxicity. Discovering novel Bacillus thuringiensis cry gene is really significant work all the time. Along with the development of biochemistry, molecular biology,genetics and other subjects, the method of identifying Bt strains' cry gene developed rapidly, including PCR-RFLP identification method (Song, 1998; Song et al., 2003), multiplex PCR identification method(Fang et al., 2007), two-step PCR identification method (Su et al., 2010), identification of molecular hybridization method (Rao et al., 2003), gene chip method (Liu and Song, 2004), protein identification method (Masson et al., 1998), genome sequencing(Zhou and Song, 2010), and so on. In this research,by applying the whole genome sequencing on Bt LTS strains, it was found that the strain contained cry gene was high similarity to cry57Aa1. After further identification, Bt δ-endotoxin gene was officially named cry57Ab1 by International Nomenclature Committee of Bt endotoxin, ultimately. The discovery of new gene cry57Ab1 enriched the resources of cry genes in China. Insecticidal activities were measured indoor, but this study suggested no insecticidal activity of expressed Cry57Ab1 protein.

At the same time, novel Cry57Ab/1Bb/57Ab protein was expressed. Compared with Cry57Ab protein,Cry57Ab/1Bb/57Ab protein insecticidal activity against Ostrinia furnacalis and Plutella xylostella reduced,almost no insecticidal activity against Spodoptera exigua and Helicoverpa armigera. All the Cry toxins contained three structural domains and shared a high degree of topological similarity. DomainⅡconsisted of three sets of antiparallel β-sheets, each terminated with a loop. The beta sheets were packed around a central hydrophobic core forming a so-called betaprism structure. Results of site-directed mutagenesis and truncation analysis provided strong evidence for the involvement of domainsⅡand Ⅲ in receptor binding and insecticidal activity (Ibrahim et al., 2010).Different strategies were used to enhance toxicity of Cry toxins, such as domain swapping between domainⅠ-Ⅱof Cry1Ia and domain Ⅲ of Cry1Ba resulted hybrid toxin Cry1Ia-1Ba which showed 7.5 times toxicity than that of Cry1Ba against colorado potato beetle (CPB) (Naimov et al., 2001). Deletion of 42 residues of amino acids at N-terminal of Cry2Aa toxin resulted in 2.0-fold increased toxicity against cotton ball worm (Mandal et al., 2007; Morse et al.,2001). Modification of Cry3A contained a chymotrypsin/cathepsin G protease recognition site in the loop between α-helix 3 and α-helix 4 of domainⅠ, which resulted in consistent activity of the toxin against neonate western corn rootworm binding of the activated toxin to gut receptors (Walters et al.,2008). Fusion of domainⅠ-Ⅱ of Cry1Ab toxin with domain Ⅲ from Cry1C resulted in increased toxicity to S. exigua compared to the parental Cry toxins(Maagd et al., 1996). Combination of domainⅠ-Ⅱ of Cry1Ba with domain Ⅲ of Cry1Ca broadened the spectrum of activity towards S. exigua and M. sexta(Maagd et al., 2000).

Previous studies demonstrated that among the three domains of various Cry toxins, the greatest amino acid variation was observed in domainⅡ, suggesting that it was involved in toxin specificity. In fact,domain Ⅱ was known to be an important region for Cry toxin specificity to target insects. However,the mode of action of Cry toxins is still a matter of hypotheses. The contribution of the surface-exposed loops in domainⅡto insect toxicity had been examined in a number of site-directed mutagenesis studies (Lee et al., 1996; Gomez et al., 2003; Tuntitippawan et al.,2005). DomainⅠis the most conserved among Cry toxins. DomainⅡand the beginning of Domain Ⅲ are the least-conserved. At the beginning of this study,it could be thought that the low pesticidal activity of Cry57Ab was due to lacking of domainⅡ, but the experiment showed that insecticidal activity of hybrid protein Cry57Ab/1Bb/57Ab was not ideal either. This result might be that the spatial structure of Cry57Ab protein had been altered after the domain substitution,causing the loops mechanism destroyed. But for specific reasons, it still needed further exploration.

Conclusions

By application of whole-genome sequencing technology, cry57Ab1 new gene was successfully cloned from BtLTS290 strain. Overlapping extension PCR was used to get heterozygous protein Cry57Ab/1Bb/57Ab, the change resulted in which the protein had a slight increase in insecticidal activity against Spodoptera exigua than Cry57Ab protein, but had slightly decline against Helicoverpa armigera,Ostrinia furnacalis and Plutella xylostella. DomainⅡof Cry toxin played a key role on affecting host specificity, the loops appeared to be key elements in receptor recognition, binding and specificity.

Conflict of lnterest

The authors declare that they have no conflicts of interest.