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A Chimeric Protein Encoded by Synthetic Genes Shows Toxicity to Helicoverpa armigera and Spodotera littoralis Larvae.

Byline: Zaheer Abbas, Yusuf Zafar, Sher Afzal Khan and Zahid Mukhtar

Abstract

Insects have natural potential to develop resistance against chemical insecticides. Several resistance strategies have been suggested including biopesticides and use of two dissimilar toxins. Advances in molecular biology techniques have now allowed construction of chimeric proteins to delay the development of resistance in insect population, but still there are chances of developing resistance in insect population against them as these fusions are based on Bacillus thuringiensis (Bt) genes only, which have some homology in their amino acid sequences, having same mode of action and derived from same bacterial origin. In the present study (omega)-ACTX-Hv1a toxin gene (Hvt) as an insect calcium channel antagonist is fused with Bt crylAc to combine both strategies (biopesticides and two dissimilar toxins) and delay the resistance in insect population.

The recombinant protein has been successfully expressed in prokaryotic system and was detected by SDS PAGE. Topical application of the 1.0 pmol purified recombinant protein to the thoracic region paralyzed and immobilized the Helicoverpa armigera and Spodoptera littoralis larvae within 2 h. 100 (Percent) mortality was observed in insects after 24 h. The LD50 was found to be 4 and 2 pmol per gram of body weight for H. armigera and S. littoralis larvae, respectively. The present study clearly indicates that this recombinant protein is highly effective against agronomical important lepidopteron insects and is an excellent candidate for use as a biopesticides or expressed heterogeneously in agricultural crops to provide long lasting resistance to insect attacks. (c) 2013 Friends Science Publishers

Keywords: Recombinant toxin; Biopesticides; Insect resistance; Multiple genes; Insecticidal proteins; Spider toxin

Introduction

Chemical insecticides have become less effective as the target insect populations develop resistance and have killed non-target population of predators and parasites that otherwise keep herbivorous insects in balance (Zhao et al.,2010). Pesticides exposure also affected the human health and organochlorine pesticides residues have also been reported in the blood of workers occupationally engaged in agriculture (Dhananjayan et al., 2012). Biopesticides are attractive alternative to the broad spectrum organophosphate insecticides. Biopesticides are fairly specific, quite lethal, safe to most beneficial insects and animals, biodegradable and do not persist in the environment and hence can delay the onset of resistance in insects unlike chemical insecticides, which are persistent in environment and quite general in their effect. Biopesticides also suit integrated pest management and integrated crop management strategies (Gupta and Dikshit, 2010).

It is well documented that many insects are susceptible to the toxic activity of B. thuringiensis. Among them lepidopterans have been exceptionally well studied, and many toxins have shown activity against them (Monerat et al., 2007). Today, a great variety of B. thuringiensis-based bioinsecticides are commercially available for the control of a wide variety of agriculture and forestry pests, including disease vectors (Sauka and Benintende, 2008). Commercial biopesticide namely XenTari consisting of Bt aizawai has been successfully used to control the larvae of Galleria mellonella (Basedow et al., 2012). Soberon and Bravo (2007); Abad et al. (2008) have described representative patents in which the insecticidal activity has been directed toward different insect pests, particularly to protect plants from pest damage. In recent years, hybrid delta-endotoxins have arisen as proteins with potential for enhanced toxic activity or improved properties.

Recent advances in molecular methodologies have allowed gene fusions and chimeric protein construction. This construction can include alteration of amino acid sequences, fusion of portions of two or more proteins together into a single recombinant protein (Rosas-Garcia, 2009). But still there are chances of developing resistance in insect population against them because these fusions are based on B. thuringiensis genes only which have some homology in their amino acid sequences, having same mode of actions and derived from same bacterial origin. It is therefore the need of time to fuse two different genes with unique mode of actions in order to develop biopesticides with durable and long lasting resistance to insect pests. Most spider venoms are rich source of insecticidal compounds and their primary role is to kill or paralyze arthropod prey.

Mukhtar et al. (2004) developed codon optimized (omega)- ACTX -Hv1a toxin gene (Hvt) for high level expression in plants. Hvt is a 37 amino acid, insect specific calcium channel antagonist from Australian funnel web spider. The peptide is toxic to a range of agriculturally important arthropods in the orders Coleoptera, Lepidoptera and Diptera but has been reported to have no effects on a number of mammals (Khan et al.,2006; Chong et al., 2007). Shah et al. (2011) cloned the Hvt gene under RSs1 and RoLC phloem specific promoters. The resulted transgenic tobacco confirmed resistance to Heliothis armigera.

In the present study the Bt cry1 Ac and Spider Hvt (ACTX) genes with unique mode of actions have been recombined. It has been demonstrated that translational fusions of Bt cry1Ac and spider Hvt (ACTX) genes paralyzed and immobilized the Helicoverpa armigera and Spodoptera littoralis larvae and is an excellent candidate for use as biopesticides or expressed heterogeneously in agricultural crops to provide long lasting resistance to insect attacks.

Materials and Methods

Gene Designing and Plasmid Construct

Translational fusion of Bt cry1Ac (patent pending) and Spider Hvt (ACTX) gene (Khan et al., 2006), was commercially synthesized from Medigenomics, Germany. To facilitate the cloning of this gene under desirable expression cassette, few restriction sites were added to the flanking regions of the gene at the 5' as well as 3' ends of the gene. Resultant vector was named as pSAK-IV (Fig.1a). The bacterial expression vector was developed at the Plant Molecular Biology and Transformation Lab at NIBGE which contained cry1Ac fused with Hvt (ACTX) gene and was named as pSAK-V (Fig. 2f).

Development of Bacterial Expression Constructs

Synthetic cry1Ac fused with the Hvt (ACTX) was amplified (Fig. 1b) from plasmid pSAK-IV (Fig. 1a) using full-length primers. The forward primer was based on cry1Ac gene 5-' GCATGGATAATAACCCTGGA-3' and reverse primer was based on Hvt gene 5-' TTAATCGCATCTTTTTACGG-3'. Total volume for PCR reaction was 50 (Mu)L. The PCR profile was optimized for the amplification of synthetic cry1Ac gene fused with the Hvt (ACTX) gene as denaturation at 94oC for 5 min, followed by 30 cycles of 94oC for 1 min, 55oC for 1 min, 72oC for 2.5 min and final extension of 72oC for 10 min. Eppendorf thermal cycler was used for PCR and amplified products were analyzed by electrophoresis on 1 (Percent) agarose gels along with 1 kbp DNA marker.

The amplified product was cloned with correct orientation in T/A cloning vector pTZ57R. The resultant vector was named as pZSTA (Fig. 1c). EcoR1-HindIII enzymes were used to lift the cry1Ac fused with Hvt (ACTX) gene from the pZSTA. The bacterial expression vector pET32a (+) was digested with EcoR1-HindIII and eluted from the gel and used as a vector for cloning.

The EcoR1-HindIII fragment of pZSTA was ligated with the pET32a (+). The resultant bacterial expression construct was named as pSAK-V (Fig. 2f).

Expression of pSAK-V in E. coli Strain BL21

The BL21 (DE) pLysS (Novagen) host containing a chromosomal copy of T7RNA polymerase gene under lacUV5 promoter was transformed with pSAK-V. The lacUV5 promoter is responsible for driving the expression of T7RNA polymerase. It is inducible by isopropyl beta D thiogalactopyranoside (IPTG). pET32a (+) carries the target gene under T7 promoter. When IPTG is added it activate the lacUV5 promoter and turn on synthesis of T7 RNA Polymerase. This in turn transcribes the gene in pET vector.

After transformation and confirmation of pSAK-V in BL21, the target gene was induced by the addition of 1 mM IPTG. Colony was picked from a freshly streaked plate and inoculated into 3 mL LB medium, in a 15 mL falcon tube, containing the ampicillin and chloramphenicol for the selection of plasmid and BL21 host strain, respectively. This 3 mL culture was incubated with shaking at 37degC for overnight and further inoculated into 50 mL of LB containing ampicillin and chloramphenicol and grown for overnight at 37degC until OD 600 nm reached 0.4-1.0 (0.6 recommended; about 3 h). Un-induced control sample was removed and to the remainder, IPTG was added from a 100 mM stock to a final concentration of 1 mM. Samples were taken at regular intervals after induction i.e., 0.5, 1.0, 1.5,2.0 and 2.5 h. The cells were harvested by centrifugation at 13,400 rpm for 1.5 min. The cells were frozen at -20degC for 30 min.

The harvested cells were allowed to thaw, sonicated and then BugBuster Protein Extraction method was used to gently disrupt the cell wall of E. coli resulting in the liberation of soluble protein. 10 (Percent) SDS polyacrylamide (SDS-PAGE) gel was prepared. The expression of target genes was assessed quickly by analysis of total cell protein on an SDS-PAGE gel followed by Coomassie blue staining and destaining.

Biological Toxicity Assays

In order to conduct the biological toxicity assay, the larvae of H. armigera and S. littoralis were taken from fields in petri plates having moist whatmann filter papers. The larvae were allowed to grow and feed on cotton leaves. Adults of larvae were then shifted to the jars having muslin cloth. Eggs of adults were then transferred from muslin cloth to the vials containing artificial insect diet. 2nd instar larvae of H. armigera and S. littoralis emerged, while incubating the vials at a temperature of 25 +- 2oC. As recombinant protein is a fusion between cry1Ac and Hvt (ACTX) gene, two separate toxicity assays were carried out.

In order to measure the toxicity of cryIAc, the recombinant protein was activated by the gut juice of silkworm following the procedure as described by Gringorten et al. (1990). The toxicity was determined by the force feeding technique as previously illustrated by Van et al. (1991). The control experiment was also carried out having only gut juice.

Toxicity of Hvt was measured by topical application of 2 (Mu)L of the recombinant protein in elution buffer to the thoracic region of 2nd instar H. armigera and S. littorallis larvae having an average weight of 3.2 +- 0.06 and 2.98 +- 0.04 mg, respectively. Each of 0.02, 0.06, 0.12, 0.25,0.5 or 1.0, 2.0, 4.0, 8.0, or 16.0 pmol of recombinant protein doses were applied, using a micropipette, to twenty larvae in order to determine the LD 50 values after twelve hours of application. Control experiment was also conducted having the thioredoxin protein only.

Results

Cloning of Cry1Ac and HVT Genes in Bacterial Expression Vector

The 1,968 bp DNA fragment containing the full length synthetic cry1Ac fused with the Hvt gene was amplified from plasmid pSAK-IV (Fig. 1a) using full-length primers. The forward primer was based on the cry1Ac 5' region while the reverse primer was designed based on the 3' region of the Hvt gene. These primers amplified 1,968 bp fragment as shown in Fig. 1b. This 1,968 bp fragment was cloned in T/A cloning vector pTZ57R and the orientation of the insert was confirmed using SacI. Since there is a unique SacI site in pTZ57R in addition to one SacI site in the insert, therefore, the digestion with SacI should produce two fragments of either 742 bp and 4,112 bp (orientation I) or 1,300 and 3,554 (Orientation II). Fig. 1d shows that upon digestion with SacI, the resultant vector produced two fragments of 742 and 4,112 bp indicating that the insert has been cloned in correct orientation I.

The resultant vector was named as pZSTA (Fig. 1c). pZSTA was digested with EcoR1-HindIII to clone the cry1Ac-Hvt (ACTX) genes in frame with the coding TAG sequence of the pET32a(+) as shown in Fig. 2a. The resultant vector was named as pSAK- V (Fig. 2b). The cloning was confirmed through digestion of pSAK-V with EcoR1 and HindIII, which should remove the insert (2,048 bp fragment) from the parent vector.

Upon digestion with EcoR1 and HindIII, pSAK-V released the 2,048 bp fragment along with the vector backbone (5,900 bp) as shown in Fig. 2d. This confirmed the cloning of cry1Ac-Hvt (ACTX) genes in pET32a (+). To further confirm the cloning in pET32a (+), pSAK-V was digested with SacI-HindIII, which should produce three fragments (i.e. vector backbone 5,900 bp, 1,318 bp and 730 bp). Fig. 2e shows that upon digestion with SacI-HindIII, pSAK-V produced three fragments of the expected sizes thus confirming the cloning of cry1Ac-Hvt genes in pET32a (+). Further confirmation of the clone was made by PCR using cry1Ac specific primers, which produced 883 bp internal fragment of the cry1Ac gene as shown in Fig. 2c indicating that the insert has successfully been cloned into pET32a (+).

pSAK-V Transformation in E. coli Strain BL21

pSAK-V was introduced into E. coli strain BL21 through electroporation. The selected bacterial colonies were analysed for the presence of pSAK-V through PCR analysis using cry1Ac specific primers as given below:5'-TGCCAACTTGCATCTCTCTG-3'and 5'-TCGGTGAATCCATGAGAACA-3'.

The amplification of 883 bp fragment (Fig. 3) confirmed the transformation of plasmid pSAK-V in E. coli strain BL21.

Expression of the Target Gene

After transformation and confirmation of pSAK-V in BL21, expression of the target gene was induced by the addition of 1 mM IPTG. Samples were taken at regular intervals after induction i.e., 0.5, 1.0, 1.5, 2.0 and 2.5 h. The samples were processed and analyzed on SDS PAGE. The intensity of desired protein (88 kDa) was increased with increase in time after induction i.e., 30, 60, 90, 120 and 150 minutes as shown in Fig. 4.

Cry1Ac Detection through Immuno-blot in Chimeric Protein

In another experiment after processing of samples, immuno- blot was done for Cry1Ac detection in translational fusion. In control sample, the total soluble protein was extracted from IPTG induced BL21 strain transformed with pET32a (+) and was used for immuno-blot. The Cry1Ac protein was not detected in control sample, while a strong signal appeared when total soluble protein from BL21 strain transformed with pSAK-V was used in the immuno-blot (Fig. 5). This shows that active domains of Cry1Ac remained intact in translational fusion.

Functional Analysis of Chimeric Protein

In order to determine the minimum toxic concentration of recombinant protein, topical application to the thoracic region of H. armigera and S. littoralis larvae was done. The minimum toxic concentration of recombinant protein was found to be 1.0 pmol for both types of insects. At 1.0 pmol the larvae showed lack of coordination among different organs, continuous head shaking, and stopped feeding and finally paralysis was noticed within two hours which is the characteristic symptoms of Hvt (ACTX) protein which shows that active domain of Hvt also have not been disturbed in chimeric protein. The LD50 was found to be 4 and 2 pmol per gram of body weight for H. armigera and S. littoralis larvae, respectively. 100 (Percent) mortality was observed after 24 h in both types of larvae. In control experiment both larvae remained unaffected, developed normally to adult stage.

In another experiment the activated recombinant protein was delivered into the midgut region of both larvae with a 0.25-mL syringe having blunted 30-gauge needle. The characteristic symptoms of the Hvt were again observed rather than the characteristic symptoms of cry1Ac, which suggested that Hvt has a more quick mode of action and more efficient to prevent damages to the agricultural crop comparatively to the cry1Ac. The larvae remained unaffected and developed normally in control experiment having gut juice solution only.

Discussion

Biopesticides are eco-friendly alternative to chemical pesticides and can be derived from natural sources such as microorganisms (Gupta and Dikshit, 2010) and animals (Nicholson, 2007). Biopesticides derived from Bacillus thuringiensis have been widely used to control agronomical important insects (Sauka and Benintende, 2008). Besheli (2007) examined the efficacy of Bt for control of Phylocnistis citrella on detached citrus leaves. The results showed positive correlation between larval mortality and Bt concentrations. It has also been observed that Bt plus mineral oil reduced the Citrus leaf minor larvae to a level non-significantly different from treatment consisting of Bt alone. LRC3 strain of B. thuringiensis containing different crystal genes on its plasmid has been widely used to control lepidopteron insects; moreover, the genes that encode these proteins are subject of intensive research (Lysyk et al.,2006).

The patented Cry7Bal from B. thuringiensis strain encoding novel toxic protein showed enhanced activity against lepidopteran insects (Sun et al., 2008). Synthetic cryIAc gene from B. thuringiensis is also used in the present study.

In order to delay the development of resistance in insect population novel chimeric protein has been expressed in the present study. Previously different chimerics have also been expressed to cover the broad host range and enhanced toxicity (Rosas-Garcia 2009). Bradfisch et al. (1998) have recombined cry1F and cryIAc for enhanced toxicity and activity against broader insect host range. Van et al. (2007) studied the functional analysis of synthetic proteins having Cry1C, Cry1B or Cry1D.

Although many chimeric proteins have been expressed but still there is a probability of developing resistance in insects against them because the genes encoding these proteins are only based on B. thuringiensis which have similar mode of actions (Schneph et al., 1998) and resistance to Bt toxins in insect population has been shown experimentally (Liu et al., 2001). The introduction of diverse and multiple genes in crop plants could reduce the resistance in insect arising from the single or similar group of genes (Zhao et al., 1997; Tabashnik et al., 2002). The present chimeric protein is based on the bacterial and spider genes which have unique mode of action from each other and expected to offer a long lasting resistance against insects. Hvt is toxic to a range of agriculturally important arthropods in the orders coleoptera, lepidoptera and diptera but has been reported to have no effects on a number of mammals (Khan et al., 2006; Chong et al., 2007).

Unlike other spider toxins, Mukherjee et al. (2006) reported that Hvt has also the ability to cause toxicity when administrated orally to American lone star tick (Amblyomma americanum). Shah et al. (2011) also examined mortality of Heliothis armigera within 72 h on detached tobacco transgenic leaves expressing Hvt under phloem specific promoters. It has also been illustrated that fusion of Hvt with other carrier proteins such as garlic lectins or snowdrop lectins tremendously increases absorbtion of toxin by midgut of insects and increase toxicity through oral administration (Fitches et al., 2004, 2008).

The chimeric protein consisting of Bt Cry IAc and Hvt (ACTX) has been successfully expressed in prokaryotic system and was detected by SDS PAGE. Functional analysis clearly indicates that this recombinant protein is highly effective against agronomical important lepidopteron insects and is an excellent candidate for use as a biopesticides or when expressed in crop plants for transgenic protection.

In summary, we performed the functional analysis of new recombinant toxin protein developed from two distinctly unrelated Bt cry1Ac and spider Hvt (ACTX) genes. Recombinant protein showed the characteristic symptoms of the spider Hvt gene during the both force feeding and topical application experiment on H. armigera and S.littoralis possibly due to the quicker mode of action of spider Hvt protein than Cry1Ac protein. The expressed Cry1Ac protein in bacterial strain BL21 was detected by immuno-blot analysis, which shows that active domains of Cry1Ac protein remained unaffected in translational fusion. Our study shows that in translational fusion the active domains of both Cry1Ac and Hvt remained intact and this translational fusion is highly effective chimeric molecule against agronomically important insects, which should be evaluated further for use as biopesticides or introduced in crop plants especially cotton for long lasting transgenic protection against herbivorous insects.

Acknowledgments

Thanks to the National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan for providing technical and financial support.

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For correspondence: zaheerpbg@yahoo.com

To cite this paper: Abbas, Z., Y. Zafar, S.A. Khan and Z. Mukhtar, 2013. A chimeric protein encoded by synthetic genes shows toxicity to Helicoverpa armigera and Spodotera litto ralis larvae. Int. J. Agric. Biol., 15: 325-330
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Author:Abbas, Zaheer; Zafar, Yusuf; Khan, Sher Afzal; Mukhtar, Zahid
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Apr 30, 2013
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