Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, aerobic, sporulating bacterium which synthesizes crystalline proteins during sporulation. These crystalline proteins are highly insecticidal at very low concentrations. The mode of action of insecticidal crystal proteins (ICP) involves a cascade of events including solubilization of the crystal, activation of the toxins by gut proteases and recognition of a binding site on the midgut brush border membrane followed by pore formation and cell lysis leading ultimately to insect death. Majority of the Cry proteins are generally are of two lengths: either 130–140 kDa or approximately 70 kDa. Three domains required for toxicity are present in the N-terminal half of the larger proteins, whereas the C-terminal half is constituted in a protoxin domain and is not found in smaller proteins. Therefore, 3′-truncated cry genes of larger proteins (such as cry1) encode for proteins with insecticidal activity (Schnepf et al. 1998). The Cry proteins have been classified as belonging to Cry 1–50 families on the basis of amino acid sequence homology (www.biols.susx.ac.uk/home/NeilCrickmore/Bt).

The most wide-spread cry1 genes encode the 130–138 kDa delta-endotoxins that form ICPs active against lepidopteran larvae. Difference in the level of toxicity and specificity exist among different Cry1A toxins due to minor variations in amino acid residues (Udayasuriyan et al. 1994; Rajamohan et al. 1996). Continuous exposure to a single kind of Bt toxin can lead to resistance development in insects. Discovery of new insecticidal protein genes is of importance for delaying the development of resistance in target insects. In the present work, we have cloned, sequenced and expressed a 3′-truncated cry1Ab gene exhibiting sequence variation from the known genes within the Cry1Ab group.

Materials and methods

Bacterial strains and plasmids

Bacillus thuringiensis (Bt) strain, A19-31 was from Prof. V. Udayasuriyan, TNAU, Coimbatore (Ramalakshmi and Udayasuriyan 2010). The Bt strain 4Q7 (acrystalliferous) and the E. coli-Bt shuttle vector pHT3101 (Lereclus et al. 1989) were originally obtained from Bacillus genetic stock centre, Ohio University, Columbus, Ohio, USA. E. coli strain DH5α was used for maintaining plasmid constructs. Plasmid pTZ57R/T (Fermentas INC) was used for DNA cloning and pHT3101 (E. coli-Bt shuttle vector) was used for gene expression in recombinant Bt strain.

Cloning and DNA sequencing of truncated cry1Ab gene from a new indigenous isolate of Bt, A19-31

Total genomic DNA was isolated as described by Kalman et al. (1993) from an indigenous isolate of Bt, A19-31, and used as template for PCR amplification. Four oligonucleotide primers specific for the 3′-truncated cry1Ab gene were designed based on the published sequence of cry1Ab1 gene. One primer pair (1AbFS1; 5′-GGAACCTCCTCAAATTTGCCATCCGC-3′, 1AbRS1; 5′-TCATTGCCTGAATTGAAGACATGAGC-3′) for screening and another pair (1AbF1; 5′-CCCCGGGCCTGGGTCAAAAATTGATATTTAG-3′, 1AbR2; 5′GCTGCAGTGCTCTTTCTAAATCATATTCTGCC-3′) were used for cloning. Sites for XmaI (CCCGGG), and PstI (CTGCAG) are in bold letters. For the amplification of the 3′-truncated cry1Ab gene, 50 ng total DNA was used as template in 25 μl reaction mixture containing 2.5 μl 10× PCR buffer (10 mM Tris/HCl; pH: 9.0, 50 mM KCl, 1.5 mM MgCl2), 100 ng each of forward and reverse primers and 0.5 units of Taq DNA polymerase. PCR cycling profiles were 1 cycle at 94°C for 2 min, 30 cycles of 94°C for 40 s, 60°C for 45 s and 72°C for 2 min, followed by a final extension step at 72°C for 7 min. The PCR product was ligated with pTZ57R/T and then transformed into E. coli strain DH5α. The resulting construct is designated as pT1Ab. The plasmid carrying cry1Ab gene was sequenced. Sequences alignment was performed in BLAST web (http://www.ncbi.nlm.nih.Gov/BLAST/) or using ClustalX.

Expression of truncated cry1Ab gene in acrystalliferous Bt strain, 4Q7

The recombinant T/A plasmids carrying 3′-truncated cry1Ab gene were digested by XmaI and PstI restriction enzymes to release the cloned DNA fragment. Simultaneously the shuttle vector pHT3101 was also digested using the same enzymes to linearize it. Both were ligated and ligation mixture was transformed into competent cells of E. coli DH5α and recombinant E. coli clones were selected on LB plates with ampicillin. The recombinant pHT plasmid carrying the truncated cry1Ab gene was isolated from recombinant E. coli clone and transformed into the acrystalliferous Bt strain 4Q7 by electroporation (Mahillon et al. 1989). Transformed Bt colonies were selected on LB agar plate containing erythromycin. Spore-crystal mixtures were prepared from recombinant and non-recombinant Bt strains 4Q7 as described by Lenin et al. (2001). Isolated spore-crystal mixtures were subjected to SDS-PAGE analysis.

Immunostrip analysis of spore-crystal mixture from recombinant Bt strain

The Bt spore-crystal mixtures isolated from 4Q7 and recombinant 4Q7 Bt strains were tested by Cry1A immunostrips (Envirologix, USA) as per the manufacturer’s instruction.

Susceptibility of H. armigera to the cloned Cry1Ab toxin

For bioassay, laboratory cultures of H. armigera and S. litura (originally initiated from field-collected larvae) were reared on a semi synthetic diet. Insect bioassays were conducted using neonate larvae of H. armigera and S. litura by surface-diet contamination method. Each treatment was replicated three times in ten units. Larval mortality was recorded periodically for 7 days.

Results and discussion

Cloning of truncated cry1Ab gene from new indigenous isolate of Bt, A19-31

Young colonies of Bacillus thuringiensis (Bt) strains A19-31 and A22-73 were screened by colony PCR with primer pair 1AbFS1 and 1AbRS1 specific to cry1Ab gene. Amplicons of expected size (~600 bp) were obtained in the case of A19-31. This indicated the presence of cry1Ab gene in A19-31 indigenous isolate whereas A22-73 used as negative control failed to produce any amplification. The 1AbF1 and 1AbR1 are corresponding to 2,100 bp DNA fragment containing 149 bp upstream region (promoter) and 1,860 bp 5′-region of cry1Ab gene (for 620 amino acids). An intact DNA fragment of 2.1 kb was amplified using 1AbF1 and 1AbR1 primers from genomic DNA of indigenous strain of Bt, A19-31. The purified PCR product was cloned into pTZ57R/T vector (T/A cloning vector). The resulting construct was named as pT1Ab. Restriction digestion of its recombinant plasmids (pT1Ab) with XmaI and PstI released fragments of expected size (Fig. 1). The recombinant clone carrying 3′-truncated cry1Ab gene was sequenced and the gene sequence was deposited in NCBI GenBank under the accession number EU357806. Alignment of 3′-truncated Cry1Ab with known Cry1Ab proteins revealed residue changes which were as follows:

Fig. 1
figure 1

Restriction analysis of recombinant plasmid pT1Ab. M molecular mass marker λ/HindIII, 1 pTAb digested with XmaI and PstI

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  • when was compared to Cry1Ab1, CryAb3, Cry1Ab4, Cry1Ab5, Cry1Ab9, Cry1Ab10, Cry1Ab12, Cry1Ab13, Cry1Ab14, Cry1Ab15, Cry1Ab18, Cry1Ab20, two residues were different: Val433Ala and Trp455Gly;

  • when was compared to Cry1Ab2, 45 residues were different: Aligned sequences are given in Fig. 2;

    Fig. 2
    figure 2

    Alignment of the amino acid sequences between Cry1Ab-19 and Cry1Ab2. Only the regions containing differences are presented. The black boxes represent the residues showing variations

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  • when was compared to Cry1Ab6, five residues were different: Val433Ala, Trp455Gly, Asn461Glu, His542Asp and His569Thr;

  • when was compared to Cry1Ab7, six residues were different: Val433Ala, Pro450Ala, Trp455Gly, Leu537Phe, Ile545Pro and Ile568Thr;

  • when was compared to Cry1Ab8, four residues were different: Ala282Gly, Leu283Ser, Val433Ala and Trp455Gly;

  • when was compared to Cry1Ab16, four residues were different: Val165Ala, Leu176Ser, Val433Ala and Trp455Gly;

  • when was compared to Cry1Ab17, four residues were different: Pro170 Ser, Val433Ala, Gly450Arg and Trp455Gly;

  • when was compared to Cry1Ab19, three residues were different: Glu385Gly, Val433Ala and Trp455Gly;

  • when was compared to Cry1Ab21, three residues were different: Pro262Gln, Val433Ala and Trp455Gly;

  • when was compared to Cry1Ab22, three residues were different: Leu176Ser, Val433Ala and Trp455Gly.

Expression of truncated cry1Ab gene of Bt strain A19-31 in an acrystalliferous Bt strain 4Q7

The 3′-truncated cry1Ab gene cloned in the pTZ57R/T vector was released by XmaI and PstI digestion and ligated to E. coli-Bt shuttle vector, pHT3101 in the same sites. Recombinant plasmids were selected based on the restriction digestion with XmaI and PstI that releases 2.1 kb cry1Ab gene and 6.7 kb pHT3101 vector (Fig. 3). The recombinant pHT3101 containing the 3′-truncated cry1Ab gene was named as pHT1Ab. The plasmid construct was electroporated into acrystalliferous Bt strain 4Q7. Spore-crystal mixtures isolated from the both recombinant 4Q7 Bt strain and non-recombinant 4Q7 Bt strain were subjected to SDS-PAGE analysis. Expression of Cry1Ab toxin was not obvious as a distinct band of expected size (~65 kDa) in the protein profile of recombinant 4Q7 strain (Fig. 4).

Fig. 3
figure 3

Restriction analysis of recombinant plasmid pHT1Ab. M molecular mass marker 1 kb, 1 pHT1Ab digested with XmaI and PstI

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Fig. 4
figure 4

SDS-PAGE analysis of spore crystal mixture isolated from 4Q7 and recombinant (Cry1Ab transformant) Bt strains. M molecular mass marker, 1 non recombinant Bt strain 4Q7, 2 recombinant Bt strain 4Q7

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However, the Cry1A immunostrip had a positive reaction to the spore crystal mixture isolated from recombinant Bt strain of 4Q7. But the spore crystal mixture of Bt strain 4Q7 did not react with the Cry1A immunostrips (Fig. 5). Thus it can be suggested that the level of expression of truncated cry1Ab in transformants of Bt is low.

Fig. 5
figure 5

Immunostrip analysis of spore crystal mixture isolated from 4Q7 and recombinant (Cry1Ab transformant) Bt strains. 1 Non recombinant Bt strain 4Q7, 2 recombinant Bt strain 4Q7

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Toxicity analysis

Minor variations in the Cry toxins can lead to significant changes on the level of toxicity and insecticidal activities of the protein (Sasaki et al. 1997). Bioassay by artificial diet contamination method with spore-crystal mixture isolated from the Cry1Ab transformant of Bt strain 4Q7 (pHT1Ab) showed 70 and 20% mortality in H. armigera and S. litura neonate, respectively. Whereas all the larvae on control diet coated with Bt strain 4Q7 were alive until the 7 days after treatment. Higher level of expression of the truncated cry1Ab or whole cry1Ab gene of the new Bt strain A19-31 in transgenic microbes or plants could result in improved level of toxicity.