Cloning of partial cry1Ac gene from an indigenous isolate of Bacillus thuringiensis

The discoveries of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity are important for the development of new products. The cry1 family genes are more toxic to the lepidopteran insects according to the previous reports. In the present study, nine indigenous isolates of Bt were used for screening of cry1 genes by PCR using degenerate primers specific to cry1 family genes. Two of the nine new isolates of Bt showed amplification with cry1 family primers. Partial cry1gene(s) was cloned from one of the cry1 positive isolate of Bt, T133. Nucleotide sequence data generated for partial cry1 gene of T133 showed 98% homology with 1420 bp of the partial cry1Ac1 gene. Deduced amino acid sequence of the partial cry1Ac of Bt strain, T133 showed two per cent variation in comparison to Cry1Ac1 by differing at 5 positions; one deletion at 442th position and five substitutions at the following positions, 296, 367, 440 and 563.


INTRODUCTION
The control of agriculture pest populations is achieved mainly by the application of chemical insecticides; the continuous use of synthetic pesticides led to serious pro- blems like environmental degradation and development of resistance in insect pest (Shelton et al., 2002).Recently, there has been a renewed interest in the development of biological alternatives to chemical pesticides.The Bacillus thuringiensis (Bt) Berliner is considered as one of the most versatile microbial insecticides.It is a gram-positive sporeforming soil bacterium .The insecticidal activity is based on the ability of the bacterium to produce large quantity of larvicidal proteins known as delta-endotoxins (Cry proteins).The Cry toxins constitute a family of related proteins that can kill insects belonging to the Lepidoptera, Coleoptera, Diptera, Hymenoptera, Homoptera and Mallophaga, as well as other invertebrates (Schnepf et al., 1998;Feitelson et al., 1999).The advancement in molecular biology led to the cloning of Bt crystal protein (cry) gene for the first time in 1981 (Schnepf and Whiteley, 1981).So far more than 445 cry genes have been successfully cloned and characterized for their insecticidal potential.
Cry1A toxins are very important because of their high toxicity to lepidopteran pests and widespread distribution among Bt strains (Li et al., 1995;Bravo et al., 1998;Uribe et al., 2003).Variation in toxicity and specificity exist among different Cry1A toxins due to minor amino acid substitutions (Tounsi et al., 1999).About 69 cry1A genes are classified into cry1Aa to cry1Ai sub-types (http:// www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).It is still essential to search for novel Bt strains that may lead to the discovery of additional insecticidal proteins with higher toxicity against wider pest range.The PCR method is proven to be a powerful tool which allows quick, simultaneous screening of many Bt samples, identification of specific insecticidal genes carried by different Bt strains, classification of cry genes and subsequent prediction of their insecticidal activities (Carozzi et al., 1991;Ben-Dov et al., 1997;Juarez et al., 1997).The amplification, subsequent cloning and sequence analysis of the cry genes from new isolates of Bt are important because they may provide new gene sequences encoding more active toxins which could be used for developing better versions of transgenic crop plants.So, the present study was undertaken for cloning and sequencing of cry gene from indigenous isolates of Bt.

Bacterial strains and plasmids
Nine indigenous Bt strains (T17, T21, T50, T75, T81, T133, T134, T142 and T147) and a reference strain, Bt (indigenous strain, 14r1) are from the collection of Bt strains maintained by the corresponding author in the Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India.Escherichia coli vector pTZ57R\T obtained from Fermentas INC.

Amplification of Bt DNA by PCR
Total genomic DNA from B. thuringiensis strains, T17, T21, T50, T75, T81, T133, T134, T142 and T147 were extracted as described earlier by Kalman et al. (1993) and used as the template for the PCR screening.The genomic DNA of a reference strain of Bt, 14r1 was used as positive control.A set of degenerate primers (JF and JR) described by Juarez-Perez et al. (1997) were used to screen the new isolates of Bt for the presence of cry1 family genes (Table 1).These primers are expected to amplify fragments of ~ 1.5 kb from the cry1 family genes.
The PCR was accomplished using an Eppendorf thermal cycler in 25 µl reaction volume containing total genomic DNA of Bt (30 ng), 2.5 µl of 10X PCR buffer (10 mM Tris-HCl; pH: 9.0, 50 mM KCl, 1.5 mM MgCl 2 ) 75 μM each of dNTPs, 50 ng each of forward and reverse primers and 0.5 Units of Taq DNA polymerase.The PCR was performed for 30 cycles as follows: 94°C for 1 min, 40°C for 45 s, 72°C for 2 min and the final extension was performed for 7 min at 72°C.

Cloning of partial cry1 fragments from Bt isolate, T133
The gel eluted PCR products of partial cry1 gene (~1.5 kb) from Bt isolate, T133 was ligated into T/A vector (pTZ57R\T Fermentas INC) as per the manufacturer's instruction.The ligated mixture was transformed into E. coli as per the standard procedure (Sambrook et al., 1989).The transformed colonies of E. coli were screened by colony PCR with M13F and M13R primers for checking the presence of insert (partial cry1 fragment of Bt isolate, T133).

Nucleotide sequencing of recombinant plasmids
The plasmid DNA was isolated from the E. coli transformants containing partial cry1 gene of Bt isolate, T133 and nucleotide sequence of recombinant plasmids was carried out by automated sequencing (Ist Base, Singapore).The sequence data was subjected to homology search through Basic Local Alignment Search Tool (BLAST) of National Centre for Biotechnological Information (NCBI) (www.ncbi.nlm.nih.gov/Blast).The deduced amino acid sequence was generated by BioEdit (Hall, 1999).

Screening of new isolates of Bt for cry1 genes
Total genomic DNA isolated from indigenous isolates of Bt was used as template for screening of cry1 genes by PCR using the degenerate primers specific to cry1 family genes.Amplification of expected size (~ 1.5 kb) of PCR products were obtained from two new Bt isolates T17 and T133, whereas amplification was not observed in the remaining seven isolates (Figure 1).

Cloning and sequence analysis of partial cry1 gene from Bt isolate, T133
The gel eluted PCR product (~1.5 kb) of partial cry1 gene  fragment from Bt isolate, T133 was cloned into pTZ57R/T vector (T/A vector).The recombinant clones (white colonies) were selected on LB agar containing X-gal, IPTG and ampicillin.Presence of insert was confirmed in recombinant E. coli colonies, by colony PCR with M13 forward and M13 reverse primers.Agarose gel electro-phoresis of DNA amplified from the transformants of E. coli showed expected size of ~1.7 kb corresponding to the sum of insert DNA of 1.5 kb and vector sequence of about 200 bp (Figure 2).Recombinant plasmid isolated from three of the E. coli clones were used to determine nucleotide sequence of the partial cry1 gene of Bt strain,  T133 by automated DNA sequencing.Nucleotide sequence data ( Acc.No. FJ794964) revealed 98% homology with 1420 bp bases in the 5' region of holotype, cry1Ac1.Deduced amino acid sequence of partial Cry1Ac gene showed one deletion at 442th position and five substitutions at the following positions, 296,367, 440 and 563 of Cry1Ac1 holotype sequence (Figure 3).

DISCUSSION
Indiscriminate use of broad-spectrum chemical insecticides has caused adverse effects to human health, other non-target organisms and has led to the build-up of chemical resistance in insect pests (Waage, 1997).Therefore, the urgent need for environmentally safe pest control is required to maintain sustainability of the environment.Deployment of integrated pest management (IPM) strategies to minimize crop production losses incurred due to insect pests can make significant contribution to food security in the developing countries.
The diversity of Bt strains facilitates isolation of new types of cry genes.PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities.Several screening projects of Bt collections from different parts of the world have been described (Juarez-Perez et al., 1997;Bravo et al., 1998;Ben-Dov et al., 1997;Thammasittirong and Attathom, 2008).In the present study, nine indigenous isolates of Bt were screened for the presence of cry1 genes through PCR with the cry1 family primers described by Juarez-Perez et al. (1997).Among the nine new isolates, only two showed amplification of expected size as in the case of the reference strain of Bt, 14R1 which is known to harbor for cry1 genes.
Most of the commercial Bt formulations used for the control of lepidopteran pests, contain toxins of Cry1A family, especially Cry1Aa, Cry1Ab and Cry1Ac proteins (Hofte and Whiteley, 1989).Cloning of the first cry gene, namely cry1A(a), was reported by Schnepf and Whiteley (1981).Recently, Xue et al. (2008) reported cloning of novel cry1Ah gene and its protein was more toxic to lepidopteran Asian corn borer.Swiecicka et al. (2008) reported that the novel Cry1Ab21 that produces a quasicuboidal crystal protein which is toxic to larvae of Trichoplusia ni.
In the present study, the partial cry1 gene fragments amplified from genomic DNA of Bt strain, T133 are cloned in T/A vector.Nucleotide sequence of partial cry1Ac gene showed 98% similarity to the already reported cry1Ac1 holotype sequence.The deduced amino acid sequences of partial Cry1Ac of Bt isolate, T133 showed difference at six positions in comparison to that of Cry1Ac1.
Result for the present study revealed the presence of new variant of cry1Ac in Bt isolate, T133.Variation of a single amino acid can significantly influence the level of toxicity in Cry proteins (Udayasuriyan et al., 1994;Liao et al., 2002).Therefore, further studies on expression of complete ORF of the novel cry1Ac cloned from the new isolate of Bt, T133 will be useful to know the insecticidal potency of its proteins.
, G or T; D = A, G or T; H = A, C or T; K = G or T; M = A or C; R = A or G and Y = T or C. Source: (Juarez-Perez et al., 1997).

Figure 2 .
Figure 2. Screening of recombinant E. coli colonies by PCR for presence of partial cry1 gene.Lane 1: 1 kb marker; lane 2: negative control; lane 3: partial cry1 gene fragment from 14r1 (positive control) and lane 4: partial cry1 gene from new isolates of Bt, T133.

Figure 3 .
Figure 3. Homology between the deduced amino acid sequence of partial cry1Ac gene of T133 and holotype, Cry1Ac1.Query: cry1Ac amino acid sequence of Bt strain, T133; Subject: cry1Ac1 amino acid sequence.Boldface letters indicate the six amino acid differences between Cry1Ac of Bt strain, T133 and Cry1Ac1 (holotype).

Table 1 .
Primers used for screening and amplification of cry1 gene fragments.