Cloning, Characterization and Diversity of Insecticidal Crystal Protein Genes of Bacillus thuringiensis Native Isolates from Soils of Andaman and Nicobar Islands

Abstract

Bt strains were isolated from soils of Andaman and Nicobar Islands and characterized by microscopic and molecular methods. Diversity was observed both in protein and cry gene profiles, where majority of the isolates showed presence of 65 kDa protein band on SDS-PAGE while rest of them showed 130, 72, 44, and 29 kDa bands. PCR analysis revealed predominance of cry1I and cry7, 8 genes in these isolates. The PCR screening strategy presented here led us to identify putative novel cry genes which could be active against Coleoptera insects. Variation in the nucleotide sequences of cry genes from the isolates suggests that the genetic diversity of Bt isolates results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats in the soils of Andaman and Nicobar islands. The implications of our studies are important from the point of view of identifying novel cry genes that could be toxic to insects other than lepidoptera.

Expression ofcry3Agene and its toxicity against Asian Gray WeevilMyllocerus undecimpustulatus undatusMarshall (Coleoptera: Curculionidae)

“…For larvae exposed to Cry3Aa protoxin treated media the mortality increased from 22 ± 2.00% (1000 ng cm −2 ) to 30.2 ± 0.63% (10,000 ng cm −2 ). Higher doses (20,000 and 30,000 ng cm −2 ) had little additional effect on mortality and produced higher variability . It is coherent with a physiological meaning (pH, proteases or changes in toxin–receptor interaction on the apical brush border of the midgut microvillae).…”

“…The cry3A gene was amplified, cloned, and sequenced. Total DNA isolated from native Bt strains from Andaman and Nicobar Islands , Jammu and Kashmir (Unpublished data) was amplified (Fig. ) using gene specific primers.…”

“…B. thuringiensis of unknown strain was obtained from the Andaman and Nicobar Island and Jammu Kashmir. pTZ57T/R (Thermo Scientific, USA) and pQE 80L (Qiagen, USA; Fig.…”

“…Hence, cloning of full toxin genes from new Bt strains and their characterization are essential to expand the use of Bt and for the management of resistance development in insects. Searching for novel sources of cry genes encoding new toxins active against coleopteran insect‐pests is important; a cry3A ‐type gene was isolated from the Andaman and Nicobar Island and Jammu Kashmir Bt strains (Unpublished data). Herein, we report the cloning and characterization of the cry3A gene, its expression in Escherichia coli cells and also its insecticidal specificities and activities towards Coleopteran insect‐pests.…”

Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm

“…The experience has shown that the resistance conferred by a single Bt gene has the potential to break down as the target insect pest mutates and adapts to defeat the Bt trait [10]. To cope with the emergence of resistant insect population, there is great interest in finding novel insect-resistant gene with higher toxicity and/or wider spectrum [11]. In this study, we tried to develop new transgenic poplar with new insect genes other than Cry1Ac , in order to cope with the potential resistant insect in the future.…”