Abstract
The crystal (Cry) proteins produced by Bacillus thuringiensis determine a particular strain's toxicity profile. This study was focused on cloning a cry1Ia gene based on amplicon restriction fragment length polymorphism (ARFLP) profile which would be helpful in developing new biopesticides with broader and higher spectrum of toxicity against Lepidoptera and Coleoptera insect pests. The present paper describes cry1Ia gene from a local isolate of Bacillus thuringiensis (B.t) CFE20(3). A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of primers based on the conserved regions of the genes to amplify 2,169 bp cry1I-type gene fragments. Amplification products were digested with the KpnI and XbaI, and new kind of cry1I-type genes was successfully identified. Escherichia coli DH5α was transformed with recombinant DNA comprising pTZ57R/T and Bt cry1I (2169 bp) amplified from a native isolate CFE20(3) for cloning. The cloned 2169 bp was sequenced and then ligated in the expression vector pQE30 for transformation of E. coli M15 and SG13009 for expression analysis. The sequence obtained shows 99% homology with known cry1Ia from B. thuringiensis subsp. Kurstaki. An expected band size of 81 KDa was observed after sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicating the expression of cry1I. The toxicity of crude recombinant Cry1I proteins was determined against third instar larvae of Diamond back moth, Plutella xylostella and Spodoptera litura. Cry1I protein was found to be effective against Plutella xylostella. Key words: Bacillus thuringiensis, cry1I, SDS-PAGE, Plutella xylostella, Spodoptera litura.
Highlights
Cloning g and expres e ssion of o Baciillus th huringiiensis cry1Ia a in Esche erichia coli an nd its insectticidal activitty
Bacillus thurringiensis was established by des signing a paiir of primers based on th he conserved d regions off the genes to o amplify
Native Bacillus thuringiensis isolate CFE20(3) and CFE25(2) which are available in Institute of Agri Biotechnology, University of Agricultural Sciences, Dharwad was used to amplify cry1Ia gene
Summary
The crystall (Cry) prote eins produce ed by Bacilllus thuringie ensis determ mine a partic cular strain's s toxicity profile. This s study was focused on cloning a crry1Ia gene ba ased on amp plicon restric ction fragme ent length polymorphiism (ARFLP) profile whiich would be helpful in developing new biopes sticides with h broader and higher spectrum of o toxicity ag gainst Lepid doptera and Coleoptera insect pests s. A PCR-re estriction fragment le ength polymo orphism metthod for iden ntification off cry1I-type g genes from B. The toxicitty of crude rrecombinantt Cry1I prote eins was de etermined against thirrd instar larv vae of Diamond back motth, Plutella x xylostella and d Spodopterra litura.
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