Cloning and characterization of an insecticidal crystal protein gene fromBacillus thuringiensis subspecieskenyae

Abstract

A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.

The synergic and antagonistic activity of Cry1Ab and Cry2Aa proteins against lepidopteran pests

“…) for Cry1Ab and F‐5′GAA TTC GGA TCC ATA TGA ATA GTG TAT TGA AT3′ and R‐5′GTC GAC GTA CGG ATCC TAC TCA AAC CTT AAT AA3′ (Mısra et al. ) for Cry2Aa were used. The PCR amplification was performed under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 52°C (50°C for Cry2Aa) for 1 min, 72°C for 3.5 min (2 min for Cry2Aa) and a final extension step at 72°C for 10 min (Mısra et al.…”

“…The PCR amplification was performed under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 52°C (50°C for Cry2Aa) for 1 min, 72°C for 3.5 min (2 min for Cry2Aa) and a final extension step at 72°C for 10 min (Mısra et al. ; Stobdan et al. ).…”

“…For the amplification of complete genes, primer pairs F-5 0 GGA TCC ATG GAT AAC AAT CCG AAC ATC3 0 and R-5 0 GTC GAC TTA TTC CTC CAT AAG AAG TAA3 0 (Stobdan et al 2004) for Cry1Ab and F-5 0 GAA TTC GGA TCC ATA TGA ATA GTG TAT TGA AT3 0 and R-5 0 GTC GAC GTA CGG ATCC TAC TCA AAC CTT AAT AA3 0 (Mısra et al 2002) for Cry2Aa were used. The PCR amplification was performed under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 52°C (50°C for Cry2Aa) for 1 min, 72°C for 3.5 min (2 min for Cry2Aa) and a final extension step at 72°C for 10 min (Mısra et al 2002;Stobdan et al 2004). Recombinant pET-cry1Ab and pET-cry2Aa plasmids were obtained through cloning cry1Ab (~3.5 kb) and cry2Aa (~1.9 kb) genes separately to pET28a(+) expression vector.…”

Genetic engineering of cotton with a novel cry2AX1 gene to impart insect resistance against Helicoverpa armigera

“…H. zea was susceptible to Bt toxins in the order Cry1Ac,Cry1Ab,Cry1Aa,and Cry2A with 63.60,89.04,159.65,and 375.78 ng/larvae, respectively. Indian populations of H. armigera were 5-to 30-fold less susceptible to cry2Aa than cry1Ac (Chakrabarti et al 1998, Babu et al 2002, Misra et al 2002. The expression level of Cry2Ab in commercialized Bt cotton ranges from 16.8 to 22.7 μg g -1 in fresh leaf tissue (Li et al 2011).…”

Determination of efficacy of some biopreparats on Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) larvae

“…kenyae kristal proteinlere göre yapılan sınıflandırmada Cry1'de yer almaktadır (Masson et al, 1992). Bt kenyae'nin HD549 ırkı, Spodoptera litura (Boisduval), Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) ve Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) türlerinin larvalarına etkili olduğu bildirilmiştir (Misra et al, 2002).…”