In Silico Analysis for Detection of CryII Gene from Local Isolates of Bacillus thuringiensis

Abstract

: Bacillus thuringiensis (Bt) is a gram-positive bacteria that can produce crystal proteins and exhibit a high insecticidal activity. One of the cry proteins is cryII gene that encoding an insecticidal protein against the Lepidoptera insect, especially in the Helicoverpa armigera species, which is a corn borer. This study aimed to obtain a suitable primer candidate to amplify and characterize cryII genes from Bacillus thuringiensis local isolates. In silico analysis was carried out for the purpose of amplification cryII gene, using several software such as BLAST to search sequence cryII gene, Bioedit for sequence alignment, SnapGene for analysis of genes within the genome annotation, and Primer3Plus for online primer design via https://primer3plus.com. A pair primer was selected to represent of cryII gene with a forward primer (cry2F) 5’- ATCTGGTCTCATAGGGGCGA-3’ and reverse primer (cry2R) 5’-CGAGCTGTCGTGTTGCTTTG-3’ with GC content percentage 55%. The purity of DNA Bacillus thuringiensis isolates B327 and B432 using nanoquant with a mark on each isolate at 1.88 and 1.99. Detection of the cryII gene from local isolates of Bt strain B327 and B432 were successfully amplified by Polymerase Chain Reaction (PCR) gradient technique using annealing temperatures at 50 and 54°C, with DNA band size of ±500 bp.