Европейский журнал экспериментальной биологии Открытый доступ

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Isolation and molecular identification of cry gene in Bacillus thuringiensis isolated from soils by semi-conserve PCR

Fatemeh Tohidi, Sadaf Toulami, Hedyeh Fazel Tolami, Fahimeh Safarnejad Tameshkel, Anosh Nourani, Seyed Reza Seyedinasab, Ali Nazemi and Mostafa Jafarpour

Bacillus thuringiensis is a gram positive and spore forming bacterium. The most of the natural habitat this bacterium is soil and it is capable of producing the diversified varieties of crystalline proteins with insecticide property. The aim of this study has been isolation of strains of Bacillus thuringiensis carrying cry gene from soils of the west Mazandaran provinces. After screening the soil of 35 regions, 12 strains of Bacillus thuringiensis were isolated by the selective minimal medium contain sodium acetate and L-serine. After DNA extraction, Bacillus thuringiensis strains were confirmed using 16srDNA PCR sequencing. Also, relative molecular weight of crystalline protein of the strain containing the cry gene was evaluated through SDS-PAGE. Out of 12 isolated strains, cry gene from 1Aa class was identified only in one strain by Semi-conserve PCR. Study of rDNA sequence of the isolated strain showed a 99% homology with IBL200 strain of the Bacillus thuringiensis. Also approximate weight of crystalline protein was measured in the range of 90 to 100 KDa. With regard to very much diversity of crystalline gene and lack of existence of accurate phenotypic methods to identify the presence of the crystalline protein using of this semi-conserve PCR method can identify the bacilli containing the crystal gene with low cost and fast method.