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Document Type : Research Paper
Abstract
This study involved two techniques for transferring important genes related to nitrogen fixation into Bacillus megaterium. Initially, morphological characteristics and molecular properties were analyzed to identify isolated bacteria. This process led to the isolation of Bacillus megaterium, Azotobacter chroococcum, and Rhizobium leguminosarum using a specific growth medium. Morphologically, the results showed that all the bacterial colonies were circular, convex, and smooth. A. chroococcum was creamy color, while B. megaterium and R. leguminosarum appeared white. The application of molecular approaches relied on identifying chromosomal DNA, specifically the nifH1 and nifV genes of A. chroococcum, and the plasmid DNA including the nodD1 and nodG genes of R. leguminosarum. Additionally, genomic DNA and two primers of B. megaterium were included. The nodD1 and nodG genes were successfully transferred from R. leguminosarum to B. megaterium, resulting in 46 colonies on agar plates of Sperber medium that contained tetracycline and ampicillin as genetic markers. Furthermore, the nifH1 and nifV genes were transferred into the transformant B. megaterium from A. chroococcum via a conjugation mechanism, leading to the growth of 170 colonies on plates of Sperber's agar medium containing erythromycin and ampicillin as genetic markers. To confirm the success of these transfers, molecular analyses were conducted to detect the nodD1 and nodG genes in the transformant B. megaterium, as well as the nifH1 and nifV genes in the conjugant B. megaterium. This was achieved using appropriate primers and PCR conditions for all the genes, followed by gel electrophoresis, which demonstrated the successful completion of both techniques.
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