Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was isolated as an intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd301. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPHU1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex planta in two such constructs BSL 12-1 and BSL 12-3.