ABSTRACT
Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.
Subject(s)
Alphaproteobacteria/genetics , Astragalus Plant/microbiology , Conjugation, Genetic , DNA Transposable Elements/genetics , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Green Fluorescent Proteins/genetics , Recombinant Proteins/geneticsABSTRACT
Astragalus sinicus (Chinese Milk vetch), a green manure leguminous plant, harbors Mesorhizobium huakuii subsp. rengei strain B3 in the root nodules. The visualization of symbiotic plasmid of strain B3 showed the presence of one sym plasmid of about 425 kbp. Curing of sym plasmid by temperature and acrydine orange was studied. Growing rhizobial cells at high temperature (37 degrees C) or treating the cells with acrydine orange at 50 mg/l eliminated sym plasmid of M. huakuii strain B3, which was confirmed by sym plasmid visualization and plant infection test of cured strains.