Establishment and screen of Cyan Fluorescent Protein labeled strains of alfalfa rhizobia

Fluorescent protein labeled strains of rhizobia play an important role in the research about the progress of strain infecting plant and forming nodules, as well as determining strain competitive capability. In the present study, assistant bacteria strain Escherichia coli pRK2073, recipient bacteria strains Sinorhizobium meliloti 12531 and Rhizobium meliloti GN5, and donor bacteria strain E. coli pMP45179 which provided cyan fluorescent protein were transformed based on triparental hybridization technology. The genetic stability of fluorescence expression and nitrogen fixation of these successful transgenic strains were evaluated on Nfree and TY medium. Then, the preliminarily screened stains were inoculated to alfalfa cv. Gannong No.5 to study their effects. The results indicated that triparental hybridization method was feasible for construction of cfp labeled rhizobia stains which provided abundant cfp labeled strains of S. meliloti 12531 and R. meliloti GN5. The strains of S. meliloti 12531cfp6 and R. meliloti GNfcfp5 had the best genetic stability with more fluorescence expression and better nodulation effects. Nfree medium with antibiotics have higher screening efficiency than the current medium with antibiotics. The large variations existed among these labeled strains which suggested that it was necessary to confirm the genetic stability of fluorescence expression, Nfixation and the effects on growth for cfp labeled strains selection.