Study On Lrp Gene Of Sinorhizobium Fredii HN01

Sinorhizobium fredii HN01 is a fast-growing Chinese isolate with fewerextracelluar polysaccher that has been adopted as a model organism for thestudy of nitrogen fixation and suited for commercial inoculation production. Inthis study, a positive clone containing lrp fragment of Sfredii HN01, wasisolated from DNA library by colony in Situ hybridization. A complete lrp genewas obtained through subcloning. Bioiformatics analysis showed that the lrpgene share 89% homology at nucleotide level and 99% homology at amino acidlevel with the reported lrp gene of Sinorhizobium meliloti 1021.With the suicide plasmid pK18mob, polar mutant GXHNLTB andnon-polar mutant GXHNLTA inactivated in lrp gene have been constructedthrough homologous recombination caused by a single crossover event. Theintegrate lrp gene was amplified by PCR method and cloned into pLAFR3resulting in a complementati0n plasmid pGXHNL100. The complementationstrains GXHNWA and GXHNWB were constructed by transformingpGXHNL 100 into the host strains GXHNLTA and GXHNLTB, respectively.Both non-polar and polar mutants of HN01 with lrp gene disrupted showed a little slower in growth with respect to their parent-type in MM mediumcontaining amino acid such as proline, leucine or serine as sole carbon andnitrogen source.Plant tests revealed that the mutants GXHNLTA and GXHNLTB showed1 day ahead of the wild-type in initial nodulation time and its result did not showobvious effects of mutation of lrp gene on nodulation efficiency ability andnitrogen-fixation.