Cloning And Functional Identification Of Symbiotic Nitrogen Fixation Genes Of Mesorhizobium Huakuii 7653R

Mesorhizobium huakuii can induce the formation of indeterminate nodules on host plant roots of Astragalus sinicus L., and shows strict host specificity. Eight thousand mutants by using Tn5-sacB insertion mutagenesis were obtained from M. huakuii 7653R. Thirty symbiotic mutants were screened by plant nodulation experiments. The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR. The results showed that five mutants contained disrupted genes which had high sequence similarity to function-unkown genes of M. loti. They were named as opa22, lpsH, dmtH, gstH and catH.Mutation in the opa22 gene failed to form nodules on Astragalus sinicus L.. Sequence analysis results indicated that opa22 encoded a protein of 257 amino acids with a predicted molecular mass of 28.1 kDa. RPS-BLAST analysis of Opa22 protein showed a sequence similarity (88.9%) to the opacity protein and related to surface antigens of bacterial outer membrane, which could mediate various pathogen-host cell interactions and promote invasion. Results of root hair curling experiments suggested that the expression of opa22 gene might occur in the stage of infection thread formation and nodule development. Its complemented stain HK24 restored the nodule formation ability of the mutant.Mutation in the lpsH gene could only form pseudonodules on Astragalus sinicus L.. DNA sequence analysis revealed that lpsH gene encoded a protein of 397 amino acids with a predicted molecular mass of 43.6 kDa. LpsH contains a putative signal peptide, 11 transmembrane helices and an O-antigen polymerase domain, which was necessary to LPS synthesis and located on the periplasmic side. SDS-PAGE showed that the lpsH mutant strain made LPS I in lower amounts than the wild type. Results of plant nodulation studies showed that lpsH gene mutant was deficient in the ability to compete for nodulation. Results from nodule ultra-structural studies revealed that cortical cells infected by lpsH mutant contained a few bacteroids and numerous senescences. In conclusion, LpsH is an integral membrane protein involved in LPS biosynthesis and plays a critical role in its interaction with the plant partner. In addition, its effect in the special symbiotic interactions was also confirmed by a double-crossover mutant HK242.Mutation in the dmtH gene resulted in the formation of pseudonodules on Astragalus sinicus L.. DNA sequence analysis results revealed that dmtH gene encoded a protein of 249 amino acids with a predicted molecular mass of 27.3 kDa. RPS-BLAST analysis of DmtH protein showed a sequence similarity (89.6%) to Demethylmenaquinone(DMK)...