Cloning And Functional Identification Of The Homologue Of Mesorhizobium Huakuii Symbiotic Opa22 Gene

opa22 gene (Genebank No.DQ 199621) is cloned and identified by our lab, involved in nodule formation by Mesorhizobium huakuii 7653R. By Blast, it is found that there are many Opa22-like secreted proteins in many rhizobia, for example:Mesorhizobium loti, Sinorhizobium medicae, Sinorhizobium meliloti, Rhizobium leguminosarum, Bradyrhizobium japonicum, and so on. Therefore, it will provide new details and foundation of signal exchange between rhizobia and their hosts to further study the functional indentification of opa22 homologues.In this study, the homologous opa22 genes of the five strains were cloned successfully. From the analysis of these Opa22 homologues stuctures and properties, it is showed that they areβ-barrel outer membrane protein (BOMP) and the members of surface_Ag_ 2 superfamily. Compared with the known genes homologously and phylogenetically, the result showed that homologous opa22 gene existed in many rhizobia and Opa22 had the similary of 99% to the homologous Opa22 in Mesorhizobium loti. Besides, the Opa22 homologues can be found in other non-rhizobia:Agrobacterium that is close relative of rhizobia; Brucella, Ochrobactrum, Rhodopseudomona which do not interact with plants. We construct phylogentic tree of Opa22 homologous protein and compare it with one of 16S rDNA. It revealed that they are very similar and identical to each other and the protein is still relatively conservative through long evolution of rhizobia.Furthurmore, the above five opa22 homologues replacement plasmids were constructed and three mutants have been aquired:S. medicae 112, B. japonicum 62, R. leguminosarumn bv. trifolii 455. Inactivation of the opa22 gene homologue of S. medicae, Bradyrhizobium japonicum made them fail to nodulate their hosts. The former mutant is capable of curling the root hair and affects the development of infection thread,but the latter mutant is not capable of curling the root hair. In contrast to the wild types, the complementary strains constructed by themselves opa22 homologues can partly restore the ability of nodulation, whereas the complementary strain of the former mutant constucted by opa22 from M.huakuii can partly restore the ability of nodulation but one of the latter mutant can not.Therefore, we have proved that the Opa22 homologues are new novel kind of nodulation outer membrane proteins and play an crucial role in the nodulation between the rhizobia and their hosts, and exist the host specificity. These results have offered important data in the dissection of the regulations of the interaction between rhzobia and hosts.