Study On Soybean Rhizobium Engineering Strain Introduced Into The NodD-lba Gene

Biological nitrogen fixation is the main source of nitrogen in natural ecosystems and has an important role in the part of agricultural production. In the process of rhizobium and plant, nodulation genes play an important role and nodD is one of the genes which belong to regulator gene.nodD gene encode NodD protein which regulate the expression of other nod structural genes. NodD protein can regulate some genes expression which perform important functions in bacteria and eukaryotes interacting. Glycine max hemoglobin present in soybean nodules has a role of oxygen buffer which around the bacteroid to prevent excessive free oxygen in contact with nitrogen-fixing enzyme nitrogenase enzyme immunoassay damage by oxygen.Thereby, It can maintain an efficient nitrogenase activity. Glycine max hemoglobin encoded by four corresponding gene (lba, lbcl, lbc2, lbc3), by the study of the laboratory that the soybean rhizobia engineered strains which introduced lba gene and nodD gene can enhance nodule nitrogenase activity.The purpose of this study is that if the tandem gene has an interaction can improve nitrogen fixation.In this study, PCR technology was used to clone nodulation gene of nodD and hemoglobin gene of lba with the template of plasmid DNA which constructed by our laboratory.The two genes were ordered joined into the plasmid vector pET-Plac which include lac promoter and the T7 terminator to constructed the plasmid vector pET-Plac-nodD-lba. The target fragment joined into the vector pTR102 which include luxAB gene to constructed the prokaryotic expression vector PTR-Plac-nodD-lba. The vector was transformed into Sinorhizobium fredii and native Rhizobia japonicum with the method of triparental hybridization to construct genetic engineering strains SF (ptr-Piac-nodD-lba) SFH (ptr-plac-nodD-lba). Western Blot indicated the genes we used can expressed normally.The results of plant experiments showed that engineering strains compared with non-inoculation and original strain and strain which transfected with empty vector can improve the height, fresh weight, dry weight, the number of nodules, nitrogenase activity and yield better.Engineering strains which introduced tandem gene have more effect than single gene,performance in nitrogenase activity with the data of 4%-7% and soybean yield with the data of 17%-22%.