Construction Of NolU Gene Mutant And Functional Studies In Mesorhizobium Amorphae CCNWGS0123

Rhizobia,which can establish a symbiont ‘nodule’ organism with the leguminous plants,are widely distributed in the soil. The fixing-nitrogen symbiont is an important part of natural biological nitrogen fixation system as well as plays an important and potential role in aiding phytoremediation of some metal contaminated soils. Mesorhizobium amorphae CCNWGS0123, isolated from root nodules of Robinia pseudoacacia growing in GanSu province lead zinc mine tailings in the northwest of China. Previous studies have demonstrated that the type Ⅲ secretion system(T3SS) plays a significant role in conduction of the symbiosis system. We proposed that the NolU was one of M. amorphae CCNWGS0123T3 SS components and constructed the nolU gene deletion mutant for studying its function.1.Construction of pK18-nolUThe upstream and downstream homology arms of nolU gene were amplified, and connected the arms through overlapping PCR. After connecting PCR production and pMD18 vector, the pK18 mobsacB vecor and recombinant fragment were digested with same restriction endonucleases and connected with T4 Ligase. Then the recombinant plasmid pK18-nolU was transformed into E.coli DH5α, and comfirmed it was correct by sequencing.2. Construction and Screening of deletion mutant of nolU geneTransformed pK18-nol U into M. amorphae CCNWGS0123 with electroporation, we got nol U gene deletion mutant. Amplification of the target gene with PCR preliminarily verified the deletion mutant, and the sequence alignment results indicated that nolU had been deleted.3. Function studies of nolU geneCompared with R. pseudoacacia inoculated with wild type strains, the number of nodules significant declined in R. pseudoacacia inoculated with mutant. However, the plant experiments and paraffin section results showed that there was no difference among the nodule form, plant height, root length and nodule internal form.The results indicated that the expression of nol U could be double regulated by fl avonoid and tts box in M. amorphae CCNWGS0123. It was infered that NolU was a novel and non-conserved T3 SS component according to sequence alignment, and nolUgene play-ed an important role in early stage of estabilishment of the rhizobia-lugume symbiotic system.