Construction Of Recombinant Rhizobia Strains With Enhanced Tolerance To Cadmium By Using Bacterial Cell Surface Display

In this study,Xanthomonas campestris ACCC10049 (inaXN) was used as an anchoring motif for bacterial surface display. A monkey metallothioneinαdomain (MT4α) was used as target protein. Fusion gene of inaXN-MT4αwas successfully displayed on the surface of Sinorhizobium fredii HN01. Under pure culturing condition, the adsorption capacity and tolerance ability to cadmium of recombinant and recipient strains were comparatively examined. By infection of recombinant strain, Soybean established symbiosis with the recombinant or recipient strains that was grown in Cd2+ polluted soils. Under this conditions, we studied Cd effects on the symbiotic system.Based on the broad-host-range vector pBBR1MCS5、pTPG-PnifH, fusion expression of the gene inaX-MT4αand MT4αwere feasible. The inaX-MT4αand MT4αwere cloned, inserted into pBBR1MCS5, pTPG-PnifH and renamed them as pBLM、pBLIM、pTNM and pTNIM, respectively. By tri-parent matting technique, four recombinant plasmids were transferred into recipient Sinorhizobium fredii HN01. Two inducible expression type of recombinant rhizobium HN01 (pTNIM)、HN01 (pTNM) and two constitutive expression type recombinant rhizobium HN01 (pBLIM)、HN01 (pBLM) were obtained, respectively.Under pure culturing condition, the adsorption capacity to cadmium of recombinant and recipient strains were comparatively examined, and it showed that the abilities of HN01 (pBLIM) were significantly improved, as such its cadmium-adsorption capacity was increased up to two-fold. The results also indicated the recombinant rhizobia demonstrated rather good static-adsorption characteristics.These recombinant strains HN01 (pBLIM), HN01 (pBLM), HN01 (pTNIM), HN01 (pTNM) and HN01 established the symbiotic relationship with soybean. The result indicated that HN01 (pBLIM), HN01 (pBLM),HN01 (pTNIM),HN01 (pTNM) and HN01 symbiosised with plants could protect the growth of plants. The symbiosis of engineering strain HN01 (pBLIM), HN01 (pBLM), HN01 (pTNIM)and HN01 (pTNM) with plants displayed much more powerful cadmium adsorption capacity and transfer ability than that of HN01. Lastly, pot plant experiments with 9μmol/L Cd concentration showed the symbionts increased Cd accumulation in nodules by 1.85 fold in hydroponic culture by infection of recombinant strain HN01 (pTNIM)...