Recombinant Expression, Identification And Function Research Of MrtR Protein In Mesorhizobium Tianshanense

In the quorum-sensing regulatory systems of rhizobia, regulation genes elaborate their functions through affecting each others. There are several LuxR/I type quorum sensing systems in different rhizobia, such as CinR/I, RaiR/I, RhiR/I, TraR/I and SinR/I etc. Our lab has found a new MrtR/I system in Mesorhizobium tianshanense. MrtI was responsible for synthesizing all AHLs detected in M.tianshanense. The expression of the quorum-sensing regulators was autoinduced, as mrtI was fully induced only when the supernatant prepared from the cell-free spent medium of the wild-type culture which contains the maximal amount of AHLs was added. Addition of supernatants from the mrtI mutant culture with no AHLs could not induce mrtI expression.The mrtR coding sequence was fused to the T7 promoter of bacteriophage T7 by PCR amplification, using pHMZ9B1 as template. The resulting PCR product was cloned into pALEx, resulting in co-expressed GST-MrtR plasmid pDJ1. pDJ1 were then transformed into E.coli BL21(DE3), constituting recombinant bacteria BL21(DE3)(pDJ1).After being induced by IPTG, the recombinant bacteria, BL21(DE3)(pDJ1) was analyzed by SDS-PAGE, showing that fusion protein GST-MrtR expressed as 58kD. After it was disrupted by sonication, SDS-PAGE experiments indicated that the fusion proteins of BL21(DE3)(pDJ1) existed as both soluble protein and inclusion body.To prepare polycolonal antibodies against MrtR, purified GST-MrtR protein was used as immunogen to immunize intraperitoneally 8-week-old BALB/c mice. The immune dose was 50μg of the purified GST-MrtR, emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the next two injections with 2-week intervals. The ELISA titer of antiserum against MrtR was about 1: 262 000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically.Specific primers were designed and synthesized according to the front of mrtI gene, and the fragment was cloned into pBSK. After double digested, the fragment was subjected to Gel-shift assay with purified GST-MrtR protein, but there was no specific moved band to be detected. It was concluded that the binding site of MrtR protein was not on the near front of mrtI or that there were other factors which could be indispensable to the intereaction between MrtR and its corresponding DNA fragment.