Cloning And Functional Identification Of The MHK-RGS1 Gene, A Novel Gene Involved In Symbiotic Nitrogen Fixation Of Mesorhizobium Huakuii 7653R.

Previous study has constructed a Tn5-sacB mutant library of Mesorhizobium huakuii 7653R, and has identified a symbiotic deficient mutant strain HK311 (nod+fix-) through pot plant nodulation experiments. This study found a gene involved in the mutation is responsible for the formation of pseudonodules on A.sinicus. This gene was termed mhk-RGS1 in this study. mhk-RGS1 sequence of the contiguous region from the Tn5 insertion site was determined by thermal asymmetric interlace PCR. Compared with the genome sequences of Mesorhizobium huakuii 7653R, it was found that there were two copies of mhk-RGS1 in Mesorhizobium huakuii 7653R, respectively named D22 and D29. Sequence analysis indicated that mhk-RGS1 only contains 251bp, showing a high sequence similarity (93%) to function-unknown gene of Rhizobium leguminosarum bv. viciae 3841 and a high homology (85%) to transposase gene of Mesorhizobium loti MAFF303099.To confirm the function of RGS1 in symbiotic nitrogen fixation, the above two copies of mhk-RGS1 homologs replacement plasmids were constructed, and two mutants MhK-D22mutKm and MhK-D29mutKm have been obtained. Results from pot plant nodulation experiments showed that both mutants of the mhk-RGS1 gene formed ineffective nodules and were deficient in the ability to fix dinitrogen, with a significant decrease in the nodule nitrogen fixation activities and impaired symbiotic phenotypes. Nodule section and electron microscopy revealed that nodule bacteroid development of both mutants were blocked, showing by a remarkably decreased-size and abnormal morphology in symbiont when compared with wild-type strain. In addition, it showed that a much larger non-bacteroid zone and premature senescence feature in the mutant nodules. Further more, quantitative PCR indicated that mhk-RGS1 gene is specifically expressed in bacteroids during symbiotic nitrogen fixation. It revealed that mhk-RGS1 is a novel gene which plays an important role in symbiotic nitrogen fixation.In order to reveal the promising regulatory mechanism of the RGS1 gene, the possible promoters of D22 coding region and D29 coding region were respectively predicted via bioinformatics analysis. By using RT-PCR, it was showed that the transcription direction of mhk-RGS1 genes were consistent with that of the structural genes located in the downstream. In the D29 coding region, mhk-RGS1 and fixABC required for rhizobial symbiotic nitrogen fixation, formed a gene cluster on the chromosome. We confirmed that the components in the gene cluster were co-transcribed as an operon. Therefore, the results provided sensible explanation for the fact that both mutants of MhK-D22mutKm and MhK-D29mutKm would dramatically decrease the capacity of nitrogenase activity when either copy of mhk-RGS1 gene was interrupted. This indicated that the expression regulation of mhk-RGS1 is closely related to symbiotic nitrogen fixation.Finally, based on the bioinformatic analysis on the coding regions of D22 and D29 for the promoter, terminator and Rfam, it was found that the mhk-RGS1 gene has some characteristics of sRNA in terms of being a small fragment and located in the transcribed spacer regions. However, the mhk-RGS1 gene had no similarity to any reported sRNA. In short, the control mode of the mhk-RGS1 needs in-depth study.