| In our previous work, a novel gene RGS1of Mesorhizobium huakuii7653R which is involved in symbiotic nitrogen fixation has been identified. The phenotype of RGS1replacement mutant in pot plant experiment shows that it forms ineffective nodules with significantly reduced nitrogen fixation activity. But its functional mechanism has not yet been characterized which needs to be studied in depth. In this continuous research work, some progresses and results have been obtained and can be summarized as follows.Based on the completed genome sequences and bioinformatics analysis, it was found that there were five copies of RGS1present in the plasmid of7653R, each of them was localized in the intergenic regions. The secondary structure analysis showed that RGS1could form one type of folded mode, displaying more than three stem-loop structures. These characteristics of RGS1were consistent with those of reported sRNA (small RNA). Meanwhile, according to two putative completed open read frames (ORFs) present in RGS1, the target protein was in vitro expressed and purified, the corresponding antibody was prepared. But the results of Western Blotting showed that there were no anticipated-sized protein to be expressed in both free-living cells and bacteroids of7653R. It indicated that the role of RGS1was not played through encoding such an unknown small protein as to be a transcription factor or functional gene in7653R.To confirm whether the RGS1was expressed as RNA and its transcription direction in the total RNA of7653R, the two sets of primers in accordance with two directions were designed and used for RT-PCR. The results showed that RGS1was expressed in both two directions. Further more, we chose the conservative sequences located in the stem loop of secondary structure and designed the specific probes. Northern Blotting found that RGS1produced a250bp fragment in the anaerobic and symbiotic conditions when the negative strand was used as a template. But100bp and200bp signal bands were detected in free-living cultures and stress conditions when the positive strand was used to as a template. The results promoted us to conclude that RGS1was a novel sRNA, which could be expressed in two strands of RGS1and being induced by some specific environmental conditions. Using5’and3’ RACE, two fragments in size of106bp and49bp in term of the full-length of RGSl transcripts were obtained. Using Real-time PCR method, the expression levels of RGS1in different growth stages were monitored. When the negative strand was used as a template, it was showed the RGS1was hardly expressed under pure free-living growth condition, but the expression level of RGSl was gradually increased during the nodulation time-course after inoculation, and it achieved the peak level at28dpi. It indicated that RGS1in the negative strand was a specific-expressed gene induced by symbiotic condition.Using Real-time PCR, the changes of structure gene expression levels between wild-type and mutant strains were determined. The result revealed that the deletion of RGSl led to down-regulation expression of downstream structure genes under free-living growth, anaerobic and symbiotic conditions, which suggested that RGS1might regulate the gene expression in the downstream. To verify the function of RGSl related to the transcription of structure genes, Northern blotting was conducted to identify the precursor of RGS1by examining the transcription changes of sRNA. The results showed that the sizes of RGS1transcription units were different in the free-living growth, anaerobic and symbiosis conditions. The transcription units became larger under symbiosis conditions. It needs further investigation on whether RGS1directly regulates the transcription of downstream structure genes by choosing a specific structural gene to probe the transcription of structure units. |
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