Study On The Symbiotic Nitrogen Fixation Function Of Sinorhizobium Meliloti SGNH Hydrolase Family Genes

The SGNH hydrolase family is a class of hydrolase with strictly conserved catalytic residues Ser,Gly,Asn and His in four conserved sequence regions,which play an important role in various of organisms.To further investigate the function of SGNH hydrolase in symbiotic nitrogen fixation,this study based on the previous work of our research group,the SGNH family hydrolase sm Est23 of Sinorhizobium meliloti 1021 and its homologous gene MCHK＿RS21235 of Mesorhizobium huakuii 7653 R in the process of symbiotic nitrogen fixation.The symbiotic nitrogen fixation function of sm Est25 and sm Est24 was also studied in S.meliloti 1021.Purpose of this study was to reveal the role of SGNH hydrolase family in nitrogen fixation of symbiotic rhizobia and leguminous plants.The results are as follows:The mutant of S.meliloti 1021 sm Est23 deletion were constructed and inoculated with two types of Medicago truncatula A17 and R108.The symbiotic phenotype in R108 showed significant decreases in aboveground fresh weight,nodule weight,and nitrogenase activity of plants.The functional complement strain could restore the symbiotic phenotype of plants,and there was no difference between the overexpression strain and the wild type strain.The results showed that sm Est23 plays a role in symbiotic nitrogen fixation,and the function of SGNH family hydrolase genes is related to symbiotic nitrogen fixation.The results of early infection showed that the deletion of sm Est23 did not affect the formation of early symbiotic infection threads and nodule primordia.The results of sm Est23 expression detected by RT-q PCR showed that the expression of sm Est23 was highest at about30 days of symbiosis,and it was speculated that sm Est23 may have a more active function in the middle stage of symbiosis.The symbiotic nitrogen fixation phenotype of the SGNH family hydrolase homologous gene MCHK＿RS21235 in M.huakuii 7653 R was similar to sm Est23,and the sm Est23 could heterogeneously compensate for the phenotypic defect caused by the deletion of MCHK＿RS21235.The results of in vitro enzyme activity assay after expression and purification of Sm Est23 and MCHK＿RS21235 proteins showed that they both had esterase activity and could hydrolyze specific substrates.The nodulation ability of 7653 R ΔMCHK＿RS21235 can be supplemented by Sm Est23 protein.sm Est23 can significantly improve the symbiotic phenotype of symbiotic plants after overexpression in M.huakuii7653 R,which brings new ideas for the development and utilization of rhizobium agents.In consideration of gene redundancy,the function of sm Est23 gene may be replaced by other genes in the same family.In this study,Crisper/cas9 technology was used to construct another two SGNH hydrolase gene mutants in the same family,namely S.meliloti 1021Δsm Est24 and 1021Δsm Est25.Furthermore,on the basis of 1021Δsm Est23,a double-process mutant named 1021Δsm Est23Δsm Est24 and 1021Δsm Est23Δsm Est25 was constructed too.After inoculation of two types of Medicago truncatula,the superposition of nitrogen fixation defects was observed in the double mutant strains,indicating that these SGNH family hydrolase genes in S.meliloti 1021 may play a synergistic role in symbiotic nitrogen fixation.