Improving Symbiotic Nodulation And Nitrogen Fixation Of Mesorhizobium Huakuii Based On Small RNA Function Investigation And Strain Rejuvenation

As a green fertilizer,the leguminous plant Astragalus sinicus is often rotated or mixed with rice to provide nitrogen for rice and reduce the application of chemical nitrogen fertilizer in agricultural production,saving costs and bringing economic benefits to agricultural production.The main research content of this study is: 1.Comparison of symbiotic nodulation and nitrogen fixation efficiency of rhizobia from different regions of Astragalus sinicus using field experiments,2.Conduct functional research on Mesorhizobium huakuii based on non-coding sRNAs.Through the above research content,we can obtain rhizobia that can improve the yield of Astragalus sinicus.The main results of this study are as follows:(1)The study examined M.huakuii 7653 R Symbiotic phenotypic characteristics of five rhizobia isolated from Astragalus sinicus,including rejuvenated strains AW-8and AW-10 of M.huakuii 7653 R and laboratory preserved strains AW7R-1,AW7R-2,and AH-20.The symbiotic phenotype of above strains show that: compared with AW7R-1,when AW-8 was inoculated with Astragalus Xinzi No.1,the aerial part weight,nodule weight,and nitrogenase activity increased by 85.55%,51.91%,and54.25%,respectively.Further sand and soil culture potted screening results showed that compared with the excellent strain AB-21 in Beijing,the excellent strain AZ-21 in Zhengzhou,and the laboratory preserved strain AW7R-1,the AW-8 strain has better the aerial part weight,nodule weight,and nitrogenase activity.The results of the field plot experiment showed that compared with the control group without urea application,the dry nitrogen content and nitrogen weight of the the aerial part plants of Astragalus inoculated with AW-8 strain increased by 9.38% and 4.15%,respectively,while the available phosphorus,available potassium,and organic matter content in the rhizosphere soil increased by 2.62%,35.84%,and 0.53%,respectively.This indicates that this condition has a promoting effect on increasing the the aerial part biomass of Astragalus and improving the nutrient elements of the plant rhizosphere soil;In summary,the AW-8 strain can be used as an efficient symbiotic nitrogen fixing strain to increase the yield of Astragalus.(2)Based on our previous work,it has been confirmed that M huakuii 7653 R interval sequence MH＿s3 and MH＿s7 is non-coding sRNA.This study is based on this,constructed non-coding sRNA MH＿s3 and MH＿s7 overexpressed strains 7653R(p SRKT-s3),7653R(p SRKT-s7),and mutant strain 7653RΔs3,7653RΔs7.The identification results of symbiotic phenotype in s7 show that: 1)Compared with the wild-type,the host plant inoculated with overexpressing strain 7653R(p SRKT-s3)showed an increase in the aerial part fresh weight,nodule number,nodule weight,and nitrogenase activity indicators,but there was no significant difference in the mutant strain,indicating MH＿s3 as a regulatory factor,overexpression of MH＿s3 promotes both nodulation and nitrogen fixation of 7653 R.2)Overexpression of strain 7653R(p SRKT-s7)did not significantly affect nodulation and nitrogen fixation,while its mutant showed a decrease in nodulation number and nitrogenase activity,indicating MH＿s7 as a regulatory factor,the absence of MH＿s7 has a negative impact on nodulation and nitrogen fixation.The M huakuii 7653 R deletion mutant strains of sRNAs molecular chaperones protein Hfq and sRNAs overexpression strain(7653RΔhfq(p SRKT-s3),7653RΔhfq(p SRKT-s7))based on the mutant were constructed.The symbiotic phenotype of above strains show that: 1)Compared with the wild-type,the the aerial part fresh weight,nodule number,nodule weight,and nitrogenase activity of the hfq deficient strain were significantly reduced,indicating that the hfq deficiency led to a decrease in the symbiotic nitrogen fixation ability of the strain;2)Compared with the hfq mutant strain,the overexpressed strain Δhfq(p SRKT-s3)and Δhfq(p SRKT-s7)significantly increased the aerial part fresh weight,nodule number,nodule weight,and nitrogenase activity.Thus,this study thinks that MH＿s3 and MH＿s7,as sRNAs,is not regulated by Hfq.In summary,this study thinks that 7653R(p SRKT-s3)is an engineering strain with high symbiotic nitrogen fixation efficiency.