Study On The Mechanism Of Sinorhizobium Meliloti SD101 Improving Salt Tolerance Of Alfalfa(medicago Sativa.l)

Alfalfa is a forage crop with strong resistance,which can not only grows on saline-alkali land,but also improves soil microbial community structure and nutrients through symbiotic nitrogen fixation by rhizobia.It has been morereported on inoculation of common rhizobia to improve the stress resistance of alfalfa,but inoculation of salt-tolerant rhizobia is less reported.A strain of Sinorhizobium Meliloti SD101 with salt tolerance has been isolated in our laboratory,and the mechanism of improving the salt tolerance of alfalfa by this strain is rarely reported.Based on this cause,the effects of SD101 inoculation on growth and development,nitrogen fixation and other physiological indexes of different alfalfa varieties were systematically studied by field experiment,pot experiment and Lenard experiment.Transcriptome and metabolome analysising were applied to reveal the mechanism of rhizobia SD101 in improving salt tolerance of alfalfa.The results are as follows:1.Plant height,hay yield,number of nodulation,dry weight of root nodulation per plant and other indicators of 20 alfalfa varieties with seed coating were comprehensively analyzed in the paper.It can be seen that WL319,Longmu 806,Junengnaiyan,4010 and Zhaodong had significant differences after inoculation.It had significant effect on nodule formation and nitrogen fixation with rhizobia SD101,but the change of 418Q was not significantly different after inoculation.These 6 varieties mentioned above were used as test materials for subsequent tests.2.Plant height,abovground biomass yield,leaf area,average nodal number per plant,nodule weight,average nitrogenase activity,Chl a,Chl b,Chl a+b,Pn,Gs and Ci of WL319,Longmu 806,Zhaodong,4010 and Junengnaiyan were significantly increased with SD101inoculation in the pot experiment and Lenard system.MDA,O2~-,CAT,GSH,Pro and SS contents were measured at 0d,3d,6d,9d and 12d.It was found that WL319,Longmu 806,Zhaodong,4010 and Junengnaiyan changed the active components.It reduced the external damage to the cell membrane of alfalfa by changing the changes of active components and improved the salt tolerance of alfalfa.But 418Q did not change significantly.3.Six alfalfa varieties were treated with different concentration of salt stress to test the sensitivity to salt.And we found that different alfalfa varieties had different sensitivity to salt.30 mmol/L Na HCO3+90 mmol/L Na Cl was the highest salt tolerance gradient for each alfalfa varieties.This provided a high salt concentration gradient for the next Lenard system test.4.The transcriptome differences of WL319 and 418Q in response to salt stress were compared on day 10 after inoculation with SD101.A total of 4 078 differentially expressed in WL319 were found,of which 2433 were up-regulated and 1645 were down-regulated.There were 1929 differentially expressed genes in 418Q,of which 1181 were up-regulated and 748down-regulated.It involved the differential gene changes of 34 transcriptomic gene families such as WRKY,C2H2,AP2/ERF,NAC,b HLH,b ZIP,GRAS,MYB and so on.The genes associated with salt tolerance,such as c30603.graph?c0,c76945.graph?c0 and c70649.graph?c0 were significantly up-regulated in WL319.The genes associated with nodule formation and nitrogen fixation ability,such as c87117.graph?c0,c68977.graph?c0,c65696.graph?c0,c67223.graph?c0,c67672.graph?c0,c68914.graph?c0 and c67435.graph?c0 were significantly up-regulated.The expression of these genes improved salt tolerance,nodule number and nitrogen fixation ability of WL319.However the defense-related gene C49813.graph?c0 was up-regulated,and the unknown function gene C64473.Graph?c0 was down-regulated in 418Q.5.KEEG analysis showed that 27 and 46 metabolic pathways annotated in WL319,and 6and 17 metabolic pathways were annotated in 418Q under pos and neg mode respectively.It found that WL319 had complex metabolic recombination in nitrogen metabolism,carotenoid biosynthesis,flavonoid biosynthesis,flavonoid and flavonol biosynthesis,and plant hormone signal transduction,which were mainly involved in the nodulation and nitrogen fixation of alfalfa.However,418Q showed the differences in nitrogen metabolism and indole alkaloid synthesis.Combined analysis of transcriptome data and metabolome products of Alfalfa in KEGG metabolic pathway showed that ko00944,ko00910 and ko00943 synthetic pathways in KEGG database were agreed with the genes C87117.graph?c0 and C68977.graph?c0,and also flavonoid,flavonoid and flavonol metabolites.The results showed that flavonoids,flavonoids and flavonols were closely related to the salt tolerance of alfalfa cultivar WL319.The speculated primary mechanism of rhizobia SD101 improving the salt tolerance of alfalfa is as follows:Under salt stress,rhizobia SD101 and alfalfa were highly compatible,invading the root hair,forming a symbiotic nodulation and nitrogen fixation relationship,which regulated the defense mechanism,carbohydrate transport and metabolism,signal transduction mechanism and translation and other processes of alfalfa.And as the key genes such as early nodulin ENOD18,early nodulin-like protein,nodule cysteine-rich(NCR)secreted peptide,nodulin expressed protein and glycoside hydrolase family protein expressed,the plant height of alfalfa increases higher.Leaf area becomes larger.The number of root nodules becomes more.And other phenomenons are appeared.The changes in physiological adaptability appear on the enzyme activity.It enhanced the photosynthesis of alfalfa,accelerated the symbiotic nitrogen fixation system,improved the growth and development ability of alfalfa,and improved the salt tolerance of alfalfa.