Identification And Functional Analysis Of Salt/Alkali Stress Response MiRNA In Medicago Sativa And Medicago Truncatula

Salt/alkali stress is a prevalent abiotic stress which is one of the main environmental factors leading to crop yield reduction.In saline soil,the growth and physiological function of plants are serverely restricted.While the the decreasing arable land and increasing food demand have challenged our breeding for crops.Alfalfa is an important leguminous forage,which enjoys the reputation of ‘king of forage' for its high yield,rich nutrition,high protein content and good palatability.It is believed that planting of salt-tolerant plants is one of the most effective and economical ways to restrict saline-alkali lands.As a kind of high-quality leguminous forage,alfalfa also has the ability to improve the soil media.Therefore,it is of great environmental and economic values to study the salt/alkali tolerance mechanism in alfalfa.Up to now,many genes involves in the regulation of salt stress have been found in plants which play indispensable roles in enhancing plant salt tolerance.In recent years,non-coding RNAs,especially miRNAs,have been emerged as an important role in many stress tolerance processes in plants with lots of miRNAs discoved to affect salt/alkali tolerance in plants.Compared to alfalfa,Medicago tuncatula functions more as an important leguminous model plant,which provides theoretical basis and reference for the research of alfalfa and other leguminous plants.To figure out the mechanism underlines salt/alkali stress response processes of alfalfa,Medicago sativa cv.Zhongmu-1 and Medicago truncatula R108 genotype were selected for the further research.Two kinds of Medicago seeds were immersed in mixed salt/alkali solution,and the differences expression of microRNAs in two alfalfa germinated were analyzed with sRNA High throughput analysis.From both Medicago spp.,we identified 685 known miRNAs and 221 noval miRNAs belonging to 156 miRNA families.81 differentially expressed miRNAs were identified after salt treatment and 129 miRNAs after alkali stress in Medicago truncatula while 24 and 72 miRNAs were detected to response to salt or alkali stress respectedly in Medicago sativa.Further analysis revealed that the target genes differentially expressed miRNAs were widely distributed,and many of them might be involved in the regulation of salt stress,which provided new evidence and research directions for the mechanism of post-transcriptional salt tolerance in plants,especially alfalfa.To further explore the mechanism of salt tolerance in Medicago sativa,we selected three kinds of miRNAs,namely mtr-mi319,mtr-mi395 and mtr-mi1510 b,which may be involved in the regulation of salt/alkali stress in Medicago truncatula.Salt tolerance study was carried out by overexpressing three kinds of miRNAs in and Medicago truncatula.Compared with wild type Arabidopsis thaliana,three kinds of miRNA transgenic seedlings did not show higher germination rate,but the time of bolting and flowering of Mtr-mi319 transgenic Arabidopsis was delayed under salt treatment,and its resistance to salt stress may be related to the expression level of mtr-mi319.Mtr-mi395 transgenic Arabidopsis thaliana also showed enhanced salt tolerance,and its leaf water-holding capacity was stro under salt treatment.In addition,the target genes of mtr-mi319 and mtr-mi395 in Medicago truncatula were verified by 5'RACE and transient gene expression in tobacco leaves.