Identification And Functional Analysis Of Salt And Alkali-tolerance Genes In Medicago Truncatula

Salinity and alkalinity have negative impacts on crop yield and quality and severely restricts agricultural production.In recent years,there have been lots of researches on salt stress which were on neutral salt.However,knowledge on alkaline stress is incomplete.Alfalfa(Medicago sativa)is a legume plant that has high protein content and good palatability.As high-quality forage,alfalfa is often referred to "the queen of forage crops".Alfalfa is resistant to salt-alkaline stress with moderate concentration.But salinization and alkalization are increasing,which has much severer effects on yield and quality of alfalfa.Alfalfa is obligate outcrossing and tetraploid species,which make genetic and genomic studies of stresses difficult.Over the past decade,Medicago truncatula has been developed into a new model legume.M.truncatula is diploid and self-fertile.It has a short life cycle and a small genome.Therefore,we can reveal the salt-tolerant mechanism of alfalfa by M.truncatula,which promotes the progress of mechanism research on alfalfa salt-alkaline tolerance.M.truncatula(ecotype R108)was used in the study.Thousands of salt and alkaline-responsive genes and miRNAs in leaves of M.truncatula were identified by mRNA and small RNA-seq.A total of 10 miRNA families were significantly responsed by identified differentially expressed miRNAs,including MtmiR156,MtmiR160,MtmiR164,MtmiR166,MtmiR169,MtmiR2111,MtmiR2592,MtmiR398,MtmiR399 and MtmiR5229.MtmiR160 and MtmiR166 expressed similarly under salt-alkaline stress.They may play a role in response to salt and alkaline stress.DEGs(differentially expressed genes)were enriched in circadian rhythm,starch and glucose metabolism,amino acid metabolism,carbon metabolism and MAPK signaling under alkaline stress;Salt-responsive DEGs were involved in MAPK signaling,plant hormone signaling,ribosomes,starch and sugar metabolism.MtmiR166 was selected from the small RNA-seq data of M truncatula wild type after saline-alkaline treatment.Compared to the control,MtmiR166 expressed differently after 1-day and 7-days salt-alkaline treatment.HD-Zip?(Homeodomain Leu Zipper ?)family genes targeted by MtmiR166 were induced under salt and alkaline stress.Transgenic plants of M.truncatula over-expressing MtmiR166 and MtREV(MtREVOLUTA)were used to investigate their roles in salt-alkaline stress.We compared the phenotype of wild type,35S:MtmiR166 and 35S:MtREV plants under salt and alkaline conditions.35S:MtmiR166 showed sensitive phenotype and more affected by salt-alkaline stress.35S:MtmiR166 displayed a higher level of MDA,lower total SOD enzyme activity and reduced expression level of MtSODl and MtSOD2 than those of wild-type plants under salt and alkaline treatment.In contrast,35S:MtREV plants displayed a salt and alkaline stress resistance phenotype with lower level of MDA,higher total SOD enzyme activity and higher relative expression of MtSODl and MtSOD2.By STTM technology,we identified STTM166 transgenic M.truncatula plants,in which the expression of MtmiR166 was dramatically reduced.These plants will be used in further stress resistance tests.