Identification And Analysis Of Differentially Genes Under Salt Stress In Leaves Of Southern Type Alfalfa Salt-tolerant Mutant

Our country has vast sea area,coastal saline land is only a small part is cultivated and the majority are wasteland and tideland,the coastal zone has a large number of saline land to be exploited,which is an important reserve land resources.Biological improvement and ecological greening of coastal saline land is an important and difficult problem in the process of coastal urbanization in China.According to the domestic and foreign experiences in recent years,selecting salt-tolerant plants is abrightest and cost-effective way to achieve biological improvement and ecological greening of coastal saline land.Alfalfa(Medicago sativa)is a leguminous plant with stronger salt tolerance,long cultivationhistories,widely cultivation in the world anda certain ornamental value,which is adesired plant for saline improving and landscaping.In this research,select salt-tolerant mutant of southern type alfalfa 'Millennium' as material for RNA-Seq analysis to filter differentially expressed genes(DEGs)and analysis whose GO function?KEGG Pathway and transcription factors,in order to explore the main biological functions and metabolic pathways involved in salt-tolerant genes.Which lay the foundation for further studing the salt stress molecular mechanism in alfalfa and cultivating new salt-tolerant varieties.The results of this research are as follows:1?Determinate physiological indicators at 0?24?48?72 and 96 h of southern type alfalfa salt-tolerant mutant seedling under 250mmol/L Na Cl,we finded that the leaf relative water content had been reduced,the Pro content?the relative electical conductivity?MDA content and SOD activity had been increased when salt stress less than 72h;the chorophyll content and SOD activity had been declined significantly,and the Pro content?the relative electical conductivity and MDA content had been rised increasely.The results showed that 250mmol/L Na Cl stress 72 h was the best time for salt tolerance response of alfalfa salt-tolerant mutant seedling,which provided the basis for the following study of salt tolerance molecular mechanism.2?By RNA-Seq,stess 72 h under 250mmol/L Na Cl,Southern type alfalfa salt-tolerant mutant seedling leaf samples detected total 30 900 genes' expression had changed,Of these,7 694 genes were defined as DEGs(4 187 genes were up-regulated,3 507 genes were down-regulated),including 422 transcription factors belong to 50 transcription families(268 transcription factors wereup-regulated,154 transcription factors were down-regulated).3?The results of q RT-PCR showed that the expression characteristics at before and after salt stress of selected randomly 8DEGs were consistent with the results of expression profilessequencing,which indicated transcriptome sequencing data is demonstrated4?GO function analysis showed that biological functions of DEGsmostly perfored in catalytic activity?binding?cell part?cell?metabolic process and cellular process.KEGG Pathway analysis showed that DEGs widely involved metabolic pathway and secondary metabolites biosynthesis.Identified candidate genes which were related to stress response,such as Ms GST?Ms L-APX?Ms GPX?Ms RLK?Ms SIK?Ms Sn RK?Ms CMLs?Ms CMO?Ms MIP?Ms P5CS1?Ms P5CS3?Ms ABCB?Ms WBC?Ms LEA3?Ms PP2 Candso on.Transcription factors analysis showed that genes with salt stress-responsive from the MYB family were observed most,followed by WRKY?NAC?b HLH,AP2-EREBP?GRAS familiesandso on.Identified candidate transcription factors which were related to stress response,such as Ms Alfin-like?Ms ERF110?Ms ANT?Msb HLH36?Ms NAI1?Msb ZIP?Ms C3H?Ms GRAS?Ms NAD?Ms MYB?Ms MYB85?Ms NAC?Ms RWP-RK?Ms Trihelix?Ms WRKYand so on.