Identification and Analysis of Differentially-expressed Genes Under Salt Stress in Leaves of Southern Type Alfalfa (Medicago sativa 'Millennium') Salt Tolerant Mutant
Abstract：Alfalfa (Medicago sativa) is widely grown and is one of the most important forage crops in the world, but its growth and biomass production are markedly reduced under salt stress. The objective of this study is to identify the inner molecular mechanisms of southern type alfalfa in response to salt stress, and mine these genes closely related to salt responsiveness. Illumina HiSeqTM 2000, a high-through transcriptome sequencing technology, was used to obtain the anscriptome differential expression data of southern type alfalfa (Medicago sativa 'Millenium') leaves under 72 h treatment at 250 mmol/L NaCl. Function and pathway of those different expression genes were also investigated using Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway biological analysis in order to obtain some of the potential target genes to salt stress. Eight randomly selected DEGs (differentially expressed genes) were used to validate the reliability of sequencing results. The results showed that after filtration of reads, a total of 60 395 324 control (CK) and 60 303 692 salt stress (ST) reads were acquired, among these reads, 54.18% and 53.77% could be precisely compared to the reference sequence of M. truncatula. After 250 mmol/L NaCl stressed for 72 h, in total, 30 900 DEGs were authenticated among which 4 187 and 3 507 were regarded as raise-and lower-regulated genes. GO analysis showed that these DEGs were mainly referred to binding, catalytic activity, cell part and cell. KEGG pathway analysis showed that these DEGs were mainly referred to biosynthesis of secondary metabolites, metabolic pathways and phenylpropanoid biosynthesis. Besides, we discovered many candidate genes, like glutathione S-transferase, superoxide dismutase [Cu-Zn] protein, L-ascorbate peroxidase, receptor-like kinase, stress-induced receptor-like kinase, sucrose nonfermenting 1(SNF1)-related kinase, Calmodulin-like protein, choline monooxygenase, delta-1-pyrroline-5-carboxylate synthetase 3, 2Ccatalytic/protein phosphatase type 2C and Trehalose-phosphate phosphatase, and many transcription factors were identified, to be related to salt tolerance, like ANTAP2-like ethylene-responsive, Transcription factor bHLH36, Transcription factor NAI1, bZIP transcription factor, Zinc finger C-x8-C-x5-C-x3-H type family protein, Nucleic acid binding transcription factor activity, Myb transcription factor, NAC transcription factor-like protein, Sequence-specific DNA-binding and WRKY family transcription factor, This study affords the initial value for the molecular mechanisms of salt tolerance in alfalfa.
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