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紫花苜蓿(Medicago sativa)是世界上被广泛种植的一种优质牧草。盐胁迫对紫花苜蓿的生长和产量具有明显抑制作用。为了理解南方型紫花苜蓿(M.sativa’Millennium’)受盐胁迫的内在分子机制,挖掘其与耐盐密切相关的功能基因。以250 mmol/L Na Cl处理72 h的南方型紫花苜蓿耐盐突变体叶片进行Illumina Hi SeqTM2000高通量转录组测序,并对所获得的差异表达基因进行基因本体(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)pathway生物信息学分析,获得可能耐盐潜在靶标基因。同时,挑选8个差异表达基因验证测序结果的可靠性。结果表明,过滤后对照(control,CK)和盐处理(salt stress,ST)样本分别保留了60 395 324和60 303 692对reads,其中54.18%和53.77%的reads能精确比对到参考序列蒺藜苜蓿(M.truncatula)上。差异表达基因(differentially expressed genes,DEGs)结果显示,在样品中共检测到30 900个基因表达发生改变,其中4 187上调表达,3 507下调表达。GO功能分析显示,差异表达基因主要表现在结合、催化活性、细胞组分和细胞等。KEGG Pathway分析显示,差异表达基因广泛涉及次生代谢、代谢途径及苯丙素的生物合成。另外,筛选了与紫花苜蓿盐胁迫应答相关的基因谷胱甘肽硫转移酶、超氧化物歧化酶Cu/Zn蛋白、L-抗坏血酸过氧化物酶、类受体蛋白激酶、诱导类受体蛋白激酶、蔗糖非发酵型蛋白激酶、类钙调素蛋白、胆碱单加氧酶、1-吡咯啉-5-羧酸合成酶、蛋白磷酸酶2C、海藻糖磷酸酯酶等和AP2类乙烯响应的转录因子、b HLH36转录因子、NAI1转录因子、b ZIP转录因子、C3H锌指蛋白、核酸结合转录因子活性、Myb转录因子、NAC转录因子蛋白、序列特异性DNA结合转录因子蛋白和WRKY转录因子等。本研究为揭示紫花苜蓿耐盐分子机制提供了基础资料。
Medicago sativa is a premium forage widely grown in the world. Salt stress had a significant inhibitory effect on the growth and yield of alfalfa. In order to understand the underlying molecular mechanism of salt stress in southern safflower (Medicago sativa L.), its functional genes closely related to salt tolerance were excavated. Southern hybrid alfalfa salt-tolerant mutant leaves treated with 250 mmol / L NaCl for 72 h were sequenced by Illumina Hi SeqTM 2000 high-throughput transcriptome analysis. Gene Ontology (GO) and Kyoto Bioinformatics analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway resulted in potential salt-tolerant target genes. At the same time, the reliability of sequencing results of 8 differentially expressed genes was selected. The results showed that 60 395 324 and 60 303 692 pairs of reads were preserved in control (CK) and salt stress (ST) samples, respectively, of which 54.18% and 53.77% of the reads were accurately aligned to the reference sequence Tribulus terrestris Alfalfa (M. truncatula). The results of differentially expressed genes (DEGs) showed that a total of 30 900 genes were detected in the samples, of which 4 187 was up-regulated and 3 507 was down-regulated. GO functional analysis showed that differentially expressed genes are mainly expressed in the binding, catalytic activity, cell components and cells. KEGG Pathway analysis showed that differentially expressed genes are widely involved in secondary metabolism, metabolic pathways, and phenylpropanoid biosynthesis. In addition, we also screened the genes glutathione S-transferase, Superoxide dismutase Cu / Zn protein, L-ascorbate peroxidase, receptor-like protein kinase, Kinases, kallikrein, choline monooxygenase, 1-pyrroline-5-carboxylic acid synthase, protein phosphatase 2C, trehalose phosphatase and the like and AP2-type ethylene B HLH36 transcription factor, NAI1 transcription factor, b ZIP transcription factor, C3H zinc finger protein, nucleic acid binding transcription factor activity, Myb transcription factor, NAC transcription factor protein, sequence specific DNA binding transcription factor protein and WRKY transcription factor Wait. This study provides the basic information for revealing the salt-tolerant molecular mechanism of alfalfa.