南方型紫花苜蓿耐盐突变体叶片盐胁迫应答差异基因鉴定与分析  

Identification and Analysis of Differentially-expressed Genes Under Salt Stress in Leaves of Southern Type Alfalfa (Medicago sativa 'Millennium') Salt Tolerant Mutant

作　　者：张婧蕾 李佳赟 王依纯 裴翠明 马进[1] 

机构地区：[1]浙江农林大学风景园林与建筑学院,临安311300

出　　处：《农业生物技术学报》2017年第10期1588-1599,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31272494);浙江省自然科学基金(No.LY16C170003)

摘　　要：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。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 transcriptomesequencing 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,

关 键 词：南方型紫花苜蓿 叶片 盐胁迫 转录组 

分 类 号：S722.3[农业科学—林木遗传育种]