摘 要 ：盐害是影响紫花苜蓿生产力的主要非生物因素之一,鉴定控制这一复杂性状的基因将为苜蓿育种计划提供关键信息。为揭示紫花苜蓿在盐胁迫下基因表达谱的变化,以紫花苜蓿Millennium为材料,对正常培养(WT_CK1)和盐胁迫(WT_N1)条件下的2个样品根系进行转录组分析,同时利用实时荧光定量PCR(qRT-PCR)技术对部分关键基因的表达特点进行验证。结果表明,紫花苜蓿根系在250 mM NaCl胁迫72 h 时,共检测到31 907个基因表达量发生了改变,2 758个基因的表达量差异达到2倍以上,包括199个转录因子,其中1 338 个表达量上调,1 420个表达量下调,这些差异表达基因功能主要涉及次生代谢、代谢途径、激素代谢及信号转导和植物病原菌互作等。qRT-PCR分析表明,6个随机选择的基因在胁迫前后的表达特点与表达谱测序结果一致。综上,紫花苜蓿根系对盐胁迫响应是一个多基因参与、多个生物代谢过程反应协同调控的过程,基因表达量的变化可能是调控的主要方式。此外,本研究候选了一系列胆汁酸:Na+共转运蛋白、晚期胚胎发生富集蛋白、谷胱甘肽-s-转移酶基因和转录因子等与紫花苜蓿盐胁迫相关的应答关键基因,为揭示紫花苜蓿耐盐分子机制奠定了基础。
Abstract:Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. Illumina RNA-sequencing was performed in two alfalfa samples of control (WT_CK1) and NaCl-treated samples(WT_N1)in order to estimate a broad spectrum of genes affected by salt stress,and the real-time fluorescent qRT-PCR technique was used to verify the expression characteristic of random selected 6 key genes. These results indicated that 31 907 genes expressed differentially in alfalfa under 250Mm NaCl stress and control at 72 h, and 2 758 of them express higher or lower more one fold under NaCl stress than control including 199 transcription factors (TFs), expression of 1 338 among the 2 758 genes were up-regulated and 1 420 were down-regulated. The functions of a large proportion of differentially expressed genes are involved in biosynthesis of secondary metabolites, metabolic process, plant hormone signal transduction, plant-pathogen interaction and so on. And expression patterns of random selected 6 genes confirmed the results of the digital gene expression profile. The study indicated that the response of alfalfa under NaCl stress involved many genes and regulated by many collaborative biological metabolism, and changes of gene expression level maybe play important roles in regulation process. And the experiment found many candidate genes which were crucial for alfalfa such as bile acid: sodium symporter,late embryogenesis abundant,glutathione S-transferase, transcription factors and so on to endure salt stress, and will help to reveal and understand the molecular mechanisms of salinity tolerance in alfalfa.