转录因子可以调节众多下游基因的表达，在植物抗逆境中起重要的调节作用。为了解析转录因子在南方型紫花苜蓿适应盐胁迫环境的分子机制，以南方型紫花苜蓿 Medicago sativa ‘Millennium’为材料，以正常培养（WT_ck1）和氯化钠（盐）胁迫（WT_N1）条件下的2个样品根系进行转录组分析，鉴定紫花苜蓿根系盐胁迫应答转录因子基因。同时，随机挑选4个转录因子差异表达基因进行实时荧光定量qRT-PCR（3次重复），验证转录组测序技术（RNA-Seq）结果的可靠性。结果表明：紫花苜蓿根系在250 mmol·L－1氯化钠胁迫下72 h，共检测到31907个基因表达量发生了改变，表达量差异达到2倍以上的基因共2758个。其中，隶属于38个转录因子家族199个转录因子在盐胁迫下差异表达，上调表达104个，下调表达95个。在各转录因子家族中，盐胁迫应答基因数量最多的是MYB基因家族，其后分别是AP2-EREBP， bHLH， WRKY， NAC和GRAS基因家族，这暗示了紫花苜蓿根系对盐胁迫响应可能是多种转录因子家族共同参与的应答过程。 qRT-PCR分析表明：4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。此外， MsERF-2b， MsbHLH， MsbZIP， MsGRAS， MsNAC， MsMGT-3a 和MsWRKY等转录因子被选为与盐胁迫应答相关的候选转录因子。该研究结果为阐明植物对盐胁迫的应答机制提供了新的线索。图3表3参36%Transcription factors (TFs), which can regulate downstream gene expression, play an important role in plant stress responses. In order to investigate the molecular mechanism of salt tolerance, the TFs of southern type alfalfa, in this study with 250 mmol·L-1 NaCl stress, Illumina RNA-sequencing was performed to evaluate the expression spectrum of transcription factors in roots of the southern alfalfa cultivar “Millennium”. Then to verify the expression of four randomly selected genes, Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR)(three repeated) was used. Results showed 31 907 differentially expressed genes, 2 758 of which showed a difference of over two fold. Among these genes, 199 transcription factors belonging to 38 TF families were up-regulated and 95 were down-regulated. Genes from the MYB family were observed most, fol-lowed by AP2-EREBP, bHLH, WRKY, NAC, and GRAS. The qRT-PCR assay of four randomly selected genes confirmed the results of RNA-Seq analysis. In addition, candidate genes such as MsERF-2b, MsbHLH, Ms-bZIP, MsC2H2, MsGRAS, MsNAC, MsMGT-3a, and MsWRKY that may be involved in salt stress responses were identified. This study indicated that multiple TF families were involved in salt stress responses in the root of southern alfalfa types, and it provided new information for further study of the mechanism of a plant ’s re-sponse to salt stress.
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