Abstract: Ethylene, as one of the important plant hormones, plays an important role in plant stress response. The ACS gene is a key rate limiting enzyme (ACC synthase) gene in the ethylene synthesis process. In this study, we laid a theoretical foundation for understanding the specific regulatory mechanism of MsWRKY33 participating in salt-tolerant response of alfalfa by exploring the regulatory relationship of MsWRKY33 transcription factor on ACS gene. We used homologous cloning, vector construction, Y1H, Y2H, qRT-PCR and other techniques to verify the transcriptional regulation of MsWRKY33 on AtACS2 and MsACS2, meanwhile, analyzed the expression pattern of MsACS2 in transgenic lines under salt stress. The results showed that the MsWRKY33 transcription factor could specifically bind to the W-box element and had transcriptional regulatory effects on AtACS2 and MsACS2;The expression level of MsWRKY33 gene in the transgenic lines was significantly higher than that in the control. The expression of MsACS2 in transgenic lines showed an increasing trend after the large amount of MsWRKY33 gene expression, further indicating that the expression of MsACS2 gene in alfalfa might be positively regulated by the MsWRKY33 transcription factor. The results indicated that the expression of MsWRKY33 could be induced when alfalfa was subjected to salt stress. The MsWRKY33 transcription factor might affect ethylene synthesis and salt stress response of alfalfa by positively regulating the expression of MsACS2.

Key words: Alfalfa, Yeast One-Hybridization technique, MsWRKY33, MsACS2, Salt tolerance

摘要： 乙烯作为重要的植物激素之一，在植物逆境胁迫应答中发挥重要作用。ACS基因是乙烯合成过程中的关键限速酶（ACC合成酶）基因，本研究通过探究紫花苜蓿MsWRKY33转录因子对ACS基因的调控关系，为MsWRKY33参与紫花苜蓿耐盐胁迫响应的具体调控机制奠定理论基础。利用同源克隆、载体构建、酵母单杂交、酵母双杂交、qRT-PCR等技术，验证MsWRKY33对AtACS2和MsACS2的转录调控，并对盐胁迫下转基因株系中MsACS2表达模式进行分析。结果显示：MsWRKY33转录因子可以与W-box元件特异性结合，且对AtACS2和MsACS2具有转录调控作用；MsWRKY33转基因株系中MsWRKY33基因的表达量显著高于对照，转基因株系中MsACS2的表达是在MsWRKY33基因大量表达后呈上升趋势，进一步说明紫花苜蓿MsACS2基因的表达可能受MsWRKY33转录因子的正向调控。因此，紫花苜蓿受到盐胁迫时可诱导MsWRKY33的表达，MsWRKY33转录因子通过正向调控MsACS2表达，可能影响乙烯合成，从而影响紫花苜蓿的盐胁迫响应。

关键词: 紫花苜蓿, 酵母单杂交, MsWRKY33, MsACS2, 耐盐性