中文摘要：

WRKY转录因子是植物特有的转录因子，广泛参与植物对多种逆境胁迫的反应。但是对紫花苜蓿中WRKY转录因子的研究还较少。本研究从紫花苜蓿中克隆了一个WRKYI类转录因子MsWRKY33。该基因CDS全长1536bp，编码512个氨基酸，结构分析显示MsWRKY33包括两个WRKY结构域和一个C2H2锌指结构（C-X4-C-X23-H-X-H），表明其属于WRKYI族WRKY转录因子。亚细胞定位预测MsWRKY33蛋白定位在细胞核。MsWRKY33基因受盐、干旱和冷胁迫诱导，暗示基因可能参与了这些逆境胁迫的调控。构建原核表达载体pET-MsWRKY33，SDS-PAGE分析表明在大肠杆菌中表达了MsWRKY33蛋白。扩增MsWRKY33编码区cDNA，以pBll21为基础载体，构建植物超表达载体pBll21-MsWRKY33。采用农杆菌介导的愈伤组织培养法转化紫花苜蓿。利用nptⅡ基因引物和载体特异引物检测抗性苗呈阳性，表明目的基因已成功导入紫花苜蓿基因组中。qRT-PCR检测发现，MsWRKY33基因在转基因株系中得到增强表达。本研究为进一步探索WRKY转录因子基因在紫花苜蓿抗逆性调控中的作用奠定了基础。

英文摘要：

Plant-specific WRKY transcription factors (TFs)are widely involved in various stress responses. However,their roles in abiotic stresses are still not well known in alfalfa (Medicago sativa ).In this study,a WRKY gene,containing 1536 bp CDS length encoding a putative protein of amino acid 512,designated as MsWRKY33,was isolated from alfalfa.The alignment results revealed that the MsWRKY33 protein contains two conserved DNA-binding domains (WRKY domain)of 60 amino acids and a C2H2 zinc finger region (C-X4-C-X23-H-X-H),falling into group I of the WRKY protein.Protein localization prediction analysis indicated that MsWRKY33 is a nuclear-targeting protein.The expression of MsWRKY33 gene was up-regulated by salin-ity (NaCl),drought (PEG)and cold temperature (4℃),indicating that MsWRKY33 gene may be involved in the regulation of environmental stress responses in alfalfa.The fragment encoding 10-239AA was inserted into pET-30α(+)to construct the expression vectors,and SDS-PAGE analysis revealed that the MsWRKY33 protein could be expressed in prokaryotic cells.The full length cDNA of MsWRKY33 was amplified from alfal-fa RNA and the plant expression vector pBI121-MsWRKY33 was constructed based on the pBI121 vector. Transgenic plants were obtained through somatic embryogenesis by Agrobacterium tumefaciens-mediated transformation.The npt Ⅱ gene and vector sequence were tested by PCR in the genome of transgenic plants and indicated that the target gene had been transferred.The qRT-PCR testing showed that the MsWRKY33 gene was over expressed in transgenic plants.This study will lay a foundation for further study of the function of the WRKY transcription factor in stress-tolerance regulation in alfalfa.