SrUGT76G1 is vital for the biosynthesis of rebaudioside A, D and M in Stevia rebaudiana Bertoni; however, its transcriptional regulatory mechanism remains unknown. In this study, the 2050-bp promoter region of SrUGT76G1 was isolated by the TAIL-PCR method, and sequence analysis revealed the presence of several W-box cis-elements, which are the recognition motifs of WRKY transcription factors. Furthermore, SrWRKY71, characterized by a typical WRKY domain and a C2H2 zinc finger-like motif, was identified as a putative transcriptional regulator of SrUGT76G1. The transcript of SrWRKY71 predominantly accumulated in leaves and was present at a lower level in stems, roots and flowers. The SrWRKY71-GFP fusion protein was specifically localized to the nucleus in tobacco epidermal cells. In addition, the N and C terminal regions of SrWRKY71 contributed to its transactivation activity. Y1H and EMSA assays validated that SrWRKY71 binds directly to W-box1 and W-box2 in the proximal promoter region of SrUGT76G1. Moreover, SrWRKY71 represses the expression level of SrUGT76G1 in both tobacco leaves and stevia callus. Taken together, the data in this study represent the first identification of an essential upstream transcription factor of SrUGT76G1 and provides new insight into the regulatory network of steviol glycoside biosynthesis in Stevia rebaudiana.

中文翻译：

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SrWRKY71转录因子负调控甜叶菊中SrUGT76G1的表达。

SrUGT76G1对于甜叶菊甜叶菊中莱鲍迪甙A，D和M的生物合成至关重要。然而，其转录调控机制仍然未知。在这项研究中，通过TAIL-PCR方法分离了SrUGT76G1的2050 bp启动子区域，并且序列分析显示存在多个W-box顺式元件，它们是WRKY转录因子的识别基序。此外，以典型的WRKY结构域和C2H2锌指状基序为特征的SrWRKY71被鉴定为SrUGT76G1的假定转录调节因子。SrWRKY71的转录本主要在叶片中积累，在茎，根和花中的含量较低。SrWRKY71-GFP融合蛋白特异地定位于烟草表皮细胞的细胞核中。此外，SrWRKY71的N和C末端区域有助于其反式激活活性。Y1H和EMSA分析证实SrWRKY71直接与SrUGT76G1的近端启动子区域中的W-box1和W-box2结合。此外，SrWRKY71抑制了SrUGT76G1在烟叶和甜菊愈伤组织中的表达水平。综上所述，本研究中的数据代表了对SrUGT76G1必需上游转录因子的首次鉴定，并提供了对甜叶菊中甜菊醇糖苷生物合成调控网络的新见解。