College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, China.
BMC Plant Biol. 2024 Oct 3;24(1):921. doi: 10.1186/s12870-024-05616-1.
The UDP-glucuronosyltransferase 91D2 (SrUGT91D2) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-β-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 (pSrUGT91D2) have not been reported.
The pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment (pSrUGT91D2-B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 (pSrUGT91D2-023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2, the full-length promoter and a series of 5' deletion fragments (P1-P7) and a 3' deletion fragment (P8) from various lines were fused with the reporter β-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2-B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2-B1188 and P8-pSrUGT91D2-023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction.
These findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.
UDP-葡糖醛酸基转移酶 91D2（SrUGT91D2）基因是甜菊糖苷（SGs）生物合成途径中的关键要素，负责在 C19 和 C13 位置形成 1,2-β-D 葡糖苷键。这一过程在瑞鲍迪甙 M（RM）和瑞鲍迪甙 D（RD）的合成中起着至关重要的作用。启动子调控基因表达，需要对其进行功能分析以理解基因表达调控。然而，目前尚未有关于 SrUGT91D2（pSrUGT91D2）启动子功能的研究报告。
从六个甜叶菊品系中分离到 pSrUGT91D2，随后的多次序列比较显示，在 GP、GX、110、1114 和 B1188 品系中存在一个 26 bp 的缺失片段（pSrUGT91D2-B1188 型），但在 023 品系中不存在（pSrUGT91D2-023 型）。生物信息学分析表明，启动子序列中存在大量的顺式调控元件（CREs），包括对脱落酸、光照、厌氧条件、生长素、干旱、低温和茉莉酸甲酯响应的元件。为了验证 pSrUGT91D2 的活性，我们将全长启动子和来自不同品系的一系列 5'缺失片段（P1-P7）和 3'缺失片段（P8）与报告基因β-葡糖苷酸酶（GUS）基因融合，构建了植物表达载体 pCAMBIA1300-pro∷GUS。通过瞬时转化在烟草叶片中检测这些基因的转录活性。GUS 组织染色分析和酶活性测定表明，全长启动子和截短的 SrUGT91D2 都能够在烟草叶片中启动 GUS 表达。有趣的是，含有缺失片段（301 bp）的 P8-pSrUGT91D2-B1188 表现出更强的驱动 GUS 基因表达的能力。对 P8-pSrUGT91D2-B1188 和 P8-pSrUGT91D2-023 在外源激素（脱落酸和吲哚-3-乙酸）和光照下的瞬时表达研究表明，缺失区域对于 P8 表现出转录活性是必需的，因为它对外源脱落酸（ABA）、吲哚-3-乙酸（IAA）和光照诱导表现出强烈的响应。
这些发现有助于深入了解 SrUGT91D2 基因上游区域的调控机制，并为进一步研究 pSrUGT91D2 的 CREs 与相关转录因子之间的相互作用提供了理论依据。
