Identification and analysis of the upstream MYB regulator for the terpenoid synthase gene TPS72 in Rhododendron ovatum

Jiang Jie, Cao Yuqing, Liu Ruiyue, Zhao Boguo, Zhou Hong, Xia Yiping, Wang Xiuyun

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0

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南京林业大学学报（自然科学版）
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Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0 DOI: 10.12302/j.issn.1000-2006.202606007

Identification and analysis of the upstream MYB regulator for the terpenoid synthase gene TPS72 in Rhododendron ovatum

  • Jiang Jie1, Cao Yuqing1, Liu Ruiyue1, Zhao Boguo2, Zhou Hong1, Xia Yiping1, Wang Xiuyun1,*
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Abstract

【Objective】 Rhododendron ovatum is a representative aromatic germplasm in the genus Rhododendron, and its floral scent formation is closely related to terpenoid biosynthesis. The terpene synthase gene RoTPS72 encodes an α-farnesene synthase and serves as a key structural gene involved in the floral scent production of R. ovatum; however, its upstream transcriptional regulatory mechanism remains unclear. This study aims to identify the upstream transcription factors regulating RoTPS72 expression, thereby providing theoretical basis and genetic resources for dissecting the regulatory network of floral scent synthesis in Rhododendron.【Method】 In this study, the α-farnesene synthase-encoding gene RoTPS72 was used as the target. Putative upstream transcription factors were screened through promoter cis-regulatory element prediction and transcriptome co-expression network analysis. The regulatory interaction was subsequently validated using yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay (Dual-LUC). 【Result】 The promoter region of RoTPS72 contains multiple MYB-binding related elements, among which TAACCA, CAACAG, and CAACTG were selected as the key candidate motifs. Combined co-expression and expression pattern analysis identified RoMYB2 as a candidate upstream regulator of RoTPS72. Yeast one-hybrid assay confirmed the interaction between RoMYB2 and the RoTPS72 promoter. EMSA further demonstrated that RoMYB2 directly binds to the TAACCA and CAACAG motifs within the promoter, whereas no stable and specific binding was detected with the CAACTG motif. Dual-luciferase reporter assay revealed that RoMYB2 significantly enhances the activity of the RoTPS72 promoter. 【Conclusion】This study identifies RoMYB2 as a positive upstream regulator of the α-farnesene synthase-encoding gene RoTPS72, which activates its transcription by directly binding to the TAACCA and CAACAG motifs in the promoter region. This finding provides direct evidence for understanding the transcriptional regulatory mechanism of sesquiterpene floral scent components in Rhododendron ovatum and holds positive implications for aromatic breeding in Rhododendron.

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Rhododendron / Rhododendron ovatum / floral scent / MYB / terpene synthase

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Jiang Jie, Cao Yuqing, Liu Ruiyue, Zhao Boguo, Zhou Hong, Xia Yiping, Wang Xiuyun. Identification and analysis of the upstream MYB regulator for the terpenoid synthase gene TPS72 in Rhododendron ovatum[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 0 https://doi.org/10.12302/j.issn.1000-2006.202606007

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Funding

浙江省自然科学基金(LY24C160003); 国家自然科学基金（32371937）; 丽水绿地华东药用植物园委托横项（K横20232800）
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