Wu Xinxin, Du Tong, Li Yan, Zhuang Weibing, Kang Naixin, Zeng Jiaxin, Yan Cong, Hu Zhenzhu, Cao Zewen
School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai'an, Jiangsu, China.
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Memorial Sun Yat-Sen), Nanjing, Jiangsu, China.
PeerJ. 2025 Sep 4;13:e19975. doi: 10.7717/peerj.19975. eCollection 2025.
The floral color variegation of cultivar 'Sahong Tao' is distinctive and possesses significant ornamental value. Currently, there are no relevant reports on how MYB transcription factors (TFs) interact with promoter to regulate the flower color variegation in peach.
In this study, we screened for proteins that interact with the promoter using yeast one-hybrid (Y1H) and next-generation sequencing (NGS). The NGS data were aligned with the database (TAIR10) utilizing Python 3.10.4. PlantTFDB was employed to identify TFs, while PlantRegMap was used to predict TFs that interact with the promoter. The Y1H assay verified MYB1R1 interaction with promoter, and Y1H-AOS predicted their binding sites. The physicochemical properties, structure and interacting proteins of MYB1R1 were analyzed using bioinformatics methods. Sequence alignment and phylogenetic tree analyses of were performed. Finally, the tissue expression specificity of and in 'Sahong Tao' was examined using qRT-PCR.
The Y1H and NGS results indicate that 1,190 proteins interact with the promoter. Among these, 20 TFs were identified, including ERF, MYB, NF-YB, SBP, S1Fa-like, TCP, bHLH, LBD, ZF-HD, C3H, DBB, MYB-related, and HD-ZIP. Of the 1,190 proteins, 1,146 exhibit high similarity to homologs in , with 332 classified as RNA binding proteins and 124 as DNA binding proteins. A comparison with the NGS results identified seven TFs that align with predictions from PlantRegMap. Based on these findings, we selected MYB44 (PRUPE_6G229000, PRUPE_1G430000) and MYB1R1 (PRUPE_5G182000) as candidate members. Y1H assays demonstrated that MYB1R1 interacts with the promoter. Y1H-AOS was used to confirm 24 interaction binding sites. consists of an 897 bp full-length CDS, encoding 298 amino acids, with a predicted molecular weight of 32.49 kDa and a theoretical isoelectric point of 7.20. MYB1R1 features a typical SANT-MYB domain, and its secondary structure is predominantly composed of irregular coils. Phylogenetic analysis indicates a close evolutionary relationship between MYB1R1 from 'Sahong Tao' and both and . Promoter prediction analysis for reveals multiple hormone- and stress-related -acting elements. MYB1R1 may interact with bHLH and other proteins to perform its functions. In variegated petals, expression is higher and expression is lower compared to red petals, suggesting that MYB1R1 negatively regulates anthocyanin synthesis by interacting with LDOX. This study contributes to elucidating the function of and the regulatory mechanism of MYB- in the flower color of 'Sahong Tao'.
‘萨红桃’品种的花色斑驳独特，具有较高的观赏价值。目前，关于MYB转录因子（TFs）如何与启动子相互作用以调控桃的花色斑驳，尚无相关报道。
在本研究中，我们利用酵母单杂交（Y1H）和二代测序（NGS）筛选与启动子相互作用的蛋白质。利用Python 3.10.4将NGS数据与数据库（TAIR10）进行比对。使用PlantTFDB鉴定转录因子，同时利用PlantRegMap预测与启动子相互作用的转录因子。Y1H试验验证了MYB1R1与启动子的相互作用，Y1H - AOS预测了它们的结合位点。使用生物信息学方法分析了MYB1R1的理化性质、结构及相互作用蛋白。对进行了序列比对和系统发育树分析。最后，采用qRT - PCR检测了和在‘萨红桃’中的组织表达特异性。
Y1H和NGS结果表明，有1190种蛋白质与启动子相互作用。其中，鉴定出20个转录因子，包括ERF、MYB、NF - YB、SBP、S1Fa - like、TCP、bHLH、LBD、ZF - HD、C3H、DBB、MYB - related和HD - ZIP。在这1190种蛋白质中，有1146种与中的同源物具有高度相似性，其中332种归类为RNA结合蛋白，124种为DNA结合蛋白。与NGS结果比较，鉴定出7个与PlantRegMap预测相符的转录因子。基于这些发现，我们选择MYB44（PRUPE_6G229000，PRUPE_1G430000）和MYB1R1（PRUPE_5G182000）作为候选成员。Y1H试验表明MYB1R1与启动子相互作用。Y1H - AOS用于确认24个相互作用结合位点。由一个897 bp的全长CDS组成，编码298个氨基酸，预测分子量为32.49 kDa，理论等电点为7.20。MYB1R1具有典型的SANT - MYB结构域，其二级结构主要由不规则卷曲组成。系统发育分析表明，‘萨红桃’中的MYB1R1与和的进化关系密切。对的启动子预测分析揭示了多个与激素和胁迫相关的顺式作用元件。MYB1R1可能与bHLH及其他蛋白质相互作用以发挥其功能。在斑驳花瓣中，与红色花瓣相比，的表达较高，的表达较低，这表明MYB1R1通过与LDOX相互作用负调控花青素合成。本研究有助于阐明的功能以及MYB - 在‘萨红桃’花色中的调控机制。
