毛白杨雄花芽发育关键时期的转录组分析

Transcriptome analysis of key developmental stages of male floral buds in Populus tomentosa

摘要

【目的】解析毛白杨雄花芽从成花诱导到花粉成熟全过程的基因表达动态规律，筛选控制花发育进程的关键调控基因，以期为深入理解多年生木本植物花发育的分子机制提供理论依据，并为通过分子设计育种调控杨树开花时间和减少花粉致敏源问题提供候选基因资源。【方法】以毛白杨8个连续发育时期（MS1 ~ MS8）的雄花芽样本为材料，进行转录组测序。通过主成分分析评估样本重复性，筛选差异表达基因并统计转录因子家族。采用Mfuzz时序聚类划分表达趋势簇，并进行GO功能富集分析。选取关键簇中的转录因子进行启动子顺式作用元件预测。利用WGCNA构建共表达网络，筛选发育时期相关模块并进行GO富集分析。选取关键基因进行RT-qPCR验证，评估转录组数据准确性。【结果】（1）差异表达基因数量在发育阶段转换中呈现显著差异：从越冬休眠到花粉成熟启动（MS8 VS MS7）差异基因总数最多（上调10 863个，下调10 890个），表明休眠解除后大规模转录重编程可能是生殖发育快速推进的分子基础。（2）Mfuzz时序聚类将所有表达基因划分为10个趋势簇，其中Cluster 1在成花诱导期高表达后持续下调，富集于细胞壁合成与细胞骨架构建；Cluster 4在雄蕊原基形成期达峰值，参与核酸代谢与染色质重塑；Cluster 6在孢原细胞形成期特异性高表达，主要富集于胁迫响应与激素响应功能条目；Cluster 8在花粉成熟期高表达，显著富集花粉管生长与花粉萌发等生殖过程，揭示了不同发育阶段基因表达的时序性功能转换。（3）WGCNA筛选出3个阶段特异性共表达模块（MEpink、MEmagenta、MEbrown），分别与早期细胞壁形成与苯丙烷代谢、休眠期囊泡运输与能量供应、花粉成熟期细胞骨架与细胞外基质建成密切相关，从网络层面印证了模块功能与发育进程的协同性。（4）启动子分析表明，Cluster 1和Cluster 8中16个关键差异表达的转录因子启动子区广泛富集光响应及多种激素响应元件，暗示光信号与激素信号可能协同调控雄花芽发育。（5）RT-qPCR验证了6个花发育相关基因（ PtoMYC2、 PtoSEP2、 PtoSEP4、 PtoSOC1、 PtoSVP、 PtoTCP9）的表达趋势，与RNA-seq结果高度一致（ r > 0.9）；且不同MADS-box家族成员（ PtoSEP2、 PtoSVP、 PtoSEP4、 PtoSOC1）在雄花芽发育过程中呈现差异化的表达模式。【结论】毛白杨雄花芽发育中，休眠解除期基因表达变化最剧烈，休眠过渡期相对平稳，说明休眠解除是一个关键的转录重编程节点。本研究绘制了毛白杨雄花芽发育8个时期的动态转录图谱，揭示了基因表达的阶段性调控特征。这些结果为理解多年生木本植物花发育的阶段性调控提供了依据，也为分子育种调控杨树开花和减少花粉污染提供了候选基因。

Abstract

[Objective] To reveal the genome-wide expression dynamics of male floral buds in Populus tomentosa from floral induction to pollen maturation, and to identify key regulatory genes controlling floral development, so as to provide a theoretical basis for understanding the molecular mechanisms of flower development in perennial woody plants and to offer candidate gene resources for molecular breeding aimed at regulating flowering time and reducing pollen allergens in Populus. [Method] Male floral bud samples at eight consecutive developmental stages (MS1–MS8) of P. tomentosa were subjected to transcriptome sequencing. Principal component analysis was used to assess sample reproducibility, differentially expressed genes (DEGs) were screened, and transcription factor families were statistically analyzed. Mfuzz time-series clustering was performed to classify expression trend clusters, followed by GO enrichment analysis. Promoter cis-element prediction was carried out for transcription factors selected from key clusters. WGCNA was applied to construct co-expression networks and to screen for developmental stage-related modules, which were then analyzed by GO enrichment. Select key genes for RT-qPCR validation to assess the accuracy of RNA-seq data. [Result] (1) The number of DEGs varied markedly across developmental transitions: the comparison between MS8 and MS7 (from overwintering dormancy to pollen maturation initiation) showed the largest number of DEGs (10 863 up-regulated, 10 890 down-regulated), suggesting that massive transcriptional reprogramming after dormancy release might be the molecular basis for rapid progression of reproductive development; whereas the comparison between MS4 and MS3 (from bud swelling to dormancy transition) showed the fewest DEGs (1 480 up-regulated, 3 178 down-regulated), indicating that this stage might be relatively quiescent. (2) Mfuzz time-series clustering divided all expressed genes into 10 trend clusters. Among them, Cluster 1 was highly expressed during floral induction and then continuously down-regulated, enriched in cell wall synthesis and cytoskeleton organization; cluster 4 peaked at stamen primordium formation, involved in nucleic acid metabolism and chromatin remodeling; cluster 6 showed specific high expression at the sporogenous cell formation stage, mainly enriched in stress response and hormone response-related terms; Cluster 8 was highly expressed at the pollen maturation stage, significantly enriched in reproductive processes such as pollen tube growth and pollen germination. This reveals the temporal functional transition of gene expression at different developmental stages. (3) WGCNA identified three stage-specific co-expression modules (MEpink, MEmagenta, MEbrown), which were closely associated with early cell wall formation and phenylpropanoid metabolism, vesicle transport and energy supply during dormancy, and cytoskeleton organization and extracellular matrix formation during pollen maturation, respectively, confirming at the network level the synergy between module functions and developmental progression. (4) Promoter analysis showed that the promoter regions of 16 key differentially expressed transcription factors from clusters 1 and 8 were extensively enriched in light-responsive and multiple hormone-responsive cis-elements, implying that light and hormone signals may coordinately regulate male floral bud development. (5) RT-qPCR validated the expression trends of six flower-development-related genes (PtoMYC2, PtoSEP2, PtoSEP4, PtoSOC1, PtoSVP, PtoTCP9), which were highly consistent with RNA-seq data (r > 0.9). Different MADS-box family members (PtoSEP2、 PtoSVP、 PtoSEP4、 PtoSOC1) exhibited distinct expression patterns in male floral bud development. [Conclusion] During male floral bud development of P. tomentosa, gene expression changes most dramatically at the dormancy-release stage and is relatively stable during the dormancy-transition stage, indicating that dormancy release is a critical node of transcriptional reprogramming. This study provides a comprehensive dynamic transcriptome atlas of male floral bud development across eight stages in P. tomentosa, revealing stage-specific regulatory characteristics of gene expression. These findings provide a basis for understanding the stage-specific regulation of flower development in perennial woody plants and offer candidate genes for molecular breeding to control flowering time and reduce pollen allergy in Populus.

关键词

毛白杨 / 雄花芽 / 发育时期 / 转录组 / 时序表达 / WGCNA / 基因表达

Key words

Populus tomentosa / male floral bud / developmental stages / transcriptome / time-series expression / WGCNA / gene expression

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基金资助

国家自然科学基金项目(31800555)

中央高校基本科研业务费专项(PTJS202609)

国家重点研发计划(2024YFD2201004)