细胞周期蛋白PmCYCD3调控梅花曲枝性状形成机制研究

Prunus mume Sieb. var. tortuosa (Dragon Mei) is the only variety with tortuous trait. As a typical cultivar, Dragon Mei is well liked by people due to special tree structure, white petal and aromas, playing an importance role in landscaping. As the abnormal development in stamen and pistil result in barrenness, it is difficult to cultivate new varieties through artificial crossing. We previously cloned a PmCYCD3 gene from differentially expressed genes in the transcriptome, and overexpressed transgenic tabacco seedlings produced stem-bending phenotype, which is the first time have been found in other herbaceous plants. This project aims to use molecular biology methods to analyze the biological functions and regulatory networks of PmCYCD3 gene. Specific aims are to: (1) determine the expression pattern of PmCYCD3 gene by mRNA in situ hybridization and laser capture microdissection technology; (2) verify the gene conservation and biological functions of PmCYCD3 by plant genetic transformation; (3) verify the interaction of PmCYCD3 by yeast two-hybrid and bimolecular fluorescence complementation assay; and (4) analysis the biological functions and regulatory network of PmCYCD3 by RNA-seq from Prunus mume protoplasts of stem-differentiating xylem. The results will reveal the molecular mechanism of tortuous trait in Prunus mume, and lay a theoretical foundation for cultivating new varieties.

龙游梅是唯一具有曲枝性状的梅花品种，因其独特树姿兼具花白味香等观赏性状深受人们喜爱。然而，龙游梅雄蕊及雌蕊发育异常，授粉不实，难以通过传统杂交育种技术丰富曲枝梅花品种。前期从龙游梅转录组数据中克隆到一个梅花细胞周期蛋白PmCYCD3基因，转基因烟草产生茎弯曲表型，尚属首次发现。本课题拟利用分子生物学方法系统解析PmCYCD3基因生物学功能及调控网络。具体内容为：(1)利用原位杂交并分离特定细胞，确定PmCYCD3精细表达模式；(2)通过植物遗传转化，验证PmCYCD3基因保守性及生物学功能；(3)开展酵母双杂交及BiFC实验，确定PmCYCD3互作蛋白；(4)通过原生质体RNA-seq测序，分析PmCYCD3基因生物学功能及调控网络。本研究结果旨在揭示梅花曲枝株型形成的分子机理，为丰富梅花新品种奠定理论基础。

龙游梅是唯一具有弯曲性状的梅花品种，因其独特的树姿兼具花白味香等观赏性状而深受人们喜爱，在园林造景中具有重要地位。为了阐明龙游梅弯曲性状形成机制，本研究以龙游梅直立枝和曲枝有材料开展三年系统研究。研究明确了龙游梅曲枝和直枝在茎尖和腋芽在形态学和解剖特征具有显著差异，木质部和韧皮部的不对称发育是导致枝条弯曲生长的主要原因；利用定量PCR和原位杂交确定了PmCYCD3在分生组织、形成层等细胞分裂旺盛区域高表达；通过植物遗传转化，验证了PmCYCD3基因影响茎和叶片发育，引起下游细胞分裂与茎发育相关基因表达；酵母双杂交及BiFC实验，确定PmCYCD3互作蛋白并验证了PmHB1基因可能参与调控梅花顶端分生组织和叶腋分生组织活性，促进分枝，且与其他互作蛋白共同参与调控梅花花序维管束发育模式和次生生长过程；本研究解析了梅花曲枝株型形成的分子机理，为丰富梅花新品种奠定理论基础。