IAA和蔗糖参与调控文冠果不同位置雌蕊选择性发育的机理研究

Hermaphrodite flowers are too less to limit the yield of seeds. Flower position determines the fate of pistil development during sex differentiation. Auxin and sucrose in apical dominance plays an important role in the regulation of pistil development. However, the regulation mechanism of auxin and sucrose involved in pistil development of different position is remain unclear. Yellow horn（Xanthoceras sorbifolia Bunge）is polygamous flowers and it is andromonoecious. In this research project, we are going to use the established system of regulating pistil development in Xanthoceras sorbifolia, and use the pistil from three different positions, the top inflorescence, lateral inflorescence and the first inflorescence after removing the top inflorescence as the experimental materials. The impact of the content growth and decline, transport sequencing of auxin and sucrose on pistil development will be measured and analyzed through Spectral mass spectrometry combined with selective ion monitoring (GC-MS/SIM). Some of the key differentially expressed genes (DEGs), which is related with auxin and sucrose involved in regulating pistil development in different positions during flower sex differentiation, will be identified and screened through several new technique such as transcriptome sequencing (RNA-seq) and Quantitative Real-time PCR(qRT-PCR). And the key candidate genes will be cloned and the function be analyzed through Western Blotting，gene over-expression analysis, yeast two-hybrid system. In addition, a molecular interactions regulating network model will be constructed, and be aimed at reveal the molecular mechanism of auxin and sucrose involved in regulating the pistil selection development destiny during sex differentiation in Xanthoceras sorbifolia Bunge., thus to provide an important theoretical basis for further clarify the regulation mechanism of the flower location determined its pistil development destiny during flower sex differentiation.

花性别分化过程中，位置决定雌蕊发育的命运，顶端优势中IAA和蔗糖在调控雌蕊发育中起重要作用，但对IAA和蔗糖参与调控不同位置雌蕊发育的机理尚不清楚。文冠果为杂性花，两性花过少，限制了种子的产量。本项目利用文冠果雌蕊发育调控研究系统，以顶花序、侧花序和去顶后第一侧花序三种不同位置雌蕊为材料，采用气谱-质谱联用/选择离子监测法（GC-MS/SIM）测定分析IAA和蔗糖的含量消长和转运对雌蕊发育的影响；利用转录组测序和实时荧光定量PCR等技术鉴定和筛选IAA和蔗糖参与调控不同位置雌蕊发育的关键差异表达基因（DEGs）；采用RACE法、Western Blotting分析、过表达分析和酵母双杂交等技术克隆与分析关键候选基因功能，构建分子互作调控网络模型，旨在阐释IAA和蔗糖参与调控文冠果不同位置雌蕊选择性发育的分子机理，进而为阐明位置决定雌蕊发育命运的调控机理提供重要理论依据。

花性别分化过程中，位置决定雌蕊发育的命运，顶端优势中IAA和蔗糖在调控雌蕊发育中起重要作用，但对IAA和蔗糖参与调控不同位置雌蕊发育的机理尚不清楚。文冠果为杂性花，两性花过少，限制了种子的产量。本项目以顶生花序、侧生花序和转化花序（去顶后第一侧花序）三种不同位置雌蕊为材料，结合注射蔗糖、环剥、喷IAA及TIBA处理，采用液-质谱联用（LC-MS）和ELISA法测定不同位置雌蕊中IAA、GA3和ABA含量，利用分光光度法测定蔗糖、葡萄糖、果糖、总糖含量及蔗糖合成酶（SS）和蔗糖磷酸合成酶（SPS）的活性，结果表明三个位置雌蕊发育相互关联，去顶后促进转化花序雌蕊发育。各期蔗糖、葡萄糖、总糖含量高，有利于雌蕊发育。外源蔗糖在顶生花序、侧生花序中主导逆转IAA对蔗糖的抑制调运作用；外源IAA在侧生花序中主导逆转IAA对蔗糖的抑制调运作用。IAA在一定阈值范围内对蔗糖调运起抑制作用，否则逆转调运作用。这与顶端优势理论中IAA调运蔗糖的观点不同。顶生花序I期ABA含量高，抑制雌蕊发育。利用转录组测序和实时荧光定量PCR等技术筛选和鉴定IAA和蔗糖参与调控不同位置雌蕊发育的5个关键差异表达基因，转运抑制响应蛋白TIR1、糖不敏感基因SIS3等；采用RACE法、Western Blotting分析、过表达分析和酵母双杂交、BiFC、Co-IP和亚细胞定位等技术克隆与分析TIR1和SIS3基因功能，二者表达量较一致，在 I期上调表达，促进雌蕊发育；TIR1和SIS3有互作，推测TIR1介导Aux/IAA蛋白酶体降解和生长素调节转录的生长素受体，在细胞核中与SIS3 E3泛素蛋白连接酶互作，SIS3抑制Smad3磷酸化，避免细胞过度纤维化死亡，节约蔗糖。TIR1和SIS3共同参与IAA和蔗糖调控文冠果雌蕊发育的信号通路。研究可为重新阐释顶端优势理论及指导栽培生产提供重要理论依据和实践指导。