抗坏血酸参与乙烯信号调控棉花纤维伸长发育的分子机制研究

Ascorbic acid(Asc) promotes cell elongation development by participating extension of cell wall and ethylene signaling, however, almost no research results were reported in cotton fiber development. We performed the first report that a ascorbate peroxidase(APX) gene of ascorbic acid metabolism pathway is involved in cotton fiber development through responding ethylene signaling. Based on our recent research results, of that Asc synthesis was induced by ethylene and Asc could promote cotton fiber elongation significantly through enhancing cell wall enzymes expression, the purpose of this study is to elucidate the mechanism of Asc promoting cotton fiber elongation by participating ethylene signaling. In this study, following research works will be developed: to clone key genes of regulating Asc synthesis in L-galactose pathway during cotton fiber elongating stage; to analyze gene function regulating Asc synthesis and responding ethylene signaling, including cloning gene promoter sequences, exploring the function elements of responding ethylene signaling, and investigating gene complementary function in Arabidopsis mutants; to study the interaction between Asc and ethylene, through utilizing Asc and its inhibitor lycorine as well as ethylene and its inhibitor AVG, using Arabidopsis mutant and in vitro cotton ovule culture technologies, and detecting the content changes of Asc and ethylene respectively; to analyze key genes function in fiber elongation development by transgenic cotton. Finally, all these results are to elucidate the mechanism of Asc promoting cotton fiber elongation by participating ethylene signaling, and to provide a basis of premium quality cotton breeding.

抗坏血酸Asc可以通过影响细胞壁延展性和乙烯信号参与细胞伸长发育,然而，相关研究在棉纤维发育中鲜有报道。我们首次发现Asc代谢的APX基因通过响应乙烯信号参与纤维发育过程；Asc的合成受乙烯诱导。在前期工作基础上，本项目旨在研究抗坏血酸通过参与乙烯信号促进棉纤维伸长发育的分子机制。拟开展如下方面工作：克隆棉纤维中调控Asc合成的L-半乳糖路径中的关键基因；克隆目的基因启动子并分析其响应乙烯的功能元件，分析目的基因在拟南芥突变体中的互补功能，研究目的基因调控抗坏血酸合成和响应乙烯信号的功能；利用Asc及其合成抑制剂石蒜碱和乙烯及其合成抑制剂AVG，通过拟南芥突变体和棉花胚珠体外培养，分别检测乙烯和Asc含量的变化，研究Asc和乙烯的相互作用；转基因棉花研究目的基因在调控棉纤维发育与品质形成过程的重要功能；最终解析Asc通过参与乙烯信号促进棉纤维伸长发育的作用机，为优质棉花新材料的培育提供参。

一、项目背景.抗坏血酸(Ascorbic acid，AsA) 是一种普遍存在于植物组织的高丰度小分子抗氧化物质，与植物细胞发育密切相关。植物激素乙烯与植物细胞的扩展密切相关，并能显著促进纤维细胞的伸长发育。抗坏血酸代谢及其与乙烯相互作用的相关研究在棉纤维发育中鲜有报道。本项目旨在研究抗坏血酸通过参与乙烯信号参与棉纤维伸长发育的分子机制。本项目为棉花纤维发育的分子机制解析及优质棉花新材料的培育提供参考。.二、主要研究内容.主要开展四方面的内容：抗坏血酸合成中L-半乳糖路径关键基因的克隆表达特征分析，利用拟南芥和烟草BY-2悬浮细胞研究基因参与细胞伸长发育的功能，分析乙烯调控抗坏血酸合成基因表达特征，转基因棉花验证基因对于棉纤维发育的功能。.三、重要结果.1. 克隆获得棉纤维中调控抗坏血酸从头合成的关键基因GhGMPase和GhGGPase。GhGMPase和GhGGPase在纤维快速伸长发育的开花后5-15天的纤维组织中表达量最高。亚细胞定位分析表明GhGMPase和GhGGPase可能主要在质膜和细胞核中表达。.2. GhGMPase和GhGGPase显著促进拟南芥根发育和烟草BY-2悬浮细胞的伸长发育。构建植物过量表达载体将GhGMPase和GhGGPase基因遗传转化拟南芥哥伦比亚野生型和烟草BY-2悬浮细胞。转GhGMPase基因的拟南芥侧根发育获得了促进，转GhGGPase基因拟南芥主根显著伸长。GhGMPase和GhGGPase基因烟草BY-2悬浮细胞的伸长发育均获得了显著的促进。.3. GhGMPase和GhGGPase的表达受乙烯的诱导。.在乙烯合成前体ACC处理下，GhGMPase和GhGGPase基因的表达显著上调，GhGMPase基因在处理4h后表达量达到最高，GhGGPase基因在处理6h时表达量最高。克隆获得GhGMPase和GhGGPase基因的启动子，这两个启动子均具有较高的驱动GUS和GFP表达的活性。启动子含有乙烯响应元件，构建5’端缺失的包含不同启动子区段的载体并转化烟草，在乙烯合成前提ACC处理下，包含乙烯元件的启动子片段均不同程度受到ACC的诱导。.4. 棉花的遗传转化。.构建E6-GhGMPase过量表达载体，通过农杆菌介导的花粉管通道法转化陆地棉新陆早33号。经过筛选鉴定后的转基因棉花材料的纤维长度获得了显著的促进。