棉花GhBOP1调控蕾铃离层形成发育的分子机制研究

The shedding rate of cotton buds and bolls is normally above 60 percentage. The yield of cotton fibre should be significantly increased when the shedding rate of buds and bolls reduces. The abscission zone (AZ) forms in the site where the shedding takes place. Typically, the AZs consist of several layers of cytoplasmically condensed cells, which are anatomically divergent from the adjacent cells. The AZs can be triggered by both developmental signals and external stimuli, which many genes including signal tranduction and metabolism pathways are involed in. A GhBOP1 gene relating to AZ fromation has been identified from Gossypium hirsutum, and putative protein sequence and dormains has been deduced. GhBOP1 gene is preferential expression in AZs of cotton compared to other organs and functions in AZ formation by qPCR analysis and virus inducing gene silence. Here, we suppose that the GhBOP1 can interact with TGA transcriptional factor and other proteins to regulate the AZ differentiation, development and formation according to bioinformation.In this project, the four types of transgenic plants, including overexpression of intact and truncated GhBOP1 gene, antisense expression, and RNAi, will be developed. The function of GhBOP1 can be exhibited through the comparison of phenotype and anatomy of AZ of transgenic plants with wild type. And, according to the GUS staining and GFP subcellular localization analyis, GhBOP1 characterization is able to be further exhibited. The protein interaction analysis should reveal whether GhBOP1 is dimerism and interacts with TGA transcription factor or other protein to regulate differentiation, development and formation of AZ according to the results of Yeast two-hybrid, firefly luciferase complementation imaging and in vitro pull-down assays. After all, the molecular mechanism of GhBOP1 regulation to abscission cell differentiation must be exposed through this study. While the signal transduction and metabolic pathways concerning GhBOP1 are revealed, the transgenic plants with lower shedding rate of buds and bolls are able to be developed by molecular design breeding.

棉花蕾铃生理脱落率在60%以上，降低其脱落可以使棉花产量大幅度上升。蕾铃脱落通常先形成离层，离层细胞从分化、生长发育到程序性死亡是一个复杂过程，有许多信号传递和代谢途径基因参与。我们已从棉花中分离一个参与蕾铃离层形成的调控基因GhBOP1，初步研究表明其在离层中特异表达并参与离层形成，其可能通过与TGA转录因子或其它蛋白互作来调控离层发育相关基因的表达。本项目通过培育过量和抑制表达GhBOP1的转基因棉花株系并与野生型进行比较研究，分析该蛋白在棉花蕾铃离层细胞分化等时期的功能，观察其超微结构，了解细胞形态和排列上变化，进一步验证GhBOP1对离层形成的调控功能。同时通过蛋白互作试验明确GhBOP1的结构域组成成分和TGA等蛋白的互作关系，结合该蛋白的组织特异性GUS染色和GFP亚细胞定位等研究结果，揭示GhBOP1在棉花蕾铃离层形成调控的分子机理，为棉花高产育种提供理论和物质基础。

BOP1蛋白主要调控植物侧生器官的分化和发育，因此从棉花中克隆GhBOP1基因，并获得抑制和过量表达GhBOP1基因棉花植株，对获得转基因植株进行表型观察鉴定、分子分析、离层解剖结构观察和抗病性检测。结果表明过量表达转基因棉花株高变矮、茎间距变短、叶柄变短。而抑制表达的转基因棉花则表现为植株变高、花蕾铃增多、侧枝变长等特征。抑制表达GhBOP1基因棉花在这些离层处的细胞没有出现特异细胞分化，而过量表达GhBOP1基因棉花则过早出现离层细胞分化，造成蕾铃过早脱落等现象。同时发现GhBOP1参与棉花的抗病反应。植物对病原菌侵染的响应中，大多数基因被暂时诱导参与植物防御反应的，而具有空间诱导表达模式的基因报道的很少。在本研究中，我们发现当Gossypium hirsutum植株受到黄萎病菌(Verticillium dahliae)的攻击时，GhBOP1可以自主地从侧向器官边界部位扩展表达，这种空间诱导的表达模式增强了植物的抗病性。通过对GhBOP1转基因植株的抗性鉴定、酵母双杂交分析、荧光素酶互补显像和GUS报告等生理生化方法，全面的综合的分析了GhBOP1的功能。结果表明，GhBOP1是植物对V. dahliae是正调节因子。GhBOP1与GhTGA3的相互作用，增强GhTAG3转录因子的转录激活活性，促进下游具有TGACG顺式调控元件的抗病相关基因表达。在V. dahliae侵染条件下，GhBOP1的表达量剧烈上调的同时伴随着GhBP1的抑制。对GhBOP1的过量表达和RNAi植株的分析表明，GhBOP1可以促进并加剧茎秆中的木质素积累，增强棉花植株对V. dahliae的抗性增强。综上所述，这些结果表明，GhBOP1和其它植物的BOP蛋白功能一样，均有调控植物侧生器官分化发育的功能。但是在棉花中其不仅可以通过GhBOP1-GhTGA3模式激活促进抗病相关基因的表达。而且，促进植株的木质素积累，正调控植物对病原菌防御反应。