多胺对金线莲胚胎发育的调控作用及其机制研究

Anoectochilus roxburghii, one of perennial precious Chinese medical herbs, belongs to the Orchidaceae family. Since it is widely used in medicine, health care, beauty products and drinks, A. roxburghii is honored as “the king of medicine”, which leads to an increasingly higher market demand. The key technology has made a breakthrough in artificial cultivation of A. roxburghii. However, study on breeding of elite varieties is lagging behind. As was found in previous studies, it was severe embryo abortion that largely contributed to a lower hybrid seed setting rate of A. roxburghii. The content of polyamines in embryo fertile groups was significantly higher than that in embryo abortion groups. The ratio of (Spd+Spm)/Put, the ratio of Spm/PAs and that of embryo abortion, there was a noticeably negative correlation among them. Polyamines play a key role in the regulation of embryogenesis. Proceeding from treating the cross combination of A. roxburghii with exogenous polyamines and their synthetic inhibitors, this project plans to analyze the reaction of embryogenesis to positive and negative regulation of polyamines. The relationship among embryogenesis, polyamines metabolism and polyamines signal transduction pathways is revealed by further investigation of polyamines dynamic indexes, embryonic structure characteristics and gene expression information. The success of the project will not only lay a foundation for germplasm utilization and cultivars breeding of A. roxburghii but also provide an important reference for similar studies on other medicinal plants.

金线莲为兰科开唇兰属多年生名贵中药材，享有“药王”的美称，广泛应用于医药、保健、美容及饮用品等诸多领域，国内外市场对金线莲需求量不断增加，市场缺口逐年加大。金线莲人工栽培关键技术已取得突破，但是关于优良品种选育方面研究相对滞后。前期研究表明，胚胎败育是引起金线莲杂交结实率低的主要原因，胚可育株系中多胺的含量显著高于胚败育株系，（亚精胺+精胺）/腐胺以及精胺/多胺比值与胚胎败育率呈显著负相关，多胺在胚胎发育过程中起到关键的调控作用。本项目拟从外源多胺和多胺合成抑制剂处理金线莲杂交组合入手，解析不同胚胎发育状态对多胺正负双向调控的响应，耦合多胺动态变化指标、胚胎结构特征和多胺代谢、信号转导基因表达信息，挖掘多胺代谢与金线莲胚胎发育的内在联系，揭示金线莲胚胎发育过程中多胺信号转导途径，为金线莲种质资源利用及优良品种选育奠定基础，也为其他药用植物开展类似研究提供参考。

本项目针对金线莲杂交种质创新过程中胚胎败育的问题，从细胞、亚细胞、分子水平解析多胺代谢与金线莲胚胎发育的内在联系，为幼胚拯救技术优化提供理论基础，为金线莲杂交种质创新技术体系构建提供科学依据。.（1）金线莲种质资源评价、种内杂交及胚胎发育研究；（2）金线莲胚胎发育过程中形态结构变化研究；（3）外源多胺对金线莲开花、结实的调控作用；（4）多胺调控金线莲胚胎发育分子机制研究。.本项目可以得到以下结论：.观测金线莲花芽分化过程中的形态特征，并分析其花芽分化过程中生理变化，明确了唇瓣和合蕊柱发育的过程。将尖叶金线莲和圆叶金线莲进行种内杂交，用石蜡切片技术和荧光显微镜观察花粉-雌蕊相互作用和胚胎发育。比较外源多胺对金线莲开花、结实的调控作用。外源多胺处理使金线莲花期提前，显著提高了鲜重、花梗长、蛋白浓度、抗氧化酶活性、内源多胺（Put、Spd、Spm）的含量。内源多胺含量的升高一方面是通过多胺传递运输系统吸收的结果，另一方面可能是通过外源多胺的刺激，加速了植株体内多胺的代谢。根据金线莲转录组序列克隆得到2个ArADC和3个ArPAO基因cDNA全长序列。时空表达分析发现，在金线莲从开花到结实过程中，ArADC1在根、茎中的表达量呈下降趋势，而在叶中呈现先上升后下降；ArADC2在根、叶中的表达量呈下降趋势，而在茎中则呈现先上升后下降的趋势。ArPAO2与ArPAO1类似，在芽或花中的表达量始终最高，伴随着金线莲开花、结实呈现下降趋势。ArPAO3在叶芽期时根和叶中表达较高，在花芽期时花芽中表达量最高。研究了多胺处理对金线莲转录组的影响，从中筛选得到23条ArWRKY序列。响应表达分析结果显示，ArWRKY5、ArWRKY8、ArWRKY9、ArWRKY13与ArWRKY23的表达能被外源亚精胺诱导上调，而ArWRKY19与ArWRKY20的表达下调。时空表达分析发现，ArWRKY5与ArWRKY20在不同时期的花中表达量均显著高于开花期的根茎叶，说明ArWRKY5与ArWRKY20对金线莲的花的形成及发育起着重要的调控作用。.在本项目的资助下，已发表论文7篇（其中SCI收录5篇），录用待刊1篇，出版专著1部。培养硕士研究生7人，已毕业5人。

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