阿拉伯半乳糖蛋白（AGPs）调控茉莉花粉管生长的机制研究

Jasmine (Jasminum sambac Aiton) is broadly used as ornament, tea, or medicine in China and which has been the country with the largest cultivation area and the highest flower productivity in the world. Therefore, it’s very important to breed new jasmine cultivars by artificial cross. However, our previous cytological studies showed that the serious pre-fertilization barriers caused the failure of cross breeding between single and double petal phenotypes of jasmine. After pollination, the growth of pollen tubes was extremely slow or arrested which stopped the pollen tubes from reaching the ovary, leading to fertilization failure in ovules. Further investigation found that the abnormal growth of pollen tubes was closely correlated with the protein level especially the arabinogalactan proteins (AGPs). Therefore, in this project, the pollen tube behavior before and after pollination will be studied using various methods including immunolocalization, laser scanning confocal microscope (LSM), transmission electron microscope (TEM), real-time quantitative PCR, RNA-seq, bioinformatics and transgenic function research, combining with the treatment of β-glycosyl Yariv reagent that binds specifically to AGPs. We hope to study the response of jasmine pollen tubes to AGPs during different growth stages by cytological observation, protein detection and molecular biological verification, and elucidate the specific genes and functions of AGPs during the process of jasmine pollen tube growth. The research results are helpful to take effective measures to overcome the cross barriers and create new jasmine germplasms, which have a great scientific significance to guide the jasmine cross breeding.

茉莉花观赏、茶用、药用功能齐备，我国是世界上栽培面积最大和产花量最多的国家。因此，开展茉莉杂交育种非常重要。前期细胞学研究发现单、双瓣茉莉杂交存在严重的受精前障碍，表现为花粉管能萌发但在花柱中生长极慢或停滞而不能到达胚珠完成受精作用。进一步研究发现花粉管生长异常与花柱蛋白质尤其是阿拉伯半乳糖蛋白（AGPs）密切相关。为此，本项目将从杂交授粉前后的花柱和花粉管入手，结合AGPs特异结合剂处理，用细胞免疫定位、荧光标记、激光共聚焦扫描、透射电子显微、实时荧光定量PCR、RNA测序、生物信息学分析及转基因功能验证等方法，研究AGPs对茉莉花粉管生长的影响，耦合AGPs动态变化、细胞超微结构特征和基因功能，揭示AGPs在茉莉花粉管生长中的作用，确定编码AGPs的特异基因和功能，初步阐明AGPs调控茉莉花粉管生长的机制。研究结果对采取有效措施克服茉莉杂交受精前障碍，指导茉莉杂交育种有重要的科学意义。

茉莉花具有较高的观赏、茶用和药用价值，我国是世界上栽培面积最大和产花量最多的国家，开展杂交育种非常重要。前期研究发现单、双瓣茉莉杂交存在严重的受精前障碍，表现为花粉管在花柱中生长极慢或停滞而不能到达胚珠完成受精作用。进一步研究发现花粉管生长异常与阿拉伯半乳糖蛋白（AGPs）密切相关。为此，本项目对花粉发育、柱头和花粉管的超微结构特征进行了观察和比较，发现柱头可授性没有问题，但花粉管在柱头中生长异常；采用离体培养技术，研究了AGPs与其抑制剂βGlcY对茉莉花粉萌发和花粉管生长的影响，发现AGPs对茉莉花粉离体萌发和花粉管生长有重要作用；进而开展了单、双瓣茉莉的杂交工作，对授粉后柱头进行取样，进行转录组和蛋白组测序并进行联合分析，发现和花粉萌发与花粉管生长密切相关的差异表达AGPs基因13个，并筛选出关键基因JsFLA3；克隆了JsFLA3基因全长，进行表达特异性和亚细胞定位分析，发现JsFLA3在雄蕊的表达量最高，定位在细胞质膜-细胞壁上；构建JsFLA3超表达载体转化拟南芥，对转基因株系进行了表型分析，发现过表达转基因植株有严重的结实障碍，表现为花丝变短、花药矮于花柱、角果变短、每角果结实数下降等表型特征；进一步研究发现过表达JsFLA3导致拟南芥花粉败育、花粉管生长异常；为了揭示转基因株系结实障碍的机理，采用RNA-seq对JsFLA3过表达和野生型植株的转录组数据进行了比较分析，构建了共表达网络，明确了差异表达基因和共表达基因。此外，项目还对茉莉内参基因进行了筛选，对3种瓣型茉莉基因组大小进行了测定与比较，对茉莉叶绿体基因组和全基因组进行了测序分析；同时，还利用HS-SPME和GC-MS技术对茉莉花发育过程中次生代谢产物进行了测定分析；基于茉莉基因组和花发育转录组信息，对次生代谢产物合成途径进行分析，并对其中的关键基因进行了表达分析。研究结果不仅丰富了茉莉花生殖发育生物学信息，有重要的理论意义，还对采取有效措施克服茉莉杂交受精前障碍、指导茉莉杂交育种有重要的科学价值。