切花小菊花粉败育的细胞与分子机理研究

Cut chrysanthemum is one of the four most popular cut flowers in the world, and can be grouped into standard cut chrysanthemum and spray cut chrysanthemum according to its inflorescence type. In recent years, spray cut chrysanthemum has become more and more popular in the world, but its excessive pollen dispersal during flowering stage can significantly reduce its ornamental value and shorten its vase life. In addition, it is harmful to people, in particular those with pollen allergies. We thus regarded low pollen dispersal and no pollen dispersal as a vital breeding objective of cut spray chrysanthemum several years ago. After several years of hard work, we obtained some imporant progress. For example, we found that pollen abortion is a key reason for no pollen dispersal of cut spray chrysanthemum and such result has been publised in BMC Plant Biology. However, the mechanism of pollen abortion is still unclear. Based on our good foundation, we will investigate stage of pollen abortion and cellular characteristics of pollen abortion by some techniques in cellular biology in this project in order to reveal mechanisms of pollen abortion. Then we will determine genes related to pollen abortion and reveal their regulatory network during pollen abortion using some techniques in proteomics and molecular biology. The results will help unravel cellular and molecular mechanisms of pollen abortion and enrich basic theory in plant reproductive biology. More importantly, the results will provide valuable data for development of cut spray chrysanthemum with low or non-dispersed pollen in the near future. Therefore, the study in this project is of great scientific significance and prospective application.

切花菊是世界四大切花之一，根据花序类型可分为切花大菊和切花小菊，其中切花小菊近年来发展迅速，已成为切花市场的重要组成部分。然而，切花小菊开放中大量散粉会降低其观赏价值和缩短其观赏寿命，同时也会对花粉过敏者健康造成负面影响。为此，近年来我们开始把培育散粉少和不散粉切花小菊作为一个重要育种目标。经过几年的努力,取得了一些重要进展，如发现花粉败育是切花小菊不散粉的一个主要原因，相关结果已在BMC Plant Biology上发表，但花粉败育机理尚不清楚。为此，本项目将在现有良好研究基础上，利用细胞生物学技术研究花粉败育阶段及细胞学特征，再用蛋白质组学及分子生物学等技术研究花粉败育相关基因类型及其调控花粉败育的途径。预期结果将有助于揭示菊花花粉败育深层机理和丰富植物生殖生物学的基础理论知识，更为重要的是将为培育散粉少和不散粉切花小菊新品种提供重要理论参考。因此，本研究具有重要的科学意义和应用前景。

在本项目中，我们首先利用细胞生物学技术对花粉败育切花小菊小孢子发生与花粉发育各个阶段进行了研究，发现了四分体小孢子时期是切花小菊花粉败育的关键时期，四分体小孢子败育伴随着绒毡层降解。在此基础上，以切花小菊小孢子四分体败育前后组织为对材料，开展了蛋白质组学和转录组学研究，获得了大量蛋白质组学和转录组学数据。经过生物信息学分析，发现花粉败育组织中半胱氨酸蛋白酶、天冬氨酸蛋白酶、metacaspase、CmACOS5、CmAMS、CmMS188等胞程序性死亡相关的蛋白或基因的表达量明显上调。克隆了菊花花粉败育基因CmACOS5以及其启动子，对基因表达特性进行了研究，构建了pORE_R4-LAT52::CmACOS5-GFP基因表达载体和pCrispr-CmACOS5/Cas9基因敲除载体，并开展了转基因研究。此外，我们建立了用于评价切花小菊花粉污染程度的花药开裂程度评价技术体系。最后，我们还开展了无花粉污染菊花育种研究，获得了一些无花粉污染菊花新种质。通过本研究，我们发现了四分体是花粉败育的关键阶段，花粉败育与绒毡层降解提前有关，克隆了花粉败育的关键基因，获得了一些无花粉污染菊花新种质。项目负责人以通讯作者在Plant Molecular Biology等期刊上发表了8篇论文（SCI论文5篇），培养了5名研究生，申请了2项国家发明专利，获得了1项国家技术发明奖二等奖。因此，本项目研究丰富了菊花花粉败育的理论知识，为培育无花粉污染菊花、乃至其他无花粉污染花卉(如百合)新品种奠定了重要基础。