FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei
FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei
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DOI：
10.1186/1746-4811-8-44
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发表时间：
2012-10-17
期刊：
影响因子：
5.1
通讯作者：
Becker, Joerg D.
中科院分区：
文献类型：
--
作者：
Borges, Filipe;Gardner, Rui;Becker, Joerg D.
背景资料：开花植物中的雄性生殖系通过单倍体单核小孢子的不对称分裂来分化，产生包围较小生殖细胞的营养细胞，其最终经历第二次有丝分裂以产生两个精细胞。营养细胞和精细胞激活不同的遗传和表观遗传机制，分别控制花粉管生长和生殖细胞特化。因此，这些过程的全面表征依赖于有效的方法来分离每个不同的细胞类型在整个male gametogenesy.Results：我们开发了稳定的转基因拟南芥系和可靠的纯化工具的基础上，荧光激活细胞分选（FACS），以分离高纯度和可行的馏分的每个细胞/细胞核类型之前和之后的花粉有丝分裂。在成熟花粉的情况下，这是通过在精子和营养核中分别表达GFP和RFP来实现的，从而产生99%纯的分选群体。利用其特征的小尺寸和自发荧光特性的小孢子，也通过FACS纯化，并被确认为98% pure.Conclusions：我们提供了简单而有效的基于FACS的拟南芥小孢子，营养核和精子细胞的纯化方案。这为随后的分子分析铺平了道路，如转录组学，DNA甲基化分析和染色质免疫沉淀，在拟南芥小配子发生的发育背景下。
Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis.Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure.Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.