Analyzing meiosis in barley.
Analyzing meiosis in barley.
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DOI：
10.1007/978-1-62703-333-6_14
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发表时间：
2013-01-01
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影响因子：
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通讯作者：
Higgins, James D
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作者：
Higgins, James D
本章详细描述了为确定大麦减数分裂过程中交叉控制所涉及的因素而开发的免疫学和细胞学技术。免疫学技术包括消化新鲜花药，然后进行染色体扩散，以去除导致不需要的背景的细胞质，同时保护脆弱的染色体结构免受损害。然后将针对减数分裂蛋白的特异性抗体与细胞核一起孵育，用与荧光染料缀合的二抗进行检测，并用宽视野或共聚焦显微镜进行可视化。在细胞学技术中，大麦花序被固定，然后解剖花药，消化细胞壁，然后传播减数分裂染色体。这两种技术都可以与特定的 DNA 探针结合使用，进行荧光原位杂交 (FISH) 来标记端粒、着丝粒和核糖体 DNA；识别 DNA 修饰，例如 5-甲基胞嘧啶；并检测 DNA 碱基类似物的掺入，例如 5-溴-2'-脱氧尿苷 (BrdU) 或 5-乙炔基-2'-脱氧尿苷 (EdU)，用于减数分裂时间进程或分析新合成的 DNA。尽管这些技术是专门为大麦开发的，但它们应该可以直接转移到其他谷类作物物种，例如小麦和水稻。
This chapter contains a detailed description of the immunological and cytological techniques developed to determine factors involved in crossover control during meiosis in barley. The immunological technique involves digesting fresh anthers followed by chromosome spreading to remove cytoplasm that causes unwanted background, whilst protecting the fragile chromosome structure from being compromised. Specific antibodies raised against meiotic proteins are then incubated with nuclei, detected with secondary antibodies conjugated to fluorescent dyes, and visualized with either wide-field or confocal microscopes. In the cytological technique, barley inflorescences are fixed, followed by dissecting out the anthers, digesting the cell walls and then spreading the meiotic chromosomes. Both techniques can be used in conjunction with specific DNA probes for fluorescent in situ hybridization (FISH) to label telomeres, centromeres, and ribosomal DNA; to identify DNA modifications such as 5-methylcytosine; and to detect the incorporation of DNA base analogues such as 5-bromo-2'-deoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU) to be used for a meiotic time-course or assaying newly synthesized DNA. Although these techniques have been specifically developed for barley, they should be directly transferable to other cereal crop species such as wheat and rice.