A method to isolate male gametic nuclei from pear pollen tubes
A method to isolate male gametic nuclei from pear pollen tubes
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一种从梨花粉管中分离雄配子核的方法
DOI：
10.1080/14620316.2013.11512971
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发表时间：
2013-01
期刊：
影响因子：
--
通讯作者：
Zhang, S. L.
中科院分区：
文献类型：
--
作者：
Gao, Y. B.;Wang, C. L.;Wu, J. Y.;Wu, J.;Zhou, H. S.;Zhang, S. L.
细胞核在调节大多数生物体的大多数生物活动中起着至关重要的作用。梨（Pyrus bretshneideri）花粉为双核，具有营养核和生殖核。为了揭示核蛋白质组，并使用荧光原位杂交（FISH）分析产生准确的DNA序列的物理图谱，它是必不可少的，以获得高质量的细胞核。分离完整细胞核的方法通常包括将它们从离心细胞中分离出来，然后用尼龙网过滤。释放细胞核是从少量组织中获得高质量材料的最关键步骤。本文介绍了一种简便、有效的梨花粉管生殖核和营养核的分离纯化方法。通过研磨花粉管释放细胞核，在孔径为40 µm的细胞过滤器上过滤，然后使其通过第二个孔径为25 µm的细胞过滤器。在这些程序和通过28%（v/v）PercollTM梯度离心纯化后，分离的细胞核表现出高水平的纯度和完整性，几乎没有碎片，如使用酶标记和4 '，6-二脒基-2-苯基吲哚（DAPI）染色所证实的。纯化的细胞核用于制备核蛋白和DNA纤维。本协议证明适合于男性配子核的分离，在体外培养的梨花粉管，核蛋白质组分析，并为FISH分析的DNA纤维的制备。
Summary The nucleus plays an essential role in regulating most biological activities in most living organisms. Pear (Pyrus bretshneideri) pollen is binucleate, possessing a vegetative nucleus and a generative nucleus. To reveal the nuclear proteome and to produce accurate physical maps of DNA sequences using fluorescence in situ hybridisation (FISH) analysis, it is essential to obtain high quality nuclei. Protocols to isolate intact nuclei usually involve liberating them from centrifuged cells, then filtering them using a nylon mesh. Releasing the nuclei is the most critical step to obtain high quality material from small amounts of tissue. Here, we describe an easy and efficient method to isolate and purify both the generative and vegetative nuclei from pear pollen tubes. The nuclei were liberated by grinding pollen tubes, filtered on a cell strainer with a pore size of 40 µm, then passing them through a second cell strainer with a mesh size of 25 µm. After these procedures, and purification through a 28% (v/v) PercollTM gradient by centrifugation, the isolated nuclei exhibited a high level of purity and integrity, with little debris, as confirmed using enzyme markers and 4’,6-diamidino-2-phenylindole (DAPI) staining. The purified nuclei were used to prepare nuclear proteins and DNA fibres. The present protocol proved suitable for the isolation of male gametic nuclei from in vitro-cultivated pear pollen tubes, for nuclear proteome analysis, and for the preparation of DNA fibres for FISH analysis.
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1080/14620316.2011.11512768
发表时间：
2011-01
期刊：
Journal of Horticultural Science & Biotechnology
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