Acquiring scanning electron microscopical, light microscopical and multiple gene sequence data from a single dinoflagellate cell
Acquiring scanning electron microscopical, light microscopical and multiple gene sequence data from a single dinoflagellate cell
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DOI：
10.1111/j.1529-8817.2006.00177.x
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发表时间：
2006-02-01
影响因子：
2.9
通讯作者：
Horiguchi, T
中科院分区：
文献类型：
--
作者：
Takano, Y;Horiguchi, T
我们已经开发了一种有用的方法来获得单个鞭毛细胞的光和扫描电子显微照片，在将细胞应用于单细胞PCR技术之前。这种方法使我们能够记录用于测序的任何细胞的详细形态信息，这对于生物体的未来鉴定非常重要，因为用于单细胞PCR的细胞通常无法保留。此外，通过同时应用多组PCR引物，我们已经成功地同时扩增和测序了多个基因（和DNA区域），甚至来自单个细胞。在本文中，我们通过使用两种不同类型的鞭毛藻来演示该技术的方法，即一种有甲的淡水物种Peridinium willei Huitfeld-Kaas，和一种无甲的海洋物种Akashiwo sanguinea (Hirasaka) Hansen和Moestrup。通过旋转细胞，即使使用扫描电镜也可以拍摄单个细胞的所有方面的照片。在这些例子中测序的基因和DNA区域包括核糖体DNA的一个区域（SSU、ITS1、5.8S、ITS2和LSU的一部分）以及线粒体DNA编码基因cox1的一部分。该技术可应用于光合和异养鞭毛藻，将加速生物多样性的研究。
We have developed a useful method to obtain light and scanning electron micrographs of a single dinoflagellate cell, prior to applying the cell to the single cell PCR technique. This method allows us to record detailed morphological information on any cell used for sequencing, which can be extremely important for the future identification of the organism, because cells used for single cell PCR usually cannot be retained. Furthermore, by applying multiple sets of PCR primers at the same time, we have successfully amplified and sequenced multiple genes (and DNA regions) simultaneously, even from a single cell. In this note, we demonstrate the methods of this technique by using two different types of dinoflagellates, i.e. an armored freshwater species, Peridinium willei Huitfeld-Kaas, and an unarmored marine species, Akashiwo sanguinea (Hirasaka) Hansen and Moestrup. By rotating the cell, photographs of all aspects of a single cell can be taken even using the SEM. The genes and DNA regions sequenced in these examples include a region of the ribosomal DNA (SSU, ITS1, 5.8S, ITS2, and part of the LSU) as well as part of the mitochondrial DNA-encoded gene, cox1. This technique can be applied to both photosynthetic and heterotrophic dinoflagellates and will accelerate biodiversity studies.