RNA/protein and RNA/DNA ratios determined by flow cytometry and their relationship to growth limitation of selected planktonic algae in culture
RNA/protein and RNA/DNA ratios determined by flow cytometry and their relationship to growth limitation of selected planktonic algae in culture
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DOI：
10.1080/09670260802578518
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发表时间：
2009-08
影响因子：
2.4
通讯作者：
A. Nicklisch;C. Steinberg
中科院分区：
文献类型：
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作者：
A. Nicklisch;C. Steinberg
RNA/蛋白质和RNA/DNA比率与许多生物体的生长速率密切相关。然而，这还没有被证明为浮游藻类，并可能是一种新的方法来确定原位生长速率的基础。因此，微小冠盘藻、无刺圆冠藻（硅藻纲）、衣原体属、通过改变日照长度、辐照度和/或营养物，使真核微藻（绿藻纲）和微小隐藻（隐藻纲）在限制下生长。使用驯化的半连续培养物测定比生长速率。将在准稳态下生长的此类培养物的细胞用戊二醛固定以保存色素自发荧光，并将固定样品的平行样品分别用SYTO RNASelect、PicoGreen或荧光素-5-ex-succinimidylester对RNA、DNA和蛋白质进行染色。使用蓝色和绿色激光激发，通过流式细胞术（FACStar PLUS）分析光合色素的染色和自发荧光。所确定的RNA/蛋白质和RNA/DNA的比例与特定的增长率的物种，无论是一种限制，但不同的物种的不同分类组之间的相关性。限制的种类和强度导致不同的色素自发荧光模式，这可以很容易地通过流式细胞术进行评估。因此，RNA，DNA，蛋白质和色素的自发荧光的测定的浮游藻类提供的信息的生长和限制状态，这提供了一个基础，即使在湖泊样品的未来评估。
RNA/protein and RNA/DNA ratios correlate closely to the growth rate for many organisms. However, this has not yet been shown for planktonic algae and could be a basis for a new approach to determine in situ growth rates. Therefore, cultures of Stephanodiscus minutulus, Cyclostephanos invisitatus (Bacillariophyceae), Chlamydomonas sp., a eukaryotic pico-alga, (Chlorophyceae) and Rhodomonas minuta (Cryptophyceae) were grown under limitation by altering daylength, irradiance, and/or nutrients. Specific growth rates were determined using acclimated semi-continuous cultures. Cells of such cultures growing in a quasi-steady state were fixed with glutaraldehyde to conserve the pigment autofluorescence and parallels of the fixed samples were stained by SYTO RNASelect, PicoGreen, or fluorescein-5-ex-succinimidylester for RNA, DNA, and protein, respectively. The staining and the autofluorescence of photosynthetic pigments were analysed by flow cytometry (FACStar PLUS) using blue and green lasers for excitation. The determined ratios of RNA/protein and RNA/DNA correlated well with the specific growth rates of the species regardless of the kind of limitation, but differed between the different taxonomic groups of the species. The kind and strength of limitation resulted in different patterns of pigment autofluorescence, which can easily be evaluated by flow cytometry. Hence, the determination of RNA, DNA, protein, and pigment autofluorescence of planktonic algae delivers information on growth and limitation state which provides a basis for future assessment even in lake samples.