Takayama pulchellum原位增长率测定的研究
STUDY ON DETECTION OF IN SITU GROWTH RATE OF TAKAYAMA PULCHELLUM
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摘要: 现今用于测定有害藻华生物原位增长率的方法很难在现场进行有效地应用,因此我们尝试采用基于rRNA、叶绿素和总蛋白质浓度的定量测定方法来估算其细胞增长率。本研究重点对Takayama pulchellum细胞株(TPXM)的rRNA含量和特定生长率的关系进行了探索。用荧光标记的肽核酸探针结合全细胞杂交方法,结合流式细胞仪和专业图像分析软件对T.pulchellum单细胞水平的rRNA进行了定量检测。结果表明不同生长阶段的T.pulchellum单细胞rRNA水平与其相应的细胞特定生长率有较好的相关关系(R2=0.7293,p0.01,n=14),单细胞蛋白质含量r、RNA水平与同步后的细胞周期变化情况也有较好的相关关系(R2=0.6984,p0.01,n=14)。上述结果提示单细胞rRNA含量可作为指示细胞周期变化和细胞特定生长率的较好指标。
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关键词:
- Takayama pulchellum /
- 肽核酸探针 /
- rRNA /
- 流式细胞仪 /
- 原位增长率
Abstract: Harmful algal blooms are getting more and more serious problems in the water ecosystem in the world than before, for its blooming areas are expanding in many places, such as freshwater and coastal water.It is important to measure the in situ growth rate to understand the dynamics of harmful algal blooms in the field, because the temporal change of algal species abundance at a fixed point can be affected by many factors such as growth (cell division), grazing mortality caused by zooplankton, sedimentation , parasite or virus infections, autolysis due to unfavourable growth conditions, etc.Moreover, cell abundance is affected by concentration or dilution due to physical processes, such as physical advection and diffusion.The abundance of a certain phytoplankton species in natural systems is a result of a large array of processes.To investigate causal relationships behind algal bloom proliferation and decline, growth rate must be measured independently of cell abundance and different loss processes.There are several methods available for estimating phytoplankton growth.The usual technique to date makes use of specific cell cycle markers such as cellular DNA content or presence of cell cycle-related proteins.The combination of growth rate measured using cell cycle analysis and the net change in cell abundancemeasured by microscopy or flow cytometry can provide both growth and loss rates for the studied species.This approach has the potential to allow the separation of population changes directly caused by factors affecting HAB growth (cell division).The PNA (Peptide nucleic acid)probe is an artificial synthetic DNA analogs, in which the sugar phosphate backbone of the DNA helix is replaced with an uncharged structurally homomorphous pseudopeptide backbone.The synthetic backbone provides PNA probes with unique hybridization characteristics such as more rapid and stronger binding to complementary targets according to the Watson and Crick basepairing rules.Takayama pulchellum was the first recorded in Japan as an ichthyotoxic dinoflagellate.It was initially described as Gymnodinium pulchellum, but recently renamed as T.pulchellum.T.pulchellum formed HABs in America and Australia, causing large numbers of fish kills, and in the recent years it blooms occurred in Xiamen Bay.The specific growth rate estimated by PNA probe in this study to measure the in situ potential growth rate of T.pulchellum is very important.Because currently available approaches for estimating harmful algal bloom species' growth rates can be difficult to apply in the field,we have been exploring the feasibility of using quantitative rRNA by peptide nucleic acid, chl a and protein concentration measurements as the basis for making such estimates.In this study, we examined the relationship between rRNA and growth rate of T.pulchellum strains (TPXM).Whole-cell hybridization with fluorescence labeled peptide nucleic acid (PNA)probe was used in conjunction with flow cytometry and the professional image analysis software to quantify rRNA on a per cell basis.The result showed that cellular rRNA levels in different growth periods were well correlated with its corresponding specific growth rates (R2=0.7293, p<0.01, n=14);cellular protein concentration, cellular rRNA level and the cell cycle also presented good correlation (R2=0.6984, p <0.01, n=14).All the results suggested that cellular rRNA level was a good indicator to analyze cell cycle and cell growth rate of T.pulchellum.-
Keywords:
- Takayama pulchellum /
- Peptide nucleic acid probe /
- RNA /
- Flow cytometry /
- In situ growth rate
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