Molecular Ecology of Marine Phytoplankton:A Practical Procedure

摘要

Molecular biological techniques are useful tools for addressing some important ecological questions for marine phytoplankton. However, the application of these techniques in this field is rather scattered and no well-established procedures are available. We have attempted to develop such a procedure with which a biological oceanograph-ic process can be studied using a key gene or protein. In our laboratory, reverse-transcription coupled with poly-merase chain reaction (RT-PCR) is used to amplify genes regulating the cell cycle and growth. After cloning and sequencing, the gene sequences obtained are used to design an antigen for antibody production. The antibodies are then tested for their specificity using Western blotting. Subsequently, immunocytochemical analysis is performed using these antibodies. The immunostaining can then be quantified and analyzed mathematically or statistically for ecological interpretation. With an immunofluorescence technique and the antibodies, we have been able to study phytoplankton growth rates and ecological responses to changing growth conditions, the genes and proteins we studied with this approach include proliferating cell nuclear antigen (PCNA), the cyclin dependent kinase p34cdc2, cyclin box, the carbon dioxide-fixing enzyme ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco), the electron transporting flavoprotein flavodoxin, and the nitrogen fixing enzyme nitrogenase. Some of the results from these studies will be presented to demonstrate the usefulness of this molecular ecological approach.