Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms.
The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.
Leblanc
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Laboratory of Molecular Plant Biology, Stazione Zoologica A. Dohrn, Naples, Italy.
Falciatore
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A
Watanabe
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Bowler
C
C
D000199:Actins / Q000235:genetics; D020418:Algal Proteins / Q000235:genetics; D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017377:Diatoms / Q000235:genetics / Q000528:radiation effects*; D005786:Gene Expression Regulation / Q000528:radiation effects*; D005796:Genes / Q000235:genetics; D008027:Light; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D000199:Actins; D020418:Algal Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D012333:RNA, Messenger; D004247:DNA