Study Of Blue Light Receptors And Genes In Laminaria Japonica Aresch

Based on the four methods for extracting RNA, an improved protocol was applied for Laminaria japonica sporophytes. A cetyltrimethylammonium bromide (CTAB)-based [2% (w/v) CTAB,100 mM Tris-HC1 (pH 8.0), 50 mM EDTA, pH 7.5, 2 M NaCl, 50 mM DTT] RNA extraction protocol was developed. The principal modifications of the protocol are: (1) high concentrations of dithiothreitol (50 mM) was applied in the extraction buffer for preventing oxidative cross-linking of nucleic acids caused by phenolics; (2) 1/4 vol of ethanol and 1/9 vol of 3 M potassium acetate (pH 4.8) were applied to precipitate polysaccharides; (3) using 1/10 vol of 3 M sodium acetate (pH 5.0) and 2 vol of ethanol precipitates RNA selectively; (4) RNA precipitation is complete in 30 min at–80℃. The CTAB-based RNA extraction protocol could yield RNA with high quality (A260/280 ratio 1.96±0.05) and high yield (68μg/g fresh weight). The isolated RNA could be used for RT-PCR to amplified some gene fragment. Besides, this method could either be used for L. japonica gametophytes RNA extraction.To target the genes of blue-light receptor to L. japonica, many degenerate primers was designed according to the cDNA and protein sequences of cryptochrome or phototropin from Arabidopsis thaliana, Chlamydomonas reinhardtii, Adiantum capillus-veneris, Mougeotia scalaris and Vaucheria frigid. RT-PCR were amplified with the primers. Four fragments were selected and cloned successfully. Sequence analysis indicated that these were not consistent with the desire sequence. Besides, a fragment of 783 bp was amplified and cloned. Sequence analysis indicated that it exihibited a high similarities with Myo inositol-1-phosphate synthase mRNA sequence. 3'-RACE of the gene was successful conducted, but 5'-RACE test was not yet finished. Further study is necessary to be carried out.