海带蓝光受体及相关基因的研究

IOCAS-IR > 海洋环流与波动重点实验室

	海带蓝光受体及相关基因的研究
其他题名	Study of blue light receptors and genes in Laminaria japonica Aresch
	姚建亭
学位类型	博士
	2008-06-09
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	海带 Rna提取蓝光受体肌醇-1-磷酸合成酶
摘要	本研究中运用四种不用的方法提取海带孢子体RNA，通过对比分析，确定了采用CTAB提取液[2% (w/v) CTAB,100 mM Tris-HC1 (pH 8.0), 50 mM EDTA, pH 7.5, 2 M NaCl, 50 mM DTT]提取RNA的方法，该方法的主要改进之处： (1) 高浓度（50 mM）的 DTT作为防止多酚氧化的还原剂加入提取液中；(2) 1/4 体积的乙醇和 1/9 体积的3 M 醋酸钾（pH 4.8）用来沉淀去除多糖；(3) 1/10倍体积的3 mol/L醋酸钠（pH 5.0）和两倍体积的乙醇选择性沉淀RNA；(4) -80℃沉淀30 min沉淀RNA。CTAB改进法提取的海带孢子体RNA质量好（A260/280 =1.96±0.05)，产量高（68 μg/g），分离的RNA可用于RT-PCR反应，扩增相关基因片段。该方法提取海带配子体RNA效果也很好。根据拟南芥(Arabidopsis thaliana)、莱茵衣藻(Chlamydomonas reinhardtii) 、铁线蕨(Adiantum capillus-veneris)、转板藻(Mougeotia scalaris)和无隔藻(Vaucheria frigida)等的蓝光受体蛋白保守序列，设计一系列简并引物，进行RT-PCR扩增，筛选并克隆测序了4条序列片段，通过比对分析，所得序列与已知的蓝光受体基因序列不一致。在所获得的序列中，一条783 bp的序列与肌醇-1-磷酸合成酶基因的片段有较高的相似性，通过RACE法克隆出该基因的3’末端，5’末端克隆没有成功，全长序列有待深入分析。
其他摘要	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 (68 μg/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, afragment 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.
页数	119
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1033
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	姚建亭. 海带蓝光受体及相关基因的研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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