根据以往试验获得的CsE1α的cDNA全长序列,利用TAIL-PCR克隆CsE1α启动子。测序验证与生物信息学分析后发现,该启动子片段长336βbp,含有2个CAAT-box,2个TATA-box,2个GATA-box,1个LTR,1个G-box等顺式作用元件。构建载体转入洋葱内表皮细胞瞬时表达,启动子可启动下游报告基因,使荧光蛋白表达于整个细胞,表明所克隆的启动子具有启动功能。CsE1α与GFP融合蛋白瞬时表达表明CsE1α定位于线粒体。本实验为下一步转基因拟南芥稳定表达,进一步研究CsE1α基因的表达调控,探讨茶树花粉抗寒的分子生物学机理奠定基础。
In this paper, full-length cDNA is identified for designing the gene-special primers in TAIL-PCR to clone the promoter of CsE1α. By sequencing and bioinformatic analyzing, we observed two CAAT-box, two TATA-box, two GATA-box, one LTR and one G-box located in the 336βbp promoter region. After constructing and transferring the transient expression vectors into the onion epidermal cells, subcellular localization of CsE1α-GFP fusion proteins is verified in mitochondria, while the promoter can activate downstream gene expressing in entire cell. This experiment may provide useful information for the subsequent stable expression in transgenic Arabidopsis and the further investigation on the expression and regulation of CsE1α gene as well as the investigation on the molecular mechanism of cold resistance in pollen of Camellia sinensis.
[1] 张振宇, 侯祥红, 孟姗姗, 等. 丙酮酸对细胞寒冷应激损伤的保护作用研究[J]. 现代生物医学进展, 2013(20): 3835-3839.
[2] Reed L J.Multienzyme complexes[J]. Accounts of Chemical Research, 1974, 7(2): 40-46.
[3] Xia Z, Watanabe S, Yamada T, et al. Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering[J]. Proceedings of the National Academy of Sciences, 2012, 109(32): E2155-E2164.
[4] Ciszak E M, Korotchkina L G, Dominiak P M, et al. Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase[J]. Journal of Biological Chemistry, 2003, 278(23): 21240-21246.
[5] 张龙雨, 袁蕾, 杨书玲, 等. 小麦雄性不育系中 TaPDC-E1α 及其调节酶基因的表达特征[J]. 作物学报, 2011, 37(4): 620-628.
[6] Yui R, Iketani S, Mikami T, et al. Atisense inhibition of mitochondrial pyruvate dehydrogenase E1[alpha] subunit in anther tapetum causes male sterility[J]. Plant Journal, 2003, 34: 57-66.
[7] Brown RM, Brown GK.Prenatal diagnosis of pyruvate dehydrogenase E1 alpha subunit deficiency[J]. Prenatal Diagnosis, 1994, 14(6): 435-441.
[8] Lissens W, De Meirleir L, Seneca S, et al. Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency[J]. Human Mutation, 2000, 15(3): 209-219.
[9] Pliss L, Mazurchuk R, Spernyak J A, et al. Brain MR imaging and proton MR spectroscopy in female mice with pyruvate dehydrogenase complex deficiency[J]. Neurochemical research, 2007, 32(4/5): 645-654.
[10] 单雷, 唐桂英, 徐平丽, 等. 花生质体型酰基载体蛋白基因5′侧翼调控序列的克隆与分析[J]. 作物学报, 2014, 40(3): 381-389.
[11] 聂丽娜, 夏兰琴, 徐兆师, 等. 植物基因启动子的克隆及其功能研究进展[J]. 植物遗传资源学报, 2008, 9(3): 385-391.
[12] 王伟东, 蒋芯, 杜昱林, 等. 低温对茶树花粉管抑制作用与NO关系的研究[J]. 园艺学报, 2013, 40(8): 1535-1540.
[13] Paterson A H, Brubaker C L, Wendel J F.A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis[J]. Plant Molecular Biology Reporter, 1993, 11(2): 122-127.
[14] Menkens A E, Schindler U, Cashmore A R.The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins[J]. Trends in biochemical sciences, 1995, 20(12): 506-510.
[15] Reyes J C, Muro-Pastor M I, Florencio F J. The GATA family of transcription factors in Arabidopsis and rice[J]. Plant Physiology, 2004, 134(4): 1718-1732.
[16] Buchel A S, Brederode F T, Bol J F, et al. Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo[J]. Plant molecular biology, 1999, 40(3): 387-396.
[17] Dunn M A, White A J, Vural S, et al. Identification of promoter elements in a low-temperature-responsive gene from barley[J]. Plant Mol Biol, 1998, 38: 551-564.
[18] Bao Y, Dharmawardhana P, Strauss S H, et al. WUS and STM-based reporter genes for studying meristem development in poplar[J]. Plant cell Reports, 2009, 28(6): 947-962.
[19] Pastuglia M, Roby D, Dumas C, et al. Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea[J]. The Plant cell, 1997, 9(1): 49-60.
[20] 岳川, 曾建明, 章志芳, 等. 茶树中植物激素研究进展[J]. 茶叶科学, 2012, 32(5): 382-392.
[21] de Kok A, Hengeveld A F, Martin A, et al. The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria [J]. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1998, 1385(2): 353-366.
[22] Mooney B P, Miernyk J A, Randall D D.The complex fate of α-ketoacids[J]. Annual review of plant biology, 2002, 53(1): 357-375.
[23] Lernmark U, Gardestrom P.Distribution of pyruvate dehydrogenase complex activities between chloroplasts and mitochondria from leaves of different species[J]. Plant physiology, 1994, 106(4): 1633-1638.
[24] Camp P J, Randall D D.Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex: A source of Acetyl-CoA and NADH for Fatty Acid biosynthesis[J]. Plant physiology, 1985, 77(3): 571-577.
[25] Smith R G, Gauthier D A, Dennis D T, et al. Malate-and pyruvate-dependent fatty acid synthesis in leucoplasts from developing castor endosperm[J]. Plant Physiology, 1992, 98(4): 1233-1238.
浙公网安备 33019902000101号 
