采用SMART-RACE技术克隆了茶树Dof（DNA binding with one finger）基因CsCDF1的全长cDNA序列,并利用在线生物信息学软件对其进行了分析。采用实时荧光定量PCR分析该Dof基因在茶树不同组织间的表达差异和昼夜表达规律,及其在氮饥饿处理2周后对不同浓度氮素诱导的响应。该cDNA序列全长1β606βbp,包含1个编码464个氨基酸的完整开放阅读框,含有高度保守的DOF结构域,推导的蛋白分子量为50.8βkDa,理论等电点（PI）为5.52;其编码的蛋白序列与茸毛烟草、马铃薯和美花烟草的CDF蛋白（Cycling dof factor）相似性分别为69%、67%、68%。系统发育分析结果表明,该基因编码的氨基酸序列与拟南芥CDF蛋白聚为一类,因此将其命名为CsCDF1;CsCDF1在3个不同茶树品种根系中的表达量均高于一芽二叶和成熟叶;在一天中,该基因的表达呈现出昼夜节律变化;在氮饥饿后重新供氮,不同组织中该基因对不同浓度氮素的响应均为上调。
The full-length cDNA of the first Dof gene (CsCDF1) was cloned from tea plant [Camellia sinensis (L.) O. Kuntze] by SMART-RACE cloning technology, and the bioinformatic analysis of the cloned gene were conducted by using online service. The expression profile of this gene in various tissues and with diurnal rhythm as well as in response to different dosage nitrogen treatment were investigated by using real-time fluorescent quantitative RT- PCR. The obtained cDNA sequence was 1β606 bp and contained a complete open reading frame encoding 464 amino acid residues with highly conserved DOF domain. The molecular weight and theoretical isoelectric point (PI) is 50.8βkDa and 5.52 respectively. The analysis by utilizing the BLAST software showed that the derived protein sequences shared separately 69%, 67% and 68% similarity with CDF (Cycling dof factor) proteins in Nicotiana to-mentosiformis, Solanum tuberosum and Nicotiana sylvestris. Phylogenetic analysis showed that the protein encoded by this gene was clustered into the same clade with Arabidopsis thaliana CDF, so it was named as CsCDF1. The expression of CsCDF1 dominated in roots rather than in one bud and two leaves or mature leaves in three phenotypes. During one day, CsCDF1 gene changed with the diurnal rhythm, and resupplying nitrogen after starvation for 2 weeks, the expression in response to different concentration of nitrogen was all raised in different tissues.
[1] Skirycz A, Reichelt M, Bu row M, et al. DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosino late biosynthesis in Arabidopsis[J]. Plant Journal, 2006, 47(1): 10-24.
[2] Yanagisawa S.A novel DNA-binding domain that may form a single zinc finger motif[J]. Nucleic Acids Research, 1995, 23(17): 3403-3410.
[3] Yanagisawa S.The Dof family of plant transcription factors[J]. Trends Plant Science, 2002, 7(12): 555-560.
[4] Kang HG, Singh KB.Characterization of salicylic acid-responsive Arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects[J]. Plant Journal, 2000, 21(4): 329-339.
[5] Diaz I, Vicente-Carbajosa J, Abraham Z, et al. The GAMYB protein from barley interacts with the DOF transcription factor BPBF and activates endosperm-specific genes during seed development[J]. Plant Journal, 2002, 29(4): 453-464.
[6] Yanagisawa S.Dof domain proteins: plant-specific transcription factors associated with diverse phenomena unique to plant[J]. Plant Cell Physiology, 2004, 45(4): 386-391.
[7] Yanagisawa S.The Dof family of plant transcription factors[J]. Trends Plant Science, 2002, 7(12): 555-560.
[8] Yanagisawa S, Izui K.Molecular cloning of two DNA-binding proteins of maize that are structurally different but interact with the same sequence motif[J]. Journal of Biological Chemistry, 1993, 268(21): 16028-16036.
[9] Lijavetzky D, Carbonero P, Vicente-Carbajosa J.et al. Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families[J]. BMC Evolutionary Biology, 2003, 3(1): 17.
[10] Chen R, Ni Z, Qin Y, et al. Isolation and characterization of TaDof1 transcription factor in wheat (Triticum aestivum L)[J]. DNA Seqence, 2005, 16(5): 358-363.
[11] Yang X, Tuskan G A, Cheng M Z, et al. Divergence of the Dof gene families in poplar Arabidopsis and rice suggests multiple modes of gene evolution after duplication[J]. Plant Physiology, 2006, 142(3): 820-830.
[12] Moreno-Risueno M A, Martinez M, Vicente-Carbajosa J, et al. The family of DOF transcription factors: from green unicellular algae to vascular plants [J]. Molecular. Genetics Genomics, 2007, 277(4): 379-390.
[13] Kushwaha H, Gupta S, Singh V K, et al. Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis[J]. Molecular Biology Report, 2011, 38(8): 5037-5053.
[14] Guo Y, Qiu L J.Genome-Wide Analysis of the Dof Transcription Factor Gene Family Reveals Soybean-Specific Duplicable and Functional Characteristics[J]. Plos One, 2013, 8(9): e76809.
[15] Yanagisawa S.Dof domain proteins: plant-specific transcription factors associated with diverse phenomena unique to plants[J]. Plant Cell Physiology, 2004, 45(4): 386-391.
[16] Park D H, Lim P O, Kim J S, et al. The Arabidopsis COG1 gene encodes a Dof domain transcriptionfactor and negatively regulates phytochrome signaling[J]. Plant Journal, 2003, 34(2): 161-171.
[17] Diaz I, Vicente-Carbajosa J, Abraham Z, et al. The GAMYB protein from barley interacts with the DOF transcription factor BPBF and activates endosperm-specific genes during seed development[J]. Plant Journal, 2002, 29(4): 453-464.
[18] Gutiérrez R A, Stokes T L, Thum K, et al. Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1[J]. Proceedings of the National Academy of Sciences, 2008, 105(12): 4939-4944.
[19] Imaizumi T, Schultz T F, Harmon F G, et al. KFK1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J]. Science, 2005, 309(5732): 293-297.
[20] Fornara F, Panigrahi K C S, Gissot L, et al. Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response[J]. Developmental cell, 2009, 17(1): 75-86.
[21] Wang H W, Zhang B, Hao Y J, et al. The soybean Dof-type transcription factorgenes GmDof4 and GmDof11 enhance lipid content in the seeds of transgenic Arabidopsis plants[J]. Plant Journal, 2007, 52(4): 716-729.
[22] Iwamoto M, Higo K, Takano M, et al. Circadian clock- and phytochrome-regulated Dof-like gene, Rdd1, is associated with grain size in rice[J]. Plant, cell & environment, 2009, 32(5): 592-603.
[23] Yang J, Yang M F, Zhang W P, et al. A putative flowering time-related Dof tran-scription factor gene, JcDof3, is controlled by the circadian clock in Jatropha curcas[J]. Plant Science, 2011,181(6): 667-674.
[24] Foyer C H, Noctor G.Photosynthetic nitrogen assimilation and associated carbon and respiratory metabolism[M]// Foyer C H, Noctor G. Advances in Photosynthesis Research. Dordrecht: Kluwer Academic Publishers, 2002: 1-22.
[25] Kumar A, Kanwal P, Gupta A K, et al. A full-length Dof1 transcription factor of finger millet and its response to a circadian cycle[J]. Plant Molecular Biology Report, 2014, 32(2): 419-427.
浙公网安备 33019902000101号 
