拟南芥转录因子CBF2的克隆与生物信息学分析

doi:10.11924/j.issn.1000-6850.2013-1118

摘要/Abstract

摘要： 本研究旨在克隆转录因子CBF2并对其进行生物信息学分析得到该基因相关信息。试验以拟南芥基因组DNA为模板，根据GenBank中拟南芥转录因子CBF2(FJ491243.1)已知序列设计引物，回收扩增的目的片段与T-easy载体连接后转入大肠杆菌(E.coli DH5α)，经蓝白斑筛选后提取重组体的质粒，经滞后筛选、PCR、酶切鉴定无误后进行测序。经生物信息学分析，该基因全长651 bp，其中编码区长648 bp，编码216个氨基酸，其蛋白的分子式为C₁₀₅₆H₁₆₂₃N₂₉₃O₃₃₃S₁₆，相对分子质量为24.26 kDa，等电点为5.00，该蛋白为亲水性非分泌型蛋白，无信号肽存在；其蛋白质二级结构以α-螺旋、β-折叠和无规卷曲为主。肽链可能含有9处丝氨酸磷酸化位点，6处苏氨酸磷酸化位点，以及2处酪氨酸磷酸化位点。本研究通过对CBF2基因的生物信息学分析，获得了该基因的相关信息，并与已有研究结果进行比对分析，推测该基因在植物抗逆过程中有重要的作用。

关键词: 可支配收入, 可支配收入

Abstract: The purpose of this study was to clone the transcription factor CBF2 and analysis of the gene related information of the bioinformatics. Arabidopsis genomic DNA was used as templates, and primers were designed according to the Arabidopsis transcription factor CBF2 (FJ491243.1) of known sequence. The recycling amplified fragments were connected with T-easy vector, and then transformed into E. coli DH5α. The recombinant plasmid after blue-white screening was sequenced after lag selection, PCR, restriction enzyme digestion. With the bioinformatics analysis, the cloned CBF2 gene was 651 bp including code segment 648 bp, encoding 216 amino acids, the predicted CBF2 protein’s molecular formula was C₁₀₅₆H₁₆₂₃N₂₉₃O₃₃₃S₁₆, whose relative molecular mass was 24.26 kDa and isoelectric point was 5.00. It was predicted that this protein was hydrophilic and non-secretary protein, and there was no signal peptide. Characteristic prediction results indicated that the secondary structure of CBF2 protein mainly includedα-helix,β-sheet and random coil. And there were night serine sites, six threonine sites, and two tyrosine sites. In this study, related information was obtained by means of bioinformatics analysis of CBF2 gene, and through comparing with previous studies, the gene played an important role in the plant resistance.

申鹏毛娟刘爱平陈佰鸿. 拟南芥转录因子CBF2的克隆与生物信息学分析[J]. 中国农学通报, 2014, 30(4): 90-95.

参考文献

[1] 左天觉,朱尊权.烟草的生产、生理与生物化学[M].上海:上海远东出版社,1993:50-52,60-61.

[2] 中国农业科学院烟草研究所.中国烟草栽培学[M].上海:上海科学技术出版社,2005:113-118,299-318.

[3] 崔红,冀浩,张华,等.不同生态区烟草叶片蛋白质组学的比较[J].生态学报,2008,28(10):4873-4880.

[4] 李晔,王潮中,赵秀香,等.铁营养对烟草幼苗生长及生理生化指标的影响[J].中国农学通报,2006,22(9):213-215.

[5] 黄中艳,王树会,朱勇,等.云南烤烟5项化学成分含量与其生态环境要素的关系[J].中国农业气象,2007,28(3):312-317.

[6] 戴冕.我国主产烟区若干气象因素与烟叶化学成分的关系研究[J].中国烟草学报,2000,6(1):27-34.

[7] 肖金香,刘正和,王燕,等.气候生态因素对烤烟产量与品质的影响及植烟措施研究[J].中国生态农业学报,2003,11(4):158-160.

[8] 韦成才,马英明,艾绥龙,等.陕南烤烟质量与气候关系研究[J].中国烟草科学,2004(3):38-41.

[9] 王彪,李天福.气象因子与烟叶化学成分关联度分析[J].云南农业大学学报,2005(5):742-745.

[10] 黎妍妍,许自成,王金平,等.湖南烟区气候因素分析及对烟叶化学成分的影响[J].中国农业气象,2007,28(3):308-311.

[11] 陈伟,王三根,唐远驹,等.不同烟区烤烟化学成分的主导气候影响因子分析[J].植物营养与肥料学报,2008,14(l):144-150.

[12] 许自成,王林,王金平,等.湖南烤烟化学成分与土壤有机质含量的关系[J].生态学杂志,2006,25(10):1186-1190.

[13] 陈杰,唐远驹.影响贵州烟叶化学成分的土壤养分因素分析[J].作物杂志,2008(1):68-71.

[14] 阎克玉,李兴波,屈剑波,等.河南烤烟(40级)水溶性总糖和还原糖含量及其相关性研究[J].郑州轻工业学院学报,1997,12(4):42-47.

[15] 李冰凌,唐香玉,代杰.烟叶中的糖类与烟叶质量[J].黑龙江烟草, 1997,(11):19-24.

[16] 中国土壤学会.土壤农业化学分析方法[M].北京:中国农业科技出版社,2000:12-13,30-33,50-58.

[17] 南京农业大学.土壤农化分析[M].北京:农业出版社,1994:55-56, 73-74,89-90,218-219.

[18] 李东亮.基于化学成分的烟草质量评价方法研究与应用[D].河南:河南农业大学,2008.

[19] 雷志栋,扬诗秀,许志荣,等.土壤特性空间变异性初步研究[J].水利学报,1985(9):10-

[1] Gilmour S J, Zarka D G, Stockinger E J, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activation as an early step cold-induced COR gene expression[ J]. The Plant Journal,1998,16(4):436.

[2] 张晗,信月芝,郭惠明,等.CBF转录因子及其在植物抗冷反应中的作用[J].核农学报,2006,20(5):406-409,448.

[3] Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J]. Proceedings of the National Academy of Sciences of USA, 1997,94:1035-1040.

[4] Gilmour S J, Zarka D G, Stockinger E J, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activator as an early step in cold–induced COR gene expression[J]. Plant J,1998,16(4):433-442.

[5] Jaglo-Ottosen K R, Gilmour S J, Zarka D G, et al. Arabidopsis CBF1 over expression induces COR genes and enhances freezing tolerance[J].Science, 1998,280(5360):104-106.

[6] Haake V, Cook D, Riechmann J L, et al. Transcription factor CBF4 is a regulation of drought adaptation in Arabidopsis[J].Plant Physiology,2002,130(2):639-648.

[7] Heish T H, Lee J T, Yang P T, et al. Heterology expression of the Arabidopsis C-Repeat/Dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato [J]. Plant Physiology, 2002(129):1086-1094.

[8] Liu Q, Kasuga M, Sakuma Y, et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pass ways in drought and low temperature-responsive gene expression, respectively, in Arabidopsis [J]. Plant Cell, 1998(10):1391-1406.

[9] Jaglo K R, Kleff S, Amundsen K L, et al. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species [J]. Plant Physiology, 2001,127(3):910-917.

[10] 沙丽娜,郭兆奎,万秀清,等.转录因子 CBF基因转化烟草抗寒性指标的检测[J].中国林副特产,2009(1):4-7.

[11] Kanaya E, Nakajima N, Morikawa K, et al. Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana[J]. The Journal of Biological Chemistry, 1999,274(23):16068-16076.

[12] Novillo F, Alonso J M, Ecker J R, Salina J. CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis [J]. Proceedings of the National Academy of Sciences of USA, 2004,101(11):3985-3990.

[13] Vogel J T, Zarka D G, Van Buskirk H A, et al. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcription of Arabidopsis [J].The Plant Journal, 2005,41(2): 195-211.

[14] 萨姆布鲁克 J, 拉塞尔 D W.分子克隆实验指南(第三版)[M].北京:科学出版社,2002:1.

[15] 傅荣昭,孙勇如,贾士荣.植物遗传转化技术手册[M].北京:中国科学技术出版社,1994:133-134.

[16] 徐春波,王勇,赵海霞,等.我国将 CBF转录因子应用于基因工程的研究进展[J].安徽农业科学, 2010,38(4):1723-1726,1816.

[17] 李新玲,杨传平,徐香玲,等.与逆境胁迫相关的 rd29A 启动子和CBF2 基因的克隆及其表达载体构建[J].安徽农业科学,2007,35 (13):3828-3829,3853.

[18] 徐蓓,郭丽香,赵昌平,等.长穗偃麦草 SnRK2.6基因克隆及生物信息学分析[J].麦类作物学报,2012,32(1):36-43.

[20] 易建华,彭新辉,邓小华,等.气候和土壤及其互作对湖南烤烟还原糖、烟碱和总氮含量的影响[J].生态学报,2010,30(16):4467-4475.

[21] 陈江华,刘建利,龙怀玉.中国烟叶矿质营养及主要化学成分含量特征研究[J].中国烟草学报,2004,10(5):20-271.

[22] 邵惠芳,许自成,李东亮,等.烤烟还原糖含量与主要挥发性香气物质及感官质量关系的统计学分析[J].中国烟草学报,2011,4(17): 8-17.