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林业科学 ›› 2015, Vol. 51 ›› Issue (3): 34-40.doi: 10.11707/j.1001-7488.20150305

• 论文与研究报告 • 上一篇    下一篇

毛竹4个B-box锌指蛋白序列和基因表达特征及PeBZF4的功能

娄永峰1, 杨丽1, 彭镇华2, 赵韩生1, 高志民1   

  1. 1. 国际竹藤中心 竹藤科学与技术重点开放实验室 北京 100102;
    2. 中国林业科学研究院林业研究所 北京 100091
  • 收稿日期:2014-05-30 修回日期:2014-07-16 出版日期:2015-03-25 发布日期:2015-04-10
  • 通讯作者: 高志民
  • 基金资助:

    "十二五"国家科技支撑计划项目"竹子优良种质选育技术研究与示范"专题"分子辅助育种技术集成与示范"(2012BAD23B0504); 国际竹藤中心基本科研业务费专项资金项目"毛竹快速生长期特异转录调控网络的构建及其应用"(1632015008)。

Protein Sequences and Expression Profiles of 4 B-box Zinc Finger Genes and Functions of PeBZF 4 from Phyllostachys edulis

Lou Yongfeng1, Yang Li1, Peng Zhenhua2, Zhao Hansheng1, Gao Zhimin1   

  1. 1. Key Laboratory on the Science and Technology of Bamboo and Rattan International Center for Bamboo and Rattan Beijing 100102;
    2. Research Institute of Forestry, Chinese Academy of Forestry Beijing 100091
  • Received:2014-05-30 Revised:2014-07-16 Online:2015-03-25 Published:2015-04-10

摘要:

【目的】 B-box锌指蛋白(BZF)在植物生长发育和应对逆境过程中发挥着重要的调控作用。强光胁迫对竹子的生长发育有着重要的影响,通过分析毛竹BZF基因编码蛋白的分子特征及组织表达模式,研究过量表达PeBZF 4 转基因植株的荧光动力学参数变化,以期为揭示竹子BZF基因对强光的应答与调节机制提供参考。【方法】 采用同源基因比对的方法直接从BambooGDB中获得毛竹BZF基因同源序列,利用专用软件和在线公共平台分析基因及其编码蛋白序列,查找各种作用元件,预测蛋白功能结构域,分析蛋白的亲水性/疏水性。采用实时定量PCR技术分析基因在不同组织中的表达情况以及强光(1 200 μmol·m-2s-1)和黑暗处理后基因在叶片中的表达变化。构建PeBZF 4 基因的过量表达载体,利用农杆菌介导法转化拟南芥,直接观察转基因植株表型,利用IMAGING-PAM荧光仪测定其叶绿素荧光参数。【结果】 从毛竹数据库获得4条不同的BZF基因同源序列,编码4个不同的锌指蛋白,分别命名为 PeBZF1,PeBZF2,PeBZF3PeBZF4。4个基因中均具有光应答顺式作用调控元件ACE和G-box,但它们所含光应答元件却有所差异,PeBZF 1 中有MNF1和Sp1,PeBZF 2 中有I-box,Sp1和boxⅡ,PeBZF3PeBZF4 中有GT1-motif,MNF1和Sp1。4个基因所编码的蛋白都具有2个B-box结构域和CHC3H2锌指结合域,属于B-box型锌指蛋白。在根、茎、叶片和叶鞘中的表达4个基因存在着明显差异,其中PeBZF 1PeBZF3 在叶片中的表达丰度最高, PeBZF2PeBZF4 在叶鞘中的表达丰度最高。强光处理后4个基因的表达均受到抑制,且随着光照时间的延长 PeBZF2PeBZF3 的表达呈逐渐下降趋势,而 PeBZF1PeBZF4 的表达却在光照1 h时比0.5 h时略微上调,随后迅速下降; 黑暗对 PeBZF1,PeBZF2PeBZF3 的表达有明显抑制作用,而PeBZF 4 的表达却显著上调,约是对照的3.2倍。过量表达PeBZF 4 的转基因植株光系统Ⅱ的实际光合效率Y(Ⅱ)和非光化学猝灭(NPQ)值都比野生型有不同程度的提高。【结论】 毛竹中有4个不同B-box型锌指蛋白基因,其表达为组成型,强光和黑暗处理4个基因的表达变化表明它们参与了光胁迫的应答调节。过量表达PeBZF 4 基因能够提高转基因植株的Y(Ⅱ)和NPQ值,证明PeBZF 4 能够增强其热耗散能力,提高强光下的光合效率。因此,BZF可能与竹子抵抗强光胁迫有关。

关键词: 毛竹, 锌指蛋白基因, 光胁迫, 基因表达

Abstract:

【Objective】 Zinc finger proteins containing B-box (BZF) play important roles in regulating growth and development, and response to stresses in plants. Light stress has a significant impact on the growth and development of bamboo. Studies on protein sequence of BZF and expression profiles of BZF genes in Phyllostachys edulis and fluorescence parameters of transgenic plants with over expressed PeBZF 4 were conducted in order to reveal the response and regulatory mechanisms of BZF genes in bamboo under high intensity of light.【Method】Homologous gene alignment method was used to obtain bamboo BZF sequences from BambooGDB directly. The analysis and comparison of gene and protein, the prediction of acting elements, protein functional domains, hydrophilicity and hydrophobicity of proteins were performed using special software and online public platform. Real-time quantitative PCR was used for the analysis of gene expression in different tissues, and the changes of gene expression in leaf after the treatments of high intensity of light (1 200 μmol·m-2s-1) and darkness. The over expression vector of PeBZF 4 was constructed and transferred into Arabidopsis thaliana mediated by Agrobacterium tumefanciens. The phenotype of transgenic plants was observed directly and the fluorescence parameters were measured using IMAGING-PAM.【Result】Four homologue BZF genes were obtained from BambooGDB, and designed as PeBZF 1, PeBZF2, PeBZF3 and PeBZF4 respectively. Cis-acting element analysis showed that four PeBZFs all had light-responsive cis-acting regulatory elements and light responsive elements, but the light-responsive elements were different, including MNF1 and Sp1 in PeBZF 1, I-box, Sp1 and boxⅡin PeBZF 2, GT1-motif, MNF1 and Sp1 in PeBZF 3 and PeBZF 4. The analysis of protein structure showed that the proteins encoded by these genes all contained two B-box domains and CHC3H2 binding motif, indicating that they were B-box zinc finger proteins. Tissue specific expression indicated that the levels of these PeBZFs were obviously different in leaf, sheath, stem and root, among which the highest level of PeBZF 1 and PeBZF3 was found in leaf, and that of PeBZF 2 and PeBZF 4 was in sheath. All the expression of these PeBZFs were inhibited by high intensity of light (1 200 μmol·m-2s-1). The expression of PeBZF 2 and PeBZF3 showed a gradual downward trend with the extension of irradiation time, while that of PeBZF 1 and PeBZF4 raised slightly after 1 h treatment compared with 0.5 h treatment, followed by a rapid decline. The expression of PeBZF 1, PeBZF2 and PeBZF3 were significantly inhibited by darkness, while that of PeBZF 4 was significantly upregulated about 3.2 times of that in control. The actual photosynthetic efficiency (Y(Ⅱ)) and non-photochemical quenching (NPQ) of transgenic plants with over expressed PeBZF 4 were higher than those of wild type.【Conclusion】 There were four different B-box-type zinc finger protein genes expressed constitutively in Ph. edulis, the expression changes of 4 genes under high intensity of light and dark treatments suggested that they were involved in regulation of light response to stress. The values of Y (Ⅱ) and NPQ increased in transgenic plants with over expressed PeBZF 4, indicating that PeBZF 4 could improve the thermal dissipation capability and photochemical efficiency of transgenic plants. This result indicated that BZF might be associated with bamboo resistance to light stress.

Key words: Phyllostachys edulis, B-box zinc finger protein gene, light stress, gene expression

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