欢迎访问林业科学,今天是

林业科学 ›› 2016, Vol. 52 ›› Issue (1): 55-61.doi: 10.11707/j.1001-7488.20160107

• 论文与研究报告 • 上一篇    下一篇

不同长度ThVHAc1基因启动子片段分离及活性分析

杨桂燕, 郭宇聪, 张凤娇, 赵震, 高彩球   

  1. 林木遗传育种国家重点实验室 东北林业大学 哈尔滨 150040
  • 收稿日期:2014-12-29 修回日期:2015-09-01 出版日期:2016-01-25 发布日期:2016-02-26
  • 通讯作者: 高彩球
  • 基金资助:
    国家自然科学基金项目(31370676);教育部"新世纪优秀人才支持计划"(NCET-13-0709)。

Isolation and Activity Analysis of Different Length ThVHAc1 Promoters

Yang Guiyan, Guo Yucong, Zhang Fengjiao, Zhao Zhen, Gao Caiqiu   

  1. State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University Harbin 150040
  • Received:2014-12-29 Revised:2015-09-01 Online:2016-01-25 Published:2016-02-26

摘要: [目的] VHAc基因 (V-ATPase c亚基)是V-ATPase的重要亚基,能响应盐、重金属等胁迫,过表达刚毛柽柳ThVHAc1基因的酵母能提高抗CdCl2耐NaCl能力。本研究拟通过分离ThVHAc1基因不同长度启动子片段并对其胁迫后活性进行分析,以进一步探讨ThVHAc1基因响应CdCl2和NaCl胁迫的机制。[方法] 根据ThVHAc1基因启动子中含有的Dof顺式作用元件的分布,将ThVHAc1基因上游启动子分为205 bp(-1- -205),504 bp(-1- -504)和781 bp(-1- -781)3个不同长度片段。将这些不同长度启动子片段分别替换pCAMBIA1301载体上的CaMV35S启动子,以驱动GUS基因表达,构建植物表达载体,利用农杆菌介导法转化拟南芥。对4周龄的T4代转基因拟南芥分别进行H2O(非胁迫处理,对照)、100 mmol·L-1 NaCl和150μmol·L-1 CdCl2胁迫处理,比较不同转基因株系的GUS染色和GUS酶活性。[结果] 正常生长条件下,CaMV35S株系在根、茎、叶中均有GUS染色; 3个启动子片段转基因株系均能在不同组织观察到GUS染色,且在根、茎、叶中有一定差异,但整体上GUS酶活性表现为781> 504> 205。NaCl胁迫下,CaMV35S株系的GUS染色及酶活性与正常生长条件相比无明显变化,但3个启动子片段转基因株系的GUS染色及酶活性均显著降低,且781株系的GUS酶活性分别为205和504株系的2.73和2.07倍;同时,3个启动子片段转基因株系的组织表达特性发生了一些改变,如,504株系在老叶中表达明显增强,嫩叶中减弱,根中无明显变化。CdCl2胁迫下各转基因株系的GUS染色和酶活性变化趋势与NaCl胁迫相似。CdCl2胁迫下, 205,504,781株系的GUS酶活性分别为非胁迫时的52.4%,57.9%和80.9%;胁迫后的组织表达活性也发生了一些改变, 205株系的GUS染色在老叶较深、嫩叶较浅,504株系则在各部分的表达较均匀,781株系大多叶片的GUS表达减弱;但781株系的GUS酶活性仍然最高,分别为205和504株系的2.67和2.07倍。[结论] ThVHAc1基因启动子片段的驱动活性与长度呈正相关;各不同片段启动子在根、茎、叶中的GUS染色及活性具有一定差异,体现在不同启动子片段的表达活性具有一定的组织特异性。NaCl和CdCl2胁迫对ThVHAc1基因启动子的驱动能力具有一定影响,胁迫后各启动子片段转基因株系GUS酶活性均显著降低,但长片段受胁迫的影响程度低于短片段。Dof元件的数量在3个启动子片段中依次减少,表明Dof元件可能对NaCl和CdCl2胁迫有一定的调节作用。同时,NaCl和CdCl2胁迫对ThVHAc1基因启动子的组织表达特性具有一定的影响。

关键词: 刚毛柽柳, 拟南芥, ThVHAc1基因, 启动子, GUS酶活性, NaCl胁迫, CdCl2胁迫

Abstract: [Objective] VHAc is an important subunit of V-ATPase which responds to salt and heavy metal stresses. In previous studies, we have identified that overexpression of Tamarix hispida ThVHAc1 in yeast can improve its tolerance to NaCl and CdCl2. In present study, we further explore the mechanism of ThVHAc1 in response to NaCl and CdCl2 by comparing the expression activities of different lengths of ThVHAc1 promoters under stresses.[Method] According to the distribution of Dof cis-element in the ThVHAc1 promoter, ThVHAc1 promoter was divided into three segments including 205 bp (-1- -205), 504 bp (-1- -504) and 781 bp (-1- -781). The CaMV35S promoter in pCAMBIA1301 was replaced by these three different lengths of ThVHAc1 promoter (205, 504 and 781 bp), respectively. And these recombined constructs were transformed into Arabidopsis thaliana using Agrobacterium-mediated method. The T4 lines of the transgenic plants at 4 weeks old were treated with 100 mmol·L-1 NaCl, 150 μmol·L-1 CdCl2 and H2O (non stress treatment as control) respectively for 30 min to compare the GUS staining and activities of transgenic plants. [Result] Under normal growing conditions, the root, stem and leaf of CaMV35S seedling showed GUS staining. The GUS staining was also observed in different tissues of the three transgenic lines expressing promoter segments, and there were some differences among root, stem and leaf tissues. However, the total GUS activities of these three promoter segment lines were 781> 504> 205. Under NaCl stress, the GUS staining and activity of CaMV35S line were not obviously different from that growing in normal condition (no stress), however, the GUS staining and activity of three promoter segments transgenic lines were significantly decreased, the GUS activity of the 781 line was 2.73-fold of the 205 line and 2.07-fold of the 504 line. Meanwhile, the tissue expression of transgenic lines of the three promoter segments were changed, for instance, old leaves of the 504 line showed increased GUS activity while the young leaves were decreased, and root showed no evident changes. Under CdCl2 stress, all transgenic lines showed patterns of GUS staining and activities similar to NaCl stress, the GUS activities of 205, 504, and 781 were 52.4%, 57.9%, 80.9% of those under no stress, respectively. The post-stress tissue expression were also changed for CdCl2 stress, the GUS staining of 205 line were darker in old leaves and lighter in young leaves, the GUS staining of 504 line was uniform among different tissues, the GUS staining in most leaves of the line 781 were lighter. However, the GUS activity of the line 781 was still the highest, 2.67 and 2.07 folds of the line 205 and the line 504 respectively.[Conclusion] The driving activity of ThVHAc1 promoter was positively correlated with its fragment length. The GUS staining and activity of the three promoter segments were different in the roots, stems and leaves of the transgenic seedlings, indicating a certain extent of tissue specificity of different promoter segments. NaCl and CdCl2 stresses generated certain influence on the driving activity of ThVHAc1 promoters, the post-stress GUS activities of the three promoter segments in the transgenic lines were significantly decreased, but the impact was weaker on long length promoter than on short ones. The number of Dof motif was successively reduced in turn in the three promoter segments of 781, 504, 205, indicating Dof motif may play some roles in regulating NaCl and CdCl2 stresses. Meanwhile, the tissue expression of ThVHAc1 promoter was more or less affected by NaCl and CdCl2 stresses.

Key words: Tamarix hispida, Arabidopsis thaliana, ThVHAc1 gene, promoter, GUS activity, NaCl stress, CdCl2 stress

中图分类号: