• 论文与研究报告 •

### 不同长度ThVHAc1基因启动子片段分离及活性分析

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

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.