DOF转录因子AtDof1.7 RNA干扰载体的构建及拟南芥的遗传转化

doi:10.3724/SP.J.1006.2011.01196

摘要/Abstract

摘要： DOF (DNA binding with one finger)转录因子是植物特有的转录因子家族，含有一个独特的富含Cys残基的单锌指DNA结合区域，在植物生长发育中参与多种生物学过程。本研究根据拟南芥AtDof1.7基因(GenBank登录号为AT1G51700)序列设计含有不同酶切位点的特异性扩增引物，以拟南芥总DNA为模板，扩增AtDof1.7基因片段，将AtDof1.7基因正向反向分别插入表达载体的相应位置，构建成AtDof1.7基因的RNA干扰载体pADOF1。利用改良的floral-dip方法将干扰载体pADOF1成功转入野生型拟南芥，经草甘膦抗性筛选和PCR检测获得5株阳性转基因植株。利用RT-PCR技术和气相色谱法分别分析了AtDof1.7基因的表达和种子脂肪酸组成，结果表明5株转基因植株中AtDof1.7基因的表达量不同程度低于野生型植株，种子油酸含量明显上升，亚麻酸含量明显下降，说明AtDof1.7转录因子与拟南芥种子脂肪酸代谢途径有一定的关系，为进一步研究其在脂肪酸代谢过程中的调控作用以及在油菜中研究该类转录因子的功能奠定了基础。

关键词: DOF转录因子, AtDof1.7, 拟南芥, RNA干扰载体, 脂肪酸代谢途径

Abstract: The Dof (DNA binding with one finger) transcription factorsare members of a family of plant-specific transcription factors that have a highly conserved DNA-binding domain, namely Dof domain which contains a single C2C2-type zinc-finger-like motif and specifically recognizes an (A/T)AAAG sequence as the recognition core. It suggests that the Dof transcription factors play diverse roles in specific biological processes in plants. Members of this protein family in plants are found to be involved in the gene regulation of many processes, but the roles in fatty acid metabolism are rarely reported. To investigate whether AtDof1.7 Dof transcription factor can regulate fatty acid metabolism, on the basis of the sequence of the gene (GenBank accession No. AT1G51700), we designed the specific primer containing different enzyme sites. With the template of Arabidopsis thaliana DNA, the AtDof1.7 gene fragment was isolated, which wasinserted into the expression vector by forward and reverse ways respectively. The RNA interference vector of pADOF1 containing AtDof1.7 gene fragment was constructed. By floral-dip method, pADOF1 was successfully transformed into wide-type Arabidopsis thaliana. Glyphosate resistance screening and PCR detection showed that five positive transgenic plants were obtained. The result of RT-PCR showed that transgenic plants had lower expression level of AtDof1.7 gene than the wild type. Fatty acid content was analyzed by gas chromatography which showed that the content of oleic acid increased and the content of linolenic acid decreased drastically in each transgenic plant compared with wide-type plants. These results indicated that AtDof1.7 transcription factor has certain relation with fatty acid metabolic pathway in Arabidopsis thaliana seed which provides a good foundation for further study on the function of AtDof1.7 transcription factor in fatty acid metabolic regulation.

Key words: DOF transcription factors, AtDof1.7, Arabidopsis thaliana, RNA interference vector, Fatty acid metabolic pathway

尹明智, 官梅, 肖钢, 李栒, 官春云. DOF转录因子AtDof1.7 RNA干扰载体的构建及拟南芥的遗传转化[J]. 作物学报, 2011, 37(07): 1196-1204.

YIN Meng-Zhi, GUAN Mei, XIAO Gang, LI Quan, GUAN Chun-Yun. RNAi Vector Construction of AtDof1.7 Transcription Factors and Genetic Transformation into Arabidopsis thaliana[J]. Acta Agron Sin, 2011, 37(07): 1196-1204.

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