茶树锌转运蛋白基因CsMTP1的克隆和功能鉴定

Cloning and functional identification of CsMTP1, a zinc transporter gene from tea plant(Camellia sinensis)

阳离子扩散促进蛋白(cation diffusion facilitator protein,CDF)是一类重要的金属转运蛋白,对维持细胞中Zn、Fe和Mn等元素的动态平衡起着重要作用。本研究利用RT-PCR技术,从茶树中克隆得到1个CDF家族基因,命名为CsMTP1(GenBank登录号:MN061493)。该基因开放阅读框长度为1353 bp,编码450个氨基酸,蛋白质相对分子质量为49.20 kDa,理论等电点5.99;不稳定指数为38.68,属于稳定蛋白;N端序列保守性较低,C端序列保守性相对较高;与猕猴桃(Actinidia chinensis)和木薯(Manihot esculenta)的序列相似性较高;位于细胞质膜,共含有6个跨膜结构域。系统发育树分析表明,CsMTP1与山萮菜(Eutrema salsugineum)、拟南芥(Arabidopsis thaliana)、菥蓂(Thlaspi arvense)和欧洲油菜(Brassica napus)的进化关系较近,属于Zn-CDF亚族。启动子分析发现,CsMTP1启动子序列中含有多个脱落酸和茉莉酸甲酯响应元件、光响应元件及MYB结合位点。实时荧光定量PCR分析发现,高浓度Al处理条件下,CsMTP1在茶树根部和第1叶中的转录变化相反,在根中先升后降,而在第1叶中先降后升;高浓度Mn处理诱导了CsMTP1在根中和第1叶中的转录;高浓度Fe和高浓度Zn处理均抑制了CsMTP1在茶树根中的转录,并不同程度地诱导了该基因在茶树第1叶中的转录。这表明,CsMTP1对高浓度Al、Fe、Zn和Mn有不同的响应模式。酵母功能互补实验证实CsMTP1具有转运Zn的功能。

Cation diffusion facilitator protein(CDF)is an important metal transporter and plays an important role in maintaining homeostasis of Zn,Fe and Mn in cells.In this study,CsMTP1 and its promoter(GenBank accession No.MN061493)were isolated from Camellia sinensis.The open reading frame(ORF)of CsMTP1 was 1353 bp,encoding 450 amino acids.Bioinformatics analyses showed that the molecular weight of the predicted protein was 49.20 kDa and the theoretic isoelectric point was 5.99.Besides,its instability index was 38.68,belonging to stable protein.The N-terminal sequence of CsMTP1 protein was less conserved compared with the C-terminal sequence.Homologous alignment showed that the predicted CsMTP1 protein had a high sequence similarity with Actinidia chinensis and Manihot esculenta.CsMTP1 was localized in plasma membrane and had six transmembrane domains.Phylogenetic tree analysis showed that CsMTP1 had a close genetic relationship with Eutrema salsugineum,Arabidopsis thaliana,Thlaspi arvense,and Brassica napus,which was grouped to Zn-CDF subfamily.Promoter analysis revealed that the CsMTP1 promoter contained several abscisic acid and methyl jasmonate response elements,photo-responsive elements,and MYB binding sites.Quantitative real-time PCR found that the transcription level of CsMTP1 in roots and the first leaves showed the opposite trend exposed to high concentration of Al:first increased and then decreased in roots,while first decreased and then increased in the first leaves.High concentration of Mn induced the transcription of CsMTP1 in roots and the first leaves.High concentration of Fe or Zn inhibited the transcription of CsMTP1 in roots,while induced the transcription of CsMTP1 in the first leaves to varying degrees.The results indicated that CsMTP1 had different transcription profiles under high concentrations of Al,Fe,Zn,and Mn.Additionally,heterologous expression in yeast showed that CsMTP1 was able to complement the hypersensitivity of mutant strains to excessive amount of Zn.