Physiological and functional analysis of a family of tetracycline transporter-like proteins in Arabidopsis

在对车前维管组织的转录组分析中，已经鉴定出一个四环素类转运蛋白(Tet-TP-like)的基因。定量RT-PCRS结果表明，该基因在大车前的维管系统中特异表达。拟南芥有一个由三个Tet-TP类似基因组成的小家族，它们位于第二染色体上(At2g16970、At2g16980、At2g16990)，第四个基因位于第五染色体(At5g42210)上，只是远亲关系较远，而且要小得多。与车前Tet-TP-like基因关系最密切的基因At2g16970的启动子驱动GUS-Report基因的高特异性表达，该基因也仅在拟南芥的维管系统中表达。一个AT2g16970 k.o.纯合子系在其第一个外显子上携带T-DNA插入，表现出严重的表型，导致产生不能产生可育种子的微小莲座丛。在这个项目中，我们的目标是鉴定拟南芥的Tet-TP样蛋白家族和车前的Tet-TP样蛋白。我们将建立和分析可诱导的amiRNA系、Tet-TP类基因的过度表达株和启动子-GUS/GFP系，并将分析这些植物的宏观、微观和代谢物表型。此外，我们将针对不同的Tet-TP样蛋白产生特异性抗血清，并使用GFP标记的cDNA进行瞬时表达分析，以确定细胞特异性和亚细胞定位。功能分析将在不同的异源表达系统(酵母和非洲爪哇)中进行。

During transcriptome analyses of the vascular tissue of Plantago major the cDNA of a tetracycline transporter-like (Tet-TP-like) protein has been identified. Quantitative RT-PCRs revealed that this gene is specifically expressed in the vasculature of Plantago major. Arabidopsis possesses a small family of three Tet-TP-like genes that are located next to each other on chromosome II (At2g16970, At2g16980, At2g16990) and a fourth, only distantly related and much smaller gene on chromosome V (At5g42210). The promoter of At2g16970, the gene most closely related to the Tet-TP-like gene from Plantago drives expression of the GUS-reporter gene with high specificity also only in the vasculature of Arabidopsis thaliana. A homozygous At2g16970 k.o.-line that carries T-DNA insertion in its 1st exon shows a severe phenotype that results in tiny rosettes that to not produce fertile seeds. In this project we aim to characterize the family of Tet-TP-like proteins of Arabidopsis thaliana and the Tet-TP-like protein from Plantago major. We will generate and analyze inducible amiRNA-lines, over-expressors of Tet-TP-like genes and promoter- GUS/GFP lines and will analyze these plants for macroscopic, microscopic and metabolite phenotypes. Moreover, we will generate specific antisera against the different Tet-TP-like proteins and perform transient expression analyses with GFPtagged cDNAs to determine the cell-specific and subcellular localization. Functional analyses will be performed in different heterologous expression systems (yeast and Xenopus).