Characterization of Organelle Metabolite Transporters involved in Nucleotide Metabolism

参与核苷酸代谢的细胞器代谢物转运蛋白的表征

• 批准号: 452173586
• 负责人: Professor Dr. Claus-Peter Witte
• 金额: --
• 依托单位: Institut für Pflanzenernährung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/452173586?language=en
• 关键词: Characterization; Organelle; Metabolite; Transporters; involved

Reactions of nucleotide metabolism take place in all organelles of the cell, and metabolic pathways of nucleotide metabolism can be distributed to different organelles. Some transporters that translocate metabolites of nucleotide metabolism across intracellular membranes have been described, but many transport processes are still not understood in molecular detail.This project aims to identify and characterize the plastid transporter for inosine monophosphate (IMP) - an end product of purine biosynthesis - and the plastid transporter for ribose - a product of nucleotide degradation - in both Phaseolus vulgaris and Arabidopsis thaliana. To identify possible transporters with these functions, a comparative analysis of the nodule and root transcriptomes of four legume species was carried out. Two legumes that export fixed nitrogen in the form of amides from the nodules and two legumes that export fixed nitrogen in the form of ureides were compared. Genes whose products are required in purine and ureide biosynthesis are likely to be active specifically in nodules from ureide producers. This assumption was confirmed on the basis of many known genes that are involved in these processes. Genes for possible IMP transporters (IMPEX) and a possible ribose transporter (RITAP) of P. vulgaris were found in this data set. Homologous genes were also identified in A. thaliana.Further investigations were initially carried out with genetic variants of Arabidopsis and showed that in an IMPEX mutant the concentrations of nucleotides are deregulated, which particularly affects IMP. Two independent RITAP mutants in Arabidopsis accumulated ribose, which was enhanced specifically in the mutants by external administration of uridine – a nucleoside hydrolyzed to uracil and ribose in the cytosol.In this project, these transporters are to be further characterized by metabolite analyses of genetic variants, also employing inducible lines in Arabidopsis. We will also investigate the function of the P. vulgaris transporters, including their role in ureide production, in transgenic adventitious roots (hairy roots) and nodules in which the corresponding genes were mutated by CRISPR / Cas9. In promoter-reporter and promoter-gene-reporter studies with fluorescent reporters in transgenic nodules, the tissue-specific promoter activity and the subcellular localization of the new transporters as well as of the known ureide biosynthetic enzymes will be investigated in order to develop a new model for the metabolic pathway of ureide biosynthesis in the nodule of P. vulgaris. Complementation studies with the plant transporters in mutants of Escherichia coli and studies on their substrate specificity and transport kinetics after expression in E. coli will complete the functional description.

核苷酸代谢的反应发生在细胞的所有细胞器中，并且核苷酸代谢的代谢途径可以分布到不同的细胞器。已经描述了一些转运核苷酸代谢的代谢物穿过细胞内膜的转运蛋白，但许多转运过程的分子细节仍不清楚。本项目旨在鉴定和表征嘌呤生物合成的终产物--肌苷一磷酸（IMP）的质体转运蛋白和核苷酸降解产物--核糖的质体转运蛋白。在菜豆和拟南芥中均存在。为了确定可能的运输与这些功能，根瘤和根转录组的四个豆科植物进行了比较分析。比较了两种以酰胺形式从结核中输出固定氮的豆科植物和两种以酰脲形式输出固定氮的豆科植物。嘌呤和酰脲生物合成所需产物的基因可能在酰脲生产者的根瘤中特异性地活跃。这一假设得到了证实的基础上，许多已知的基因参与这些过程。在该数据集中发现了P. vulgaris的可能的IMP转运蛋白（IMPEX）和可能的核糖转运蛋白（RITAP）的基因。在A.进一步的研究最初是用拟南芥的遗传变体进行的，并且显示在IMPEX突变体中核苷酸的浓度被解除调节，这特别影响IMP。拟南芥中的两个独立的RITAP突变体积累核糖，这在突变体中通过外部施用尿苷（在胞质溶胶中水解为尿嘧啶和核糖的核苷）而特异性地增强。这些转运蛋白将通过遗传变异体的代谢物分析进一步表征，也使用拟南芥中的诱导系。我们还将研究P. vulgaris转运蛋白的功能，包括它们在转基因不定根（毛状根）和根瘤中的酰脲产生中的作用，其中相应的基因被CRISPR / Cas9突变。 在转基因根瘤的荧光报告基因启动子-报告基因和启动子-基因-报告基因研究中，将研究新转运蛋白以及已知酰脲生物合成酶的组织特异性启动子活性和亚细胞定位，以开发一种新的酰脲生物合成代谢途径的模型。在大肠杆菌突变体中与植物转运蛋白的互补性研究以及在大肠杆菌中表达后的底物特异性和转运动力学研究。大肠杆菌中完成功能描述。