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Genetic analyses of response mechanisms for aluminum stress in plant

植物铝胁迫响应机制的遗传分析

基本信息

批准号：
16580046
负责人：
EZAKI Bunichi
金额：
$ 2.37万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580046/
关键词：
aluminum (Al) stress gene response mechanism Al stress induced gene transcription factor glutathione S-transferase oxidative stress signal transduction Arabidopsis thaliana Ring Zinc finger protein Homeobox-Leucine Zipper 転写調節因子アルミニ

项目摘要

1. Construction of cDNA library to isolate and characterize several candidate clones encoding transcription factor(s) which are related for the gene expression of the AtGST11 gene under aluminum (Al) stress.Using mRNA molecules derived from Al treated Arabidopsis thaliana, cDNA library was constructed. The constracted cDNA library were applied for a 3 times repeated screening designated "Biopanning". Finally, we could isolate 50 candidates clones in the third screening.2. Molecular genetical analysis of the isolated clonesIsolated 50 clones were applied to DNA sequencing and could be classified into 8 genes. Ring Zn finger protein (#13) and Homeobox-Leucine Zipper protein 6 (#43) which have been already reported as transcription factors were included in these clones. One clone (#4) which encodes an unknown protein was also included. Unfortunately, other 5 genes seemed not to be related to gene-expression of the AtGST11 gene.3. Gel shift assayTo confirm whether the three clones (#4, 13 and 43) actually can bind to the promoter region of the AtGST11 gene, gel shift assay was performed using the purified three proteins. These proteins were extracted and purified by His-tag affinity column before the gel shift assay. The result suggested that all of these proteins can bind to the promoter.4. New approach (One hybrid assay) for an isolation of transcription factorsIn the last year of this project (2006), we also started a new approach to isolate transcription factor(s) which are related to the gene-expression of either the AtGST1 or AtGST11 gene under Al stress by "Yeast One Hybrid System"., We could isolate four new candidates from this screening.

1. 构建cDNA文库，分离和鉴定与铝胁迫下AtGST11基因表达相关的转录因子候选克隆。利用Al处理的拟南芥mRNA分子构建cDNA文库。构建的cDNA文库进行3次重复筛选，命名为“Biopanning”。最后，我们可以在第三次筛选中分离出50个候选克隆。分离克隆的分子遗传学分析分离的50个克隆应用DNA测序，可分为8个基因。已报道的转录因子Ring Zn finger protein（#13）和Homeobox-Leucine Zipper protein 6（#43）也包含在这些克隆中。其中还包括一个编码未知蛋白质的克隆（#4）。不幸的是，其他5个基因似乎与AtGST11基因的基因表达无关。为了确认这3个克隆（#4、13和43）是否真的能与AtGST11基因的启动子区域结合，我们用纯化的3个蛋白进行了凝胶转移实验。这些蛋白在凝胶转移实验前通过His-tag亲和柱提取和纯化。结果表明，这些蛋白均能与启动子结合。在本项目的最后一年（2006年），我们也开始了一种新的方法，通过“酵母一杂交系统”分离与铝胁迫下AtGST1或AtGST11基因表达相关的转录因子。我们可以从这次筛选中找出四个新的候选人。