Genetic analyses of response mechanisms for aluminum stress in plant

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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.