Figure 1 Structural formula of AbA, CAS#127785-64-2

Research has found that the AUR1 gene of Saccharomyces cerevisiae and the AURA gene of Aspergillus nidulans are homologous, both encoding IPC synthase. Therefore, mutations in these two genes can confer strong AbA resistance to strains, such as the mutated gene AUR1-C. AbA is very suitable as a drug selection marker for screening positive clones, and it does not require condition optimization, with a very low background. AbA resistance is also an ideal reporter for yeast single/double hybrid studies and is compatible with yeast hybrid systems carrying corresponding resistance.

What is the Yeast Single/Double Hybrid System

The yeast two-hybrid system (Yeast two-hybrid assay) was created by Fields and Song and others based on the characteristics of eukaryotic transcriptional regulation. It can quickly and directly analyze the interactions between known proteins and is widely used in the study of antigen-antibody interactions, the discovery of new proteins and new protein functions, screening for drug targets, and establishing genomic protein linkage maps. The principle of yeast two-hybrid is that the transcriptional activator of eukaryotes contains two different structural domains: the DNA binding domain (DNA binding domain, DNA-BD) and the DNA transcription activation domain (Activation domain, AD), which can be independently separated without affecting each other's function. BD and AD alone cannot activate the transcriptional response, only when the two are spatially close enough, they exhibit the activity of a complete transcriptional activator, allowing the downstream gene to be transcribed. By constructing fusion plasmids of the two proteins under study (protein X and protein Y) with BD and AD domains respectively and expressing them in the same yeast cell, if there is no interaction between the two proteins, the reporter gene will not be transcribed; if the two proteins interact, the BD and AD domains will be spatially close, thus the reporter gene is transcribed.

Figure 2 Principle diagram of yeast two-hybrid ^[1]

Yeast one-hybrid technology is a tool for studying nucleic acid-protein interactions, developed on the basis of yeast two-hybrid, and is widely used to study the expression regulation of genes in eukaryotic cells, such as identifying whether there is an interaction between known DNA and known proteins; isolating new proteins that bind to the target cis-regulatory element or other short DNA binding sites; accurately locating the DNA binding sites that have been proven to interact and analyzing the DNA binding domains of proteins. Its basic principle is to construct the known cis-acting element upstream of the most basic promoter (minimal promoter, Pmin) and connect the reporter gene downstream of Pmin. The cDNA encoding the transcription factor to be tested is fused with the yeast AD domain expression vector and introduced into yeast cells. If the product of this gene can bind to the cis-acting element, it can activate the Pmin promoter, allowing the reporter gene to be expressed.

Figure 3 Principle diagram of yeast one-hybrid ^[2]

For yeast single/double hybrid studies, Yeasen offers product 60231ES Aureobasidin A (AbA), a solution of AbA dissolved in methanol with a purity of ≥ 97% and a concentration of 1 mg/mL. Yeasen recommends an AbA inhibitory concentration of 100-1000 ng/mL in yeast single/double hybrid experiments, and the specific working concentration depends on the sensitivity of the host cells (see the following table, Minimum inhibitory concentrations (MIC) of AbA for various yeast strains).

Application Case

To study the binding site of GmWRKY31 protein on the promoter of GmSAGT1 gene, in the yeast one-hybrid experiment, the target DNA sequence was inserted into the pBait-AbAi vector containing the uracil reporter gene Ura3, located upstream of the AUR1-C gene. After yeast transformation with the corresponding plasmid, it was suspended in 0.9% NaCl solution, and the OD600 was adjusted to 0.005. Subsequently, 100 μL of the sample was spread on plates containing 500 ng/mL AbA, inverted, and cultured at 30℃ for 3-5 days^[3].

Figure 3 Interaction of GmWRKY31 protein with yeast strains containing fragments F16, F20, F73, delF16, delF20, or delF73.

Published articles with our reagents

[1] Shunan Zhang, Yuyi Zhang, Kangning Li,, et al. Nitrogen Mediates Flowering Time and Nitrogen Use Efficiency via Floral Regulators in Rice,Current Biology,Volume 31, Issue 4, 2021, https://doi.org/10.1016/j.cub.2020.10.095. (IF:10.834)

[2] Jiaying Kuang, Yingchun Xu, Yidan Liu, et al. An NnSnRK1-centered regulatory network of shade-induced early termination of flowering in lotus,Environmental and Experimental Botany,Volume 221,2024,105725, https://doi.org/10.1016/j.envexpbot.2024.105725. (IF:5.7)

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Reference documentation

[1] Paiano A, et al. Yeast Two-Hybrid Assay to Identify Interacting Proteins.Curr Protoc Protein Sci. 2019 Feb;95(1):e70.

[2] John S, et al. Yeast one-hybrid assays: a historical and technical perspective.Methods. 2012 August;57(4): 441-447.

[3] Dong H, et al. Transcriptome analysis of soybean WRKY TFs in response to Peronospora manshurica infection. Genomics. 2019 Dec;111(6):1412-1422.