Yeast One-Hybrid

Service Introduction

The yeast one-hybrid technique is a tool developed based on yeast two-hybrid systems to study nucleic acid-protein interactions. It is widely used to investigate the regulation of gene expression in eukaryotic cells, such as identifying DNA binding sites, discovering potential binding protein genes, and analyzing information about DNA-binding domains.

pHis2 system yeast one-hybrid:

The pHis2 yeast one-hybrid system uses two plasmids, pHis2 and pGADT7-Rec2. The selectable marker for plasmid pHis2 is Trp, which is used to express the pBait-His2 construct (the full-length promoter cloned into pHis2); the selectable marker for plasmid pGADT7-Rec2 is LEU, which is used to express the AD (GAL4 C-terminal amino acids 768–881) fused with the target protein (Prey). When the prey protein binds to the bait sequence (Bait DNA), GAL4 AD activates the expression of HIS, allowing growth on medium containing 3-AT. This system is mainly used to study the interaction between full-length promoters and transcription factors.

pAbAi system yeast one-hybrid:

The pAbAi yeast one-hybrid system uses two plasmids, pAbAi and pGADT7, in combination. The selectable marker for plasmid pAbAi is Ura, which is used to express the pBait-pAbAi construct (1–3 copies of bait DNA sequences tandemly cloned into pAbAi); the selectable marker for plasmid pGADT7 is LEU, which is used to express the fusion protein of AD (GAL4 C-terminal amino acids 768–881) and the target protein (Prey). When the prey protein binds to the bait DNA sequence, GAL4 AD activates the expression of AbAi, allowing growth on media containing AbA. This enables the determination of DNA–protein interactions and is primarily used to study the interactions between promoter cis-acting elements and transcription factors.

pLacZi Yeast One-Hybrid System:

The pLacZi yeast one-hybrid system uses two plasmids, pLacZi and pB42AD, together. The screening marker for plasmid pLacZi is Ura.The pB42AD plasmid has the selection marker Trp and expresses a fusion protein of AD and the target protein (Prey) when galactose is used as the carbon source. When the prey protein binds to the bait sequence (BaitDNA), AD activates LacZi expression, which can result in a color change on X-gal-containing medium, thereby confirming the interaction between DNA and protein. This system is mainly suitable for studying the interactions between cis-acting elements of promoters and transcription factors that are toxic to yeast growth.

Service Advantages

▶Many years of experience in library construction, effectively ensuring library quality;

▶Three-frame uniformization technology, effectively ensuring correct gene translation and uniform gene coverage in  

the library;

▶One-stop bioinformatics analysis support, providing reliable experimental conclusions;

▶Direct detection of interactions between proteins without relying on other molecules or cell involvement;

▶Protein-protein interaction strength can be quantified through the expression level of reporter genes, further understanding the dynamics and functions of the interactions.

Service Process

Yeast one-hybrid library construction

Yeast one-hybrid library screening

Yeast one-hybrid validation

Article Case

(1) pHis2 Systematic Yeast One-Hybrid

Identification and characterization of TCP transcription factor GmTCP670 associated with soybean development

academic journal：Scientific Reports

Y1H assay of the interaction between GmTCP670 transcription factor and GGNCCC. (A) Diagrams of pGADT7+GmTCP670 prey vectors. (B) Diagram of pHIS2+GGNCCC bait vectors. (C) Y1H validation of

GmTCP670 binding to GGACCC.

(2) pAbAi Systematic Yeast One-Hybrid

An InDel in the Promoter of Al-ACTIVATED MALATE TRANSPORTER9 Selected during Tomato Domestication

Determines Fruit Malate Contents and Aluminum Tolerance

academic journal：The Plant Cell

(A)Diagram of the Sl-ALMT9 genomic region. Arrows indicate W-box in the promoter and the eighth W-box circled with a black box indicates the location of indel_3. The red boxes represent the coding regions. The thin red line and thick red line represent the introns and 39 UTR, respectively. (B) Diagram of the sequence of the eighth W-box in the Sl-ALMT9HMH and Sl-ALMT9LMH genotypes, respectively. (C) Yeast-one hybrid (Y1H) assay of Sl-WRKY42 binding to Sl-ALMT9 promoter fragments. The bait vectors Sl-ALMT9HMHpro and Sl-ALMT9LMHpro (the fragments containing the region 1523 bp upstream of the Sl-ALMT9 initiation codon) and the prey vector containing Sl-WRKY42 were introduced into yeast strain Y187, and interaction between bait and prey enhanced ABA resistance. Yeast cells were grown on SD-Leu media with various concentrations of ABA (0, 10, 15, and 20 mM). The bait vector (Sl-ALMT9HMHpro and Sl-ALMT9LMHpro) + pGADT7 were also transformed into Y187 as a negative control.

(3) pLacZi Systematic Yeast One-Hybrid

The bHLH transcription factor PIL1 orchestrates starch synthesis in key cereal crops

academic journal：nature communications

Yeast one-hybrid assay showing that TaPIL1-5D bound the E-box other than mE-box (mutant E-box) motif.

Applicable Fields

▶Transcription factor research: Yeast one-hybrid can be used to study the specific binding of transcription factors to DNA sequences. By constructing a bait containing promoter or enhancer sequences and testing interactions with transcription factors in the prey, the interactions between transcription factors and gene regulatory sequences can be identified and verified.

▶DNA-binding protein identification: Yeast one-hybrid can be used to identify proteins that interact with specific DNA sequences. By constructing a bait containing a DNA-binding sequence and testing interactions with proteins in the prey, proteins that specifically bind to that DNA sequence can be screened and verified.

▶Drug screening and discovery: It can be used to find drugs targeting specific transcription factor-DNA interactions, which is especially applicable for transcription factor targets that are traditionally considered difficult to drug.