Screening And Identification Of Interaction Proteins With Wheat DREB/ERF Transcription Factors

Plants can perceive stress signals under drought, salt and low-temperature conditions, transduce them into transcription factors; and ultimately activate the stress-related genes. Transcripton factors play critical roles in regulating transcriptional levels. Lots of studies demonstrated that DREB/ERFs widely existed in plants, which can activate the downstream stress-related gene expression and increase tolerence to drought, salt and low-temperature.Transcription factors interacted with other proteins during the whole regulating process. Ham et al. screened cDNA library by yeast two-hybrid method using the Tsil as bait. The experiments showed the interaction of the Tsil with Tsip1 enhanced the regulation to the downstream stress-related genes. Therefore studying crop stress-related transcription factor interaction proteins can further facilitate learning the mechanism of the transcription, and providing data for improving transgenic crops.We screened the putative interaction proteins of the W17 and DREB3 by yeast two-hybrid methods. Major results are as follows:1. Construction of the cDNA library:the cDNA was synthesized by SMART method. There is a smear band from 200 bp to 2 Kb, and the major band is at 700 bp position.2. Screening of cDNA library using yeast two-hybrid method:W17, cDNA and AD plasmids were transformed into the yeast competent cells together. The efficiency of the transformation and the size of the insert fragment satisfied the quality of the yeast library. Sequencing of the positive clones can be classified into two groups:post-translated modified proteins, transcriptional complex proteins.3. Identification of protein interaction:To investigate the interaction of W17 with target proteins, a BD vector fusing W17 (BD-W17) and an AD vector fusing target proteins were transformed into yeast cells. The yeast transformants with BD-W17 and AD-HSP90 as well as yeast transformants with BD-W17 and AD-PPR were able to grow on selective media lacking Trp, Leu, His and Ade in the presence of 5 mM 3-AT, whereas the transformants with BD and/or AD empty vector (s) did not grow under the same conditions, indicating that W17 could interact physically with HSP90 and PPR in vitro.4. Isolation and expression analysis of the STK gene:a STK full length sequence was cloned by EST assembling and 5'RACE method. Homology analysis indicated there is a 200 AA conserved domain in N terminus of the protein, and a ATP binding motif in C terminus. The expression patterns showed that STK might be involved in the stress regulation pathway.5. Prokaryotic expression and western blot of transcription factor TaBSl gene in wheat. Transcription factors play an important role in improving resistance in plants. A novel transcription factor gene, named TaBS1, was isolated from wheat. In order to investigate the role of TaBS1 in response to stresses and the structure, function and activity of proteins, the TaBS1 gene was constructed into the prokaryotic expression vector pET-28a(+). The fusion protein His- TaBS1 was induced to expression using 1 mmol/L of IPTG. The fusion protein His-TaBS1 was detected by Western blot. This study laid a basis for investigation of protein function, activity, and structure.