The Cloning And Expression Of Wheat Endosperm-specific Transcription Factor Gene WPBF And Its DNA Binding Property Research

Wheat (Triticum aestivum L.) that is widely grown around the world is one of mankind'searliest cultivated plants and35%-40%of people worldwide live on it. With the improvementof people's living conditions, people attach more and more importance to wheat qualityimprovement. Wheat High Molecular Weight Glutenins (HMW-GS) are importantcomponents of wheat seed storage proteins and they form complex highpolymers with otherstorage proteins such as gliadins. The highpolymers can determine the flexibility andextensibility of the gluten, thus affect the processing quality of wheat. Studying the expressionregulation mechanism of HMW-GS is not only an important theoretical issue, but haveimportant guiding significance to the genetic improvement of wheat quality and yield.Some studies showed that WPBF can specifically bind to Prolamin box in Gliadin andLMW-GS promoters and may participate in the expression regulation of them. there is norelevant reports on which WPBF participates in HMW-GS expression regulation. we clonedWPBF and optimized its prokaryotic expression and purification conditions. We also verifiedits DNA binding properties and prepared its Dof domain proteins. In this study, we mainlyobtain the following conclusions:1. We cloned WPBF from the endosperm of wheat Shaanyou225by RT-PCR. Sequencingresults showed that the coding region of WPBF in Shaanyou225is highly consistent withChinese Spring and3338, only two or three amino acid residues are different among the threeproteins and the corresponding sequence of Dof domain is exactly consistent. Thephylogenetic analysis showed that BPBF in barley, WPBF in wheat, MPBF in corn and twoDof protein in sorghum (DAA34027.1and DAA34022.1) are in the same evolutionary branch.whereas RPBF is in another adjacent evolutionary branch.2. Compared with recombinant His-tagged WPBF, Recombinant MBP-and His-taggedWPBF existed mainly in soluble form when it was expressed at33℃in Origami B (DE3) strain, and the recombinant protein accounted for48%of the total cell protein. The aboveexperiments indicated that The combination of MBP tag and Origami B (DE3) host cansignificantly improve the solubility of recombinant WPBF.3. There existed a mass of impurity proteins when we purified recombinant WPBF byAmylose or Ni-NTA column alone, so we purified recombinant protein by Amylose andNi-NTA column successively and ultimately obtained the pure recombinant WPBF.4. We analyzed the binding property between recombinant WPBF and two types ofProlamin-Like boxes including "TGCAAAG" and "TGCAAG"respectively in HMW-GSpromoters. The results show that recombinant WPBF could bind specifically to two types ofProlamin-Like boxes, However, the binding strength of the former was higher than the latter.5. We cloned the coding sequence of Dof domain from WPBF, then we expressed it in E.Coli and purified the recombinant protein. We removed the fusion labels including MBP-andHis-tag in N terminal of recombinant Dof domain by TEV protease and finally obtained theoff-label target protein. We compared the effects of20×TEV-A buffer and10×TEV-B bufferfor TEV protease digestion and found that TEV protease can digest substrates well on twoconditions.