Screening Of Proteins Binding To OsHsfB2c Promoter By Yeast One-hybrid System

High temperature, drought and salinity stresses have become the important detrimental factors to affect the yield and quality of rice, therefore research on stress-resistance has become an important issue. The role of transcription factors is very important in the regulation of plant responses to high temperature, drought and salinity stresses through regulating the expression of downstream genes. In the previous study, a number of highly heat-responsive genes were screened out. Further studyon OsHsfB2c promoter revealed that the promoter is highly responsive to high temperature and drought, and has specific expression in young panicle.In this study, the upstream regulatory proteins binding to OsHsfB2c promoter were screened using yeast-one-hybrid technology. The main results of this study are as follows:1:The bioinformatics analysis with PlantCARE on the 1500 bp sequence upstream of ATG of OsHsfB2c gene showed that the promoter sequence contains core conserved elements such as CAAT box, TATA box, and also many stress-responsive elements such as HSE, G-box, Spl, GC-motif and ABRE.2:Based on the distribution of stress-responsive elements, four fragments of OsHsfB2c promoter region were cloned into bait vector pAbAi+Bait, and transformed into Y1H gold, respectively, and successfully constructed bait yeast strains were obtained through SD/-Ura plate screening and PCR verification.3:The constructed bait yeast strains were transformed with plasmids from rice cDNA library. Total 47 positive clones were detected on the SD/-Leu plate containing 600 ng/mL AbAr. The final 21 positive clones were identified by removing the empty vectors and clones with insertion length less than 800 bp. Further sequencing and bioinformatics analysis revealed that these cDNA encode 7 classes of binding proteins mainly involved in transcription initiation, protein degradation, abiotic stress response and some unknown functions.