Screening Of Interacting Proteins With Transcription Factor OsDREBA5-1 Using Yeast Two-hybrid System

Plant type is an important agronomic trait that closely related to rice production. Leaf type is a crucial component of plant type and erect leaves enhance light capture for photosynthesis and enable more dense plantings for increased yield. Leaf angle is an important factor affecting the development of erect leave. Illustration of leaf angle morphogenesis will provide theoretical foundation for high yield rice breeding through improving plant leaf type. In our previous study, we found that OsDREBA5-1, a DREB transcription factor, involved in rice leaf angle regulation. Overexpression of OsDREBA5-1, leaded to increased leaf angle, while decreased its expression by RNAi resulted in an opposite phenotype. Based on the previous studies, we screen the interacting proteins of OsDREBA5-1 by yeast two-hybrid assay. The results will help us to well understand the molecular mechanism of OsDREBA5-1 in leaf angle control. The main findings are as follows:1. Construction of yeast two-hybrid recombinant expression vectorsOsDREBA5-1 full-length CDS and its truncations were constructed as baits for Yeast Two-Hybrid System screening. The baits were pGBKT7-OsDREBA5-1, pGBKT7-OsDREBA5-1-fl, pGBKT7-OsDREBA5-1-f2, pGBKT7-OsDREBA5-1-f3, pGBKT7-OsDREBA5-1-f4, pGBKT7-OsDREBA5-1-f5 and pGBKT7-OsDREBA5-1-f6.2. Testing bait for toxicity and autoactivationTo detect bait for toxicity and autoactivation, yeasts containing different bait vector were screened on the plate of SD/-Trp and SD/-Trp/X-a-Gal nutritional deficient medium, respectively. Results of toxicity testing showed that bait proteins did not affect the normal growth of yeast Y2HGold. Results of autoactivation testing showed that bait plasmids pGBKT7-OsDREBA5-1, pGBKT7-OsDREBA5-1-f5 and pGBKT7-OsDREBA5-1-f6 have autoactivation activity, while pGBKT7-OsDREBA5-1-f1, pGBKT7-OsDREBA5-1-f2, pGBKT7-OsDREBA5-1-f3 and pGBKT7-OsDREBA5-1-f4 did not. Therefore pGBKT7-OsDREBA5-1-f2 was chosen as bait plasmid for screening interaction proteins of OsDREBA5-1.3. Screening of interaction proteins of OsDREBA5-1Using pGBKT7-OsDREBA5-1-f2 as bait, a total of 43 positive colonies have been screened. These colonies grew normally on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/-X-a-gal/-AbAr agar plates, and meanwhile colonies turned blue on SD/-Trp/-Leu/-His/-Ade/-X-a-gal/-AbAr agar plate, all of which demonstrated that those 43 colonies were really positive colonies.4. Bioinformatics analysis of candidate genesPlasmids of 43 positive colonies were sequenced and bioinformatics analyzed. The results show that cDNA in the 43 colonies were matched with 6 protein encoding genes.