Functional Analysis of Rice (Oryza sativa) TFⅡB-related Factor OsBRF1
CHEN Xin-Hui, CHEN Guang-Na, NING He, CHEN Jia-Luo, WANG Si-Yi, ZHU Cheng, YANG Su*
College of Life Sciences/Key Laboratory of Specialty Agri-Products Quality and Hazard Controlling Technology of Zhejiang Province, China Jiliang University, Hangzhou 310018, China
Abstract In most eukaryotes, transcription factor ⅡB (TFⅡB) is a general transcription factor of RNA polymerase Ⅱ in transcription of DNA into RNA. TFⅡB-related factor 1 (BRF1) serves as the general transcription factor for RNA polymerase Ⅲ, responsible for synthesizing tRNA and 5S rRNA. In Arabidopsis thaliana, AtBRF1 and AtBRF2 participate in gametogenesis in a functionally redundant manner. However, the function of BRF1 in rice (Oryza sativa), an important monocotyledonous model plant and staple crop, has not been reported yet. In this study, OsBRF1 gene promoter-driven β-glucuronidase (GUS) expression vector was constructed. The results of GUS staining showed that OsBRF1 exhibited high expression levels in root, leaf, leaf sheath, stigma, mature root, mature leaf, panicle, pollen, endosperm, and anther, but it was not expressed in the glume. The fusion expression vector of OsBRF1 and GFP was transfected into Nicotiana benthamiana leaves via Agrobacterium tumefaciens, and it was found that OsBRF1 protein predominantly localized in the nucleus. Unable to obtain homozygous frameshift mutant plants using CRISPR/Cas9 gene editing methods indicated that OsBRF1 gene was homozygous lethal. RNAi-mediated silencing led to a significant reduction in pollen activity and fertility (P<0.05), suggesting a potential role of OsBRF1 in regulating rice fertility by modulating pollen viability. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated interaction between OsBRF1 and OsTBP1~3 (TBP: TATA-binding protein), indicating its potential role as a general transcription factor. This study provides basic material for further exploration of the gene function and regulatory mechanisms of OsBRF1.
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