Preliminary Identification Of Target Genes Of Rice Transcription Factor OsBP-73 And Identification Of Proteins Interacted With Rice Genes OsRRM And Its Homologue OsRRMT

This dissertation focuses on preliminary identification of target Genes of rice transcription factor OsBP-73 and identification of proteins interacted with OsRRM and its homologue OsRRMT.1. Preliminary Identification of Target Genes of Rice Transcription Factor OsBP-73The interactions between transcription factors and their DNA binding sites are an integral part of transcriptional regulatory networks. They control the coordinated expression of thousands of genes during normal growth and in response to external stimuli. Identifying the targets of transcription factors is important for understanding cellular processes.Previous data showed that a 31bp (from -840bp to -810bp) DNA fragment located at 5' upstream region of rice waxy gene could interact with nuclear protein extracted from developing endosperm of rice. And this 31bp DNA sequence was used as a bait to screen a rice cDNA library with a yeast one-hybrid, three groups of cDNA clones were isolated. One of them is pC73, the correspondent rice gene of pC73 was named as OsBP-73(Oryza sativa Binding Protein). OsBP-73 is expressed in developing endosperm of rice, root, stem and leaf. Using double-stranded RNA interference, we observed a dramatic decrease of whole plant growth. Those results suggest that OsBP-73 protein not only regulate the rice Wx gene, but also may play an important role in the regulation of rice growth. My work was to focus on identification of target genes of this rice transcription factor OsBP-73. The protein p73 (including DNA binding domain) and p512 (including full length ORF) of OsBP-73, fused with a 6xhis.tag, was expressed in E.coli cells and purified with Ni-NTA. The target genes of p73 were identified in rice genome-wide by using a pull-down assay, and 22 candidate genes were obtained. It will facilitate the study of rice transcriptional network(s) in which OsBP-73 participates.2. Identification of proteins interacted with OsRRM and its homologue OsRRMT OsRRM, a rice gene expressed specifically in rice endosperm, was identified bygene trap method. It contains two RNA recognition motifs (RRMs), one Spen paralog and ortholog C-terminal (SPOC) domain and one Leucine-Zip-like domain. OsRRM was the first reported Spen gene in rice. In animal, Spen genes usually function by protein-protein interactions. My work focused on screening of elements interacted with the proteins encoded by OsRRM and its homologue OsRRMT. We performed ayeast two-hybrid screen, with the OsRRM and OsRRMT as baits, 6 and 5 candidates were isolated from a rice cDNA library respectively. It will facilitate to elucidate the exact biological function and molecular mechanisms of OsRRM and OsRRMT in rice.