Functional Analysis Of Transcription Factors OsMYB112 In Rice(Oryza Sativa)

The MYB family of proteins is large, functionally diverse and represented in all eukaryotes. In plants, most MYB proteins function as transcription factors in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. However, R2R3-MYB subfamily is the largest and most diverse transcription factors in plants. Recently, An explosion of research have reported R2R3-MYB genes involved in many biological pathways such as cell cycle and morphogenesis, meristem formation and floral and seed development, light and hormone transduction, and responses to the stress.R2R3MYB transcription factors are known to play a wide role in regulating the phenylpropanoid pathway in plants. In this study, we report isolation, cloning and characterization of an R2R3 MYB transcription factor gene OsMYB112. OsMYB112 consists of 705 bp coding sequence corresponding to 235 amino acids. Sequence alignment revealed that the N-terminal(MYB) domain of the gene shares up to 95 % similarity with subgroup 4(Sg4) members of R2R3 Myb gene family functionally known to be lignin repressors. Highly divergent C-terminal region of the gene carried an ERF-associated amphiphilic repression(EAR) motif, another characteristic of the Sg4. The gene was phylogenetically grouped closest with ZmMYB31, a known repressor of anthocyanin biosynthetic pathway genes. In this study, forward genetic analysis was used to investigte the biological functions of OsMYB112 in rice, and the findings are:(1) Cloning of full-length cDNAs encoding OsMYB112. Total RNA of various plants and tissues was isolated by Trizol(TaKaRa) according to the manual provided and was used for reverse transcription by Moloney murine leukemia virus(M-MLV).(2) Database search and phylogenetic analysis.The deduced amino acid sequence contains two highly conserved motifs in its N-terminus, which are known to be MYB DNA-binding domains R2 and R3.(3) Vector construction and rice transformation. To construct the RNAi vector, a 214-bp cDNA fragment of OsMYB112 was amplified from the cDNA clone using the primer set RNAi-OsMYB112 F and RNAi-OsMYB112 R and was inserted into the Kpn I and Sal I sites. For overexpression, the full-length cDNA of Os MYB112 amplified with the primer set 35S-OsMYB112 F and 35S- OsMYB112 R was inserted into the KpnI and SalI sites of pU1301. To construct the translational OsMYB112-GFP fusion, the coding region of OsMYB112 was amplified using the primers GFP-OsMYB112 F and GFP-OsMYB112 R. The amplified fragment was inserted into the KpnI and Sal I sites of modified pCAMBIA1301-GFP to create 35S:: OsMYB112-GFP.(4) Subcellur localization. The resulting 35S:: OsMYB112-GFP fusion construct was used for transient expression in onion epidermal cells following Zhang[1]. The location of the introduced genes in the onion cells was observed using confocal laser scanning microscopy.(5) Anthocyanin measurement. The relative total content of rice anthocyanin was determinated. The results show that the relative total content of anthocyanin in R2 line and R6 line are 17% and 51% lower than that in wild type, respectively.