Influence Of OsWR2 On Rice Cuticle And Screening Thedownstream Genes Of OsWR2

Plant cuticle is located at the plant/airinterface and playing essential functions in plant interactions withits environment.Under normal environmental conditions,plant stomatal transpiration is the main way of water loss,account for more than 90%and the cuticlular transpiration is only less than 10%of total water lose rate.While under limited water conditions,plants would start its own defense mechanism to close the stomata in order to reduce water loss for survival,and making the cuticlular transpiration pathway the main way of plant water loss.The regulation of the plant cuticle on non-stomatal dehydration is of great significance for the maintenance of plant life.Our previous study of cuticle biosynthesis transcription factor gene OsWR2 showed that the OsWR2 overexpression in rice increased the cuticular wax and cutin content,changed the structure of rice cuticle,and enhanced tolerance to water-limited conditions.Based on our previous studies,this study was conducted to investigate the effects of OsWR2-RNAi on the composition and content of cuticle and seedlings drought tolerance.And intended to screening the downstream target gene of transcription factor OsWR2,identifying the important cis-acting elements in the promoter of target genes.The transcription factor toxicity and self activation of OsWR2 were also detected,which laid the foundation for the screening of yeast library cDNA and the new genes related to rice cuticle biosynthesis and drought tolerance.The main results are as follows:1.OsWR2-RNAi decreased the cutin,wax content and seedling drought tolerance of rice cuticleThe cuticular wax and cutin components,content,structure and plant drought resistance were compared among Ri-Os WR2,OE-Os WR2 transgenic plants and wild-type rice?WT?.As a result,OsWR2-RNAi transgenic rice exhibited reduced total cuticular wax amounts by14.8%mainly due to the decrease of aldehydes,alkanes and alcohols,lessened full cutin monomer level by 36.2%due primarily to the decrease of C16:0,C18:1?-OH and di-OH fatty acid components,and reduced seedling tolerance to water deficit.2.Interaction screening between OsWR2 and Os GL1-2 promoter by yeast one-hybrid1)Based on the dates of the microarray,the real-time PCR amplification of candidate target genes and the SMART analysis,three wax synthesis related genes OsGL1-1,OsGL1-2and OsGL1-3 were selected as candidate genes for interaction screening.Then the lowest concentration of the AbA^r which inhibits the growth of Y1H[pOsGL1-1-AbAi],Y1H[pOsGL1-2-AbAi]and Y1H[pOsGL1-3-AbAi]were screened as 800 ng/mL,450 ng/mL and 350ng/mL respectively.OsGL1-1 was eliminated for the next screening procedure as the lowest inhibition concentration of the AbA^r to Y1H[pOsGL1-1-AbAi]was so high.The fusion expression vector pGADT7-OsWR2 was constructed.Yeast one-hybrid results showed that there is a interaction between OsWR2 proteinand pOsGL1-2,but not pOsGL1-3.2)Screening and identification the functional region of OsGL1-2 promoter interacted with OsWR2 protein.Based on the analysis result of the promoter region of OsGL1-2 by online software PlantCare,the target fragment with a GCC-box on the OsGL1-2 promoter region was amplified.After screened the lowest AbA^r background concentration of YlH[pOsGL1-2?GCC?-AbAi]as 450 ng/mL.The result that the recombinant yeast Y1H[pOsGL1-2?GCC?-AbAi/pGADT7-OsWR2]can grow normally on the medium SD/-Leu/AbA^{r 450} proved the interaction between pOsGL1-2?GCC?and OsWR2 protein.3.Identification of the self-activating effect and cell toxicityon yeast strain of transcription factor OsWR2.After the full-length sequence of OsWR2 was analysed by Blast and SMART software online,the full-length 822 bp sequence and the sequence without C-terminal activation region?1-592bp?of Os WR2 were amplified by PCR respectively.The cell toxicity and self-activation analysis of the recombinant yeast strains found that both the Y2H[pGBKT7-OsWR2]and Y2H[pGBKT7-OsWR2?1-592bp?]have no toxicity to the yeast cell.While results of the full length sequence of OsWR2 activating the experssion of report gene alone but not that of OsWR2?1-592bp?identified the self-activating effect of the C-terminal sequence of OsWR2.