Identification And Characterization Of Transcription Factor Ojerf Gene From Ophiopogpn Japonicus

Mondo Grass is an important Liliaceae perennial evergreen herb, like grass, flowering plants, preferring growing in warm and humid climate. It is distributed widely in China, often used as an ornamental garden plant. It has excellent characteristics of drought, salt, shade, cold and long green period. At the same time, it has good medicinal value.The Yin and traditional Chinese medicines are widely used in clinical for the long-term. Therefore, it will have important implications for improving varieties by cloning quality gene and functional identification with modern molecular biology techniques. On the other hand, the AP2/ERF transcription factors have been cloned in many species, they are play important roles in the transmission of the signal, regulation of downstream responses gene expression and cell development. They can be combined with the GCC-box, DRE/CRT cis-acting elements involved in biotic and abiotic stress response. The research on improving plant resistance through transformation separate AP2/ERF transcription factors has become hotspots.Because of the serious global shortage of water resources and soil salinization.Drought and Salt stress have become a major limiting factor in plant growth and crop yields. Salt and drought-resistance involve a number of complex metabolic pathways and the molecular mechanism has not been known clearly. The research is still in the initial stage. Cloning drought and salt-related transcription factor and reveals its participation in signal transduction network, and study the function of the gene by transgenic technology in adversity stress, which will provide theoretical basis for breeding of new varieties of plants under environmental stresses.In this study, we cloned the transcription factor OjERF gene from Mondo Grass and identified its biological characteristics, and through transformation tobacco plants to study the mechanism of regulation of plant drought and salt tolerance. The specific experimental results are as follows:1. The ERF Class transcription factor OjERF gene was isolated by RT-PCR method from Mondo Grass.The open reading frame of the gene is1047bp encoding348amino acids. The predicted amino acid sequence analysis showed that:it contained a AP2/ERF typical domain, a putative nuclear localization signal and a transcription activation. It is a new gene in Liliaceae and N-teminal has a conserved amino MCGGAII motif, which belongs to the fourth class of the ERF transcription factor.2. RT-PCR analysis results showed that the OjERF gene which had no tissue-specific expression characteristics was expressed in the roots, stems and leaves. The expression level was leaf> stem> root. OjERF gene expression were also affected by drought, high salt, low temperature, ABA and ET, but their variation degrees are different which illustrated the OjERF gene may have acertain role in abiotic stresses and also probably through the ABA signal transduction pathways involved in the Stress Response.3. By PEG-mediated constructed PEZR (K)-LC-OjERF plasmid transformation into rice protoplast cells. Subcellular localization results showed:OjERF protein located in the nucleus.4. OjERF gene was fused into GAL4DB and transformed into yeast cells. Studies have shown that: OjERF gene had transcriptional activation ability and also can activate the reporter gene expression.5. The full-length cDNA sequence of OjERF gene, was cloned into the prokaryotic expression vector pEASY-E1Expression to construct the recombinant plasmid pEASY-E1-OjERF. After induced, expression product was detected by SDA-PAGE electrophoresis and showed that the target protein was successfully expressed in E. coli cell.6. The OjERF gene was cloned into PBI121and then transferred into tobacco by Agrobacterium infection. TO generation plants were identified by molecular method. Salt-tolerance and drought-tolerance experiments showed that under high salt stress, the chlorophyll content of transgenic tobacco is3-fold greater than that in the wild-type control; After20d drought treatment, the transgenic tobacco had lower electrolyte leakage and malondialdehyde content and higher proline content and SOD, CAT activity compared to the control, which suggest that transgenic tobacco plants have stronger drought and salt tolerance. Under normal growth conditions, resistance related gene of NtERD10B, NtERD10C, NtERF5, NtSOD and NtCAT1expression level were significantly higher than that of non-transgenic control. Overexpression of the OjERF gene can efficiently activate downstream resistance related gene expression.7. Quantitative real-time PCR results showed that:The expression level of ABA synthesis genes NtSDR was higher in the transgenic tobacco than that in the non-transgenic control. Measured by ELASA found that ABA content is in the transgenic tobacco plants were higher than wide-type,which may indicate the OjERF gene can be combined with multiple cis-acting elements and activiates the expression of stress responsive and ABA biosynthesis-related genes consequently causing ABA accumulation, and ultimately by the ABA-dependent signal transduction pathways to improve plant resistance to drought and high salt stress. These results indicated that overexpression OjERF can enhance tobacco tolerance under high salinity and drought.