Cloning And Function Analysis Of Dihydrogen Flavonoids-4-Reductase Gene From Aegilops Tauschii

Drought is a major cause of leading to crop failures. It is of great significance for breeding wheat varieties of drought resistance, for what we should explore available drought-resistant gene from wild relatives germplasm resources, and for exploring its relationship with plant drought resistance. Aegilops tauschii is the diploid ancestors of common wheat, which contains excellent genetic resources of biological and abiotic stress resistance. Previously, we found significant differences expression of dihydrogen flavonoids-4-reductase(DFR) gene of XJ2 and XJ98 through seedling drought resistance identification and transcriptome analysis. To expose the relationship between the DFR gene and plant drought resistance, we studied DFR gene from two Aegilops tauschii samples by cloning, function prediction, vitro expression and fluorescence quantitative PCR analysis, and analyzed the enzymatic dynamics of DFR that extracted from XJ2 before and after the drought stress. Through this study, we hope drought-resistant germplasm resources of Aegilops tauschii can be effectively used in developing drought-resistant wheat varieties. The experimental results are as follows:(1) Real-time fluorescent quantitative PCR analysis of DFR gene of XJ2 and XJ98 showed that consistent with the transcriptome, drought stress induced a up-regulation of DFR in XJ2 while the transcription level in XJ98 did not change significantly.(2) The cloned sequences of DFR gene from XJ2 and XJ98 showed that they both have 6 exons and 5 introns, while the DFR gene of XJ98 is a pseudogenes because of an eight-nucleotides sequence missing in exon. The DFR gene of XJ2 can be encoded 347 amino acid residues and has the highest similarity(95%) with the DFR gene of barley. The bioinformatics analysis showed that the protein that DFR gene encoded may be hydrophobic protein, and ? helix and random coil as its main secondary structure elements, and its function structure domain including the short chain dehydrogenase/reductase(SDR) family locus which belong to NADB-Rossmann family.(3) Using HPLC to detect the enzymatic reaction of DFR gene before and after the drought stress in XJ2, and results showed that there is a obvious peak of DFR in enzymatic reaction product after drought stress. It suggests that when plants under drought stress conditions DFR gene may play a role on the drought resistance of plants.(4) Prokaryotic and eukaryotic expression vector of DFR gene were constructed. SDS-PAGE electrophoresis and western blot analysis showed that the expression of DFR gene can be induced by 0.2 mmol/L IPTG and the quantity of induced protein tends to be stable after 5 h.