The Improvement Of Wheat Ubiquitin Gene Ta-Ub2 In The Abiotic Stress Tolerance Of Brachypodium Distachyon

Ubiquitin is a small, highly conserved, ubiquitously expressed eukaryotic protein. The ubiquitin-26 S proteasome system(UPS) plays an important role in the resistance of plants to abiotic stresses. Ubiquitin acts as a central component in this pathway.Our early research shows that wheat ubiquitin gene Ta-Ub2 plays an important role in the response to abiotic stress in transgenic tobacco and the overexpression of Ta-Ub2 promotes the development of transgenic tobacco. In this study, on the basis of cloning Ta-Ub2 gene, many transgenic Brachypodium plants overexpressing Ta-Ub2 under the control of the CaMV35 S and stress-inducible RD29 A promoter were constructed. We investigated the Ta-Ub2's response to abiotic stress and the effects of Ta-Ub2 on the growth and development in the transgenic Brachypodium distachyon plants. Then we researched the probable physiological and biochemical mechanism underlying the enhanced abiotic stress tolerance in the Brachypodium distachyon plants. The results are as follows:(1) Ta-Ub2 was shown to be expressed ubiquitously in all the tested tissues, including root, culm and leaf by qRT-PCR. Ta-Ub2 exhibited a highest expression level in leaf, whereas had a low expression in culm and root. The transcript level of Ta-Ub2 could be induced by PEG, NaCl and 4?C.(2) 35S:TaUb2 and RD29A:TaUb2 transgenic Brachypodium distachyon plants were obtained and identified by qRT-PCR and genomic PCR, which indicating that transgenic lines with different Ub transgene levels were obtained. Immunology analysis with ubiquitin antiserum indicated that the ubiquitin levels were not significantly alters under normal conditions. But the level of conjugated ubiquitin was significantly higher in the transgenic Brachypodium plants under drought stress.(3) Under control conditions, 35S:TaUb2 transgenic plants showed growth retardation at seedling stage compared with WT. In addition, the thousand-grain weight of these 35S:TaUb2 transgenic plants was less than that of the WT plants. However, there were no differences between RD29A:TaUb2 transgenic Brachypodium distachyon and WT plants.(4) The overexpression of Ta-Ub2 improved the drought, salt and chilling stress tolerance of transgenic Brachypodium under abiotic stresses. Under drought stress, compared with WT, all the transgenic plants had higher RWC, higher survival rate, a lower water loss rate and biomass than the WT plants. At the same time, the transgenic plants accumulated more soluble sugars and proline, compared to the WT plants. Based on the above results, osmotic adjustment by accumulation of more compatible solutes might be involved in the improved drought tolerance in transgenic Brachypodium plants.(5) Under drought stress, the transgenic plants displayed increased antioxidant tolerance with less H2O2 accumulation and O2?- production rate and lower MDA content and protein carbonylation. The transgenic plants had higher activities of antioxidant enzymes. These results indicate that the overexpression of Ta-Ub2 improved the active oxygen scavenging ability and effectively reduced the oxidative damage in transgenic Brachypodium plants.(6) By detecting the expression of stress related genes, including antioxidant related genes and stress related transcription factors genes. The results showed that most of those genes showed higher expression in transgenic plants compared with WT.In conclusion, overexpression of Ta-Ub2 affects the growth and development of moncot transgenic Brachypodium plants. The effect in the monocot Brachypodium is, however, different from that in the dicot tobacco. The overexpression of Ta-Ub2 improved the abiotic stress tolerance of transgenic Brachypodium under abiotic stresses. The functions of Ta-Ub2 in stress tolerance in the monocot Brachypodium was similar to that in the dicot tobacco. At the physiological and biochemical levels, the improvement in osmotic adjustment and active oxygen scavenging system by regulation of stress-responsive gene expression was involved in the enhanced stress tolerance of the transgenic plants; at the molecular level, Ta-Ub2 involved in the expression of stress related genes in transgenic plants.