过表达二穗短柄草BdDREB1-like基因提高烟草的抗氧化能力

Enhanced Oxidative Stress Tolerance of Transgenic Tobacco Plants by Overexpression of Brachypodium distachyon BdDREB1-like Gene

为了研究二穗短柄草BdDREB1-like基因在转基因烟草中的功能,克隆了该基因,并进行了生物信息学分析。该基因开放阅读框684 bp,编码228个氨基酸,编码蛋白的分子量为24.178 ku,只含有一个典型的AP2结构域,属于AP2/EREBP(APETALA2/ethylene-responsive element binding protein)超家族的DREB家族。多序列比对与进化分析显示它与黑麦ScCBFI的亲缘关系较近。亚细胞定位分析表明,BdDREB1-like基因定位于细胞核。将其构建到植物表达载体上,并通过农杆菌介导法转入烟草中。PCR和RT-PCR筛选鉴定显示BdDREB1-like基因不仅整合到烟草基因组中而且还能稳定表达。过氧化氢胁迫条件下,转BdDREB1-like基因烟草幼苗具有较高的发芽率。为了进一步确定BdDREB1-like基因与氧化胁迫的关系,又对一个月大的转基因烟草进行氧化胁迫(MV,甲基紫精)处理。结果表明,在正常生长条件下,T3转基因株系除了SOD含量外,测定的其他各种生理指标均与野生型无显著差异,T1转基因株系中只有POD和叶绿素含量与野生型中的有显著差异;而在氧化胁迫处理条件下,转BdDREB1-like基因的烟草植株相对于野生型具有较高的过氧化氢酶、过氧化物酶、超氧化物歧化酶和叶绿素的含量和较低的丙二醛和过氧化氢含量。这些结果表明,过表达BdDREB1-like基因增强了植物对氧化胁迫的耐受性。

In order to explore the function of BdDREB1-like gene in transgenic tobacco, it was cloned and analyzed through bioinformatics technology. The open reading frame of this gene was 684 bp, encoded 228 amino acids, and the molecular weight of its encoding protein was 24.178 ku. It contained only one typical AP2 domain and belonged to the DREB family of AP2/EREBP superfamily. Multiple sequence alignment and evolutionary analysis showed that it was closely related to Rye ScCBFI. Subcellular localization analysis showed that BdDREB1-like was located in the nucleus. It was then constructed into plant expression vector and transformed into tobacco by Agrobacterium-mediated method to further confirm its function. PCR and RT-PCR screening showed that BdDREB1-like gene was not only integrated into tobacco genome, but also expressed stably. Transgenic tobacco seedlings with BdDREB1-like gene had higher germination rate under hydrogen peroxide stress. In order to further determine the relationship between BdDREB1-like gene and oxidative stress, we treated the one-month-old transgenic tobacco plants under oxidative stress with MV(Methyl viologen). The results showed that there were no significant differences in the contents of hydrogen peroxide, catalase and other physiological indicators except for SOD between wild type and T3 transgenic line, while there were only two physiological indexes of POD and chloroplast content of T1 transgenic line, which were significantly different from those of wild type under normal growth condition. Under oxidative stress, transgenic tobacco plants with BdDREB1-like gene had lower malondialdehyde and hydrogen peroxide content, higher peroxidase activity, catalase, superoxide dismutase and chlorophyll content compared with wild type. These results suggested that overexpression of BdDREB1-like gene enhanced the tolerance of plant to oxidative stress.