Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3–A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the β-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance.

脱水应答元件结合蛋白（dehydration responsive element binding, DREB）转录因子已有研究报道参与植物胁迫应答过程。当前多数研究聚焦于草本植物中A-1和A-2亚组的DREB基因，而针对A-3至A-6亚组DREB的功能以及木本植物中该基因家族的相关研究仍较为稀缺。桑树作为兼具生态与经济价值的多年生木本植物，其胁迫生理、生化及分子生物学领域的研究尚且不足。本研究选取本课题组前期鉴定并归类至A-4亚组的桑树DREB基因MnDREB4A，开展进一步的功能表征工作。研究结果显示，MnDREB4A蛋白定位于细胞核，且可激活转录过程。MnDREB4A的启动子能够在热、冷、干旱及盐胁迫条件下，驱动下游β-葡萄糖苷酸酶（β-glucuronidase, GUS）基因高效表达，且在胁迫处理12小时后GUS染色信号最强。过表达MnDREB4A的转基因烟草在正常培养条件下即呈现出更优良的生长表型：叶片色泽更浓绿、根系更长，同时水分流失速率与衰老速率更低；在热、冷、干旱及盐胁迫环境中，该转基因烟草的胁迫耐受性显著提升，与野生型植株相比，转基因植株的叶圆片与幼苗在各类胁迫条件下均表现出更轻微的叶片萎蔫与衰老症状。进一步的检测表明，无论在正常培养还是胁迫处理条件下，转基因植株均拥有更高的水分含量与脯氨酸含量，而丙二醛含量更低。综合上述结果可知，MnDREB4A蛋白在植物胁迫耐受过程中发挥着重要的调控作用。