去甲基化酶基因AtROS1化学诱导表达载体的构建及其在烟草中的瞬时表达

植物去甲基化酶ROS1是表观遗传中一种重要的作用因子，参与基因的表达调控，与植物的生长发育及各种逆境响应过程密切相关。以拟南芥AtROS1基因为目的基因，采用“酶切-连接”的方法构建了以17 β 雌二醇为诱导剂的植物表达载体pER8 ROS1。将其转入农杆菌LBA4404菌株中，通过烟草瞬时表达技术，对该载体的诱导表达特性进行了研究。qPCR结果表明：17 β 雌二醇处理能够有效调控pER8 ROS1中目的基因的表达，17 β 雌二醇最佳诱导浓度为50～100 μmol·L-1；随着诱导时间的延长，目的基因表达量逐渐升高，在12 h后达到最高。该诱导表达载体的构建为研究植物抗逆胁迫过程中的表观遗传机制奠定了基础。

Construction of a chemical inducible expression vector of AtROS1 and its transient expression in tobacco

Plant demethylase ROS1 was an important factor in epigenetic regulation. It could activate the expression of genes and was closely related to the processes of plant development and various stress responses. In this study， AtROS1 gene from Arabidopsis thaliana was used as the target gene， and ‘digestion ligation method was applied for constructing plant expression vector pER8 ROS1 which could be induced by 17 β estradiol. Then the vector was transferred to Agrobacterium tumefaciens LBA4404， and the inducible expression characteristics were verified by the transient expression system of Nicotiana tabacum. The results of qPCR showed that 17 β estradiol could effectively induce the expression of the target gene in pER8 ROS1 and the optimal concentration of 17 β estradiol was 50 to 100 μmol·L-1. The expression level of ROS1 gene gradually increased and reached the highest level at 12 h. The construction of pER8 ROS1 laid a good foundation for the epigenetic mechanism study in plant environmental stress.