Lu Fengzhong, Wang Kexin, Yan Lamei, Peng Yalin, Qu Jingtao, Wu Jing, Cao Yang, Yang Qingqing, Fu Fengling, Yu Haoqiang
Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Chengdu, 611130 People's Republic of China.
Physiol Mol Biol Plants. 2020 Nov;26(11):2189-2197. doi: 10.1007/s12298-020-00910-2. Epub 2020 Nov 18.
The clade A members of serine/threonine protein phosphatase 2Cs (PP2Cs) play crucial roles in plant growth, development, and stress response via the ABA signaling pathway. But little is known about other PP2C clades in plants. Our previous study showed that maize the , a clade B member of , negatively regulated drought tolerance in transgenic . However, the upstream regulatory mechanism of remains unclear. In the present study, the expression of gene in maize was analyzed by quantitative real time PCR (qRT-PCR). The results showed that the expression of in shoot and root was both significantly inhibited by drought stress. Subsequently, a 2175 bp promoter of was isolated from maize genome ( ). To validate whether the promoter possess some key -element and negatively drive expression in drought stress, three 5´-deletion fragments of 1505, 1084 and 215 bp was amplified from and were fused to β-glucuronidase () and luciferase gene () to produce and constructs, and transformed into tobacco, respectively. Transient expression assays indicated that all promoters could drive and expression. The GUS and LUC activity were both significantly inhibited by PEG-6000 treatment. Notably, the - 1084 to - 215 bp promoter possess one MBS element and inhibits the expression of and under drought stress. Meanwhile, we found that the 215 bp length is enough to drive expression. These findings will provide insights into understanding the transcription-regulatory mechanism of negatively regulating drought tolerance.
丝氨酸/苏氨酸蛋白磷酸酶2C（PP2C）的A类成员通过脱落酸信号通路在植物生长、发育和胁迫响应中发挥关键作用。但对于植物中其他PP2C类知之甚少。我们之前的研究表明，玉米PP2C的B类成员ZmPP2C4在转基因植物中负调控耐旱性。然而，ZmPP2C4的上游调控机制仍不清楚。在本研究中，通过定量实时PCR（qRT-PCR）分析了ZmPP2C4基因在玉米中的表达。结果表明，干旱胁迫显著抑制了ZmPP2C4在地上部和根部的表达。随后，从玉米基因组中分离出ZmPP2C4的2175 bp启动子（pZmPP2C4）。为了验证该启动子是否具有一些关键元件并在干旱胁迫下负向驱动ZmPP2C4表达，从pZmPP2C4中扩增出1505、1084和215 bp的三个5´-缺失片段，并分别与β-葡萄糖醛酸酶（GUS）和荧光素酶基因（LUC）融合，构建p1505GUS、p1084GUS和p215GUS以及p1505LUC、p1084LUC和p215LUC构建体，并转化到烟草中。瞬时表达分析表明，所有启动子都能驱动GUS和LUC表达。PEG-6000处理显著抑制了GUS和LUC活性。值得注意的是，-1084至-215 bp启动子具有一个MBS元件，并在干旱胁迫下抑制GUS和LUC的表达。同时，我们发现215 bp的长度足以驱动GUS表达。这些发现将为理解ZmPP2C4负调控耐旱性的转录调控机制提供见解。
