过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性

本文克隆了RIN4(RPM1-interacting protein 4)在胡杨中的同源基因PeRIN4，并在拟南芥中进行过表达，通过研究转基因株系的耐盐表型、质膜H+-ATPsae活性及H+ 、Na+、K+等的动态离子流，揭示了PeRIN4基因在植物响应和适应盐胁迫环境中的作用。利用定位载体pGreen0029-PeRIN4-GFP瞬时转化拟南芥叶肉细胞原生质体的方法，对胡杨PeRIN4蛋白进行亚细胞定位，发现该蛋白定位在细胞的胞质中。耐盐表型实验结果显示，在100 mmol/L NaCl处理下，拟南芥PeRIN4过表达株系(OE1和OE8)的生存率和根长均明显高于野生型(WT)和转空载体拟南芥(VC)，说明PeRIN4基因能够提高拟南芥的耐盐性。与WT和VC相比，拟南芥PeRIN4过表达株系质膜H+-ATPsae的活性较高。动态离子流数据显示，在盐胁迫下，PeRIN4过表达株系外排H+和Na+ 离子的能力强于野生型和转空载体拟南芥，然而K+的外流却弱于WT和VC。因此，PeRIN4蛋白具有调节质膜H+-ATPsae活性的功能。拟南芥质膜H+-ATPsae活性的提高主要有两方面的作用:一是可以增强H+泵的质子动力势，驱动Na+/H+逆向转运蛋白，提高Na+外排的能力；二是抑制质膜的去极化，减少K+离子通过去极化激活的外向型K+通道(DA-KORCs)和非选择性阳离子通道(DA-NSCCs)外流，维持了K+/Na+平衡，从而提高PeRIN4转基因拟南芥的耐盐性。 

Over-expression of PeRIN4 enhanced salinity tolerance through up-regulation of PM H+-ATPase in Arabidopsis thaliana

In the study, a homolous gene encoding RIN4 protein was cloned from Populus euphratica. To clarify the role of PeRIN4 in salinity stress, PeRIN4 was introduced to Arabidopsis thaliana and plant response to NaCl was examined in transgenic plants. Salt-resistant phenotypes, activity of plasma membrane ( PM) H+-ATPase and flux profiles of H+, Na+, K+were screened in this work. Subcellular localization of PeRIN4 was determined by transforming the fusion protein of pGreen0029-PeRIN4-GFP into arabidopsis mesophyll protoplast. Results showed that PeRIN4 was localized in the cytoplasm. Seven-day-old seedlings of wild-type ( WT) , vector control ( VC) and PeRIN4-transgenic arabidopsis were exposed to 100 mmol/L NaCl saline. PeRIN4-transgenic lines showed higher survival rates and root length than WT and VC plants. These results indicated that over-expression of PeRIN4 improved salt tolerance in arabidopsis. In addition, PeRIN4-transgenic arabidopsis exhibited a greater hydrolytic activity of PM H+-ATPase compared with WT and VC. Moreover, PeRIN4-transgenic roots showed a higher H+ and Na+efflux than WT and VC, but displayed a less K+ loss under salinity conditions. We concluded that PeRIN4 could improve the activity of PM H+-ATPase under salinity environment. Under NaCl stress, the activated PM H+-ATPase could sustain an H+ gradient to drive the Na+/H+ antiport across the PM, thus enhancing Na+ exclusion, and preserve a less-depolarized membrane potential, thus restricting the K+efflux through depolarization-activated outward rectifying K+ channels and non-selective cation channels. Our data demonstrate that PeRIN4 may improve salt adaptation in transgenic arabidopsis through controlling K+/Na+ homeostasis under salinity environment.