拟南芥H~+_-焦磷酸化酶AVP1互作小GTP结合蛋白AtRAB的特性鉴定与功能分析

以拟南芥AVP1为诱饵,采用膜蛋白酵母双杂交系统筛选拟南芥cDNA文库,获得一个与AVP1互作的小GTP结合蛋白AtRAB。酵母互作试验表明,AVP1和AtRAB存在互作;双分子荧光互补实验(BiFC)证明,AVP1和AtRAB能在质膜和细胞核上发生相互作用。野生型拟南芥(WT)、AtRAB和AVP1的拟南芥突变体rab和avp1在高盐胁迫条件下,随着NaCl浓度的加大,突变体rab和avp1的表型变化相似,相对于WT都表现为根长缩短,侧根数减少;在低磷处理条件下,随着磷浓度的逐渐降低,2个突变体的表型相似,相对于WT同样表现为根长缩短,总根面积减少,侧根数减少;在相同磷浓度条件下,突变体rab对胁迫的反应比avp1强;在低钾处理条件下,随着钾浓度的降低,2个突变体的表型与在低磷胁迫处理条件下的表型相似。结果说明,AVP1和AtRAB可以在细胞膜和细胞核上相互作用,AtRAB能够影响植物对离子的吸收,AVP1和AtRAB的突变体在高盐、低磷和低钾等胁迫条件下表型变化相似,证明了2个基因都正向调控植物对高盐、低磷和低钾胁迫的反应,它们属于同一信号途径。

Characterization and Functional Analysis of a Small GTP-binding Protein AtRAB Interacting with H+-Pyrophosphatase AVP1 in Arabidopsis thaliana

H⁺-Pyrophosphatase (H⁺-PPase) is an important proton transporter in plants. It cooperates with H⁺-ATPase and transport protons in vacuole or extracellular area to maintain a constant H⁺ gradient, which enables the transport of ions and other components (e.g. amino acids, carbohydrates). In the current research, AVP1 was applied to membrane proteins-based yeast two-hybrid system, and a small GTP-binding protein AtRAB was identified by screening the Arabidopsis cDNA library. The interaction between AVP1 and AtRAB was confirmed by interaction analysis in yeast. Bimolecular fluorescent complementation (BiFC) analysis suggested that AtRAB and AVP1 interaction took place in the plasma membrane and the nucleus. Phenotypes of wild type (WT) and Arabidopsis mutants of avp1 (AVP1) and rab (AtRAB) were compared under high-salt, low-phosphorus and low-potassium conditions. Under the high salt stress, avp1 and rab presented similar phenotypes along with the increase of NaCl concentration, which were characterized by shortened root length and reduced lateral root number in comparison with the WT. The decreasing phosphorus concentration also led to the phenotypes of shortened root length, reduced lateral root number and reduced total root area in the two mutants. Particularly, stronger response was observed in rab than in avp1 under the same phosphorus concentration. The potassium treatments resulted in similar phenotypes to those in phosphorus treatment. The results indicated that AVP1 can interact with AtRAB on the plasma membrane and the nucleus and further influence the absorption of ions in plants. The two mutants of avp1 and rab showed similar phenotypes, suggesting that both AVP1 and AtRAB positively regulate plant response to high salt, low phosphorus and low potassium stresses in the same signaling pathway.