橡胶树水通道蛋白HbPIP1;4的亚细胞定位与多聚化分析

Subcellular Localization and Multimerization Analyses of HbPIP1;4,a PIP Aquaporin from Rubber Tree(Hevea brasiliensis Muell.Arg.)

质膜内在蛋白(PIP)隶属于水通道蛋白家族,其以细胞膜定位并具有高效的水分转运活性而著称。PIP高度保守,主要包含PIP1和PIP2两个亚类。天然橡胶是重要的工业原料和战略物资,其主要来源为巴西橡胶树。作为橡胶合成和储藏的特异组织,乳管与邻近的薄壁细胞之间缺乏胞间连丝,其水分平衡主要由PIP介导。通过前期研究,团队鉴定到一个PIP1亚类成员HbPIP1;4,该基因在乳管中高水平表达,然而,其在蟾蜍卵母细胞中异源表达却无水分转运活性,推测因不能有效定位到细胞膜所致。为探讨HbPIP1;4在植物体内的功能部位及作用模式,本研究构建了其亚细胞定位和双分子荧光互补(BiFC)载体。通过利用农杆菌介导法瞬时转化烟草叶片,再用激光共聚焦显微镜进行观察,发现荧光信号出现在细胞膜,这与生物信息学预测的细胞膜定位结果一致。BiFC实验进一步证实HbPIP1;4定位在细胞膜,结果同时显示,HbPIP1;4可形成同源多聚体,这与3D结构预测结果一致。上述结果为深入揭示乳管的水分平衡机制奠定了坚实的基础。但HbPIP1;4的异源互作模式还有待进一步研究。

Plasma membrane intrinsic proteins(PIPs),which belong to the aquaporin(AQP)family within the major intrinsic protein(MIP)superfamily,are known for the high water transport activity at the cell membrane.The subfamily is highly conservative and only includes two phylogenetic groups named PIP1 and PIP2.Natural rubber,an important industrial raw material and strategic material,is mainly derived from Para rubber tree(Hevea brasiliensis Muell.Arg.),a big tree originated in the Amazon basin of South America.As a special tissue for rubber synthesis and storage,the laticiferous cells possess no plasmodesmata with neighboring parenchyma cells,and the water balance is mainly mediated by PIP aquaporins.In previous studies,a highly abundant PIP gene denoted HbPIP1;4 was identified,however,no detectable water transport activity was found when heterologously expressed in Xenopus laevis oocytes,which may be due to its inability to localize to the cell membrane.To uncover the acting site and multimerization features of HbPIP1;4 in vivo,recombinant plasmids pNC-Cam1304-SubN-HbPIP1;4,pNC-BiFC-ECN-HbPIP1;4,and pNC-BiFC-ENNHbPIP1;4 for subcellular localization and bimolecular fluorescence complementation(BiFC)were successfully constructed.Agrobacterium tumefaciens transformed with above recombinant plasmids were used to infiltrate tobacco(Nicotiana benthamiana),and transiently transformed leaves were subsequently checked using laser confocal microscopy.Compared with wide distribution of fluorescence signals throughout the cell for the control pNC-Cam1304-SubN,signals of pNC-Cam1304-GFP-HbPIP1;4 were restricted to the cell membrane,supporting the plasma membrane localization of HbPIP1;4,which is consistent with the bioinformatics prediction.The result was further supported by the BiFC analysis,which also implied that HbPIP1;4 could form homomultimer,in accordance with the 3D prediction via homology modeling.The results would lay a solid foundation for further uncovering the mechanism of water balance of the laticifer.Nevertheless,PIP aquaporins that could interact with HbPIP1;4 in rubber tree remain to be further characterized.