橡胶树HbPIP2;3的亚细胞定位与多聚化分析

Subcellular Localization and Multimerization Analyses of HbPIP2;3,an Efficient Water Transporter from Hevea brasiliensis

水通道蛋白是一类高效转运水分子的膜内在蛋白,其在生物膜上以四聚体的形式起作用。天然橡胶在橡胶树的乳管细胞中特异合成,并在割胶过程以胶乳的形式被排出。胶乳系乳管细胞的胞质成分,其含水量高达70%,水分通过调节胶乳的粘稠度和乳管膨压进而影响橡胶树的产排胶能力,是决定胶乳产量的关键因素。前期研究显示,橡胶树乳管的水分平衡主要由质膜内在蛋白(plasma membrane intrinsic protein,PIP)特别是HbPIP2;3介导。为揭示HbPIP2;3调控乳管水分平衡的分子机制,本研究采用RT-PCR技术对其861 bp的编码区进行分离。序列分析显示:HbPIP2;3预测编码286个氨基酸,理论分子量为30.58 kDa,等电点为8.50,不稳定系数为31.68,总平均疏水指数为0.449,为稳定的疏水型碱性蛋白;该蛋白含有保守的MIP结构域,其中包括6个典型的跨膜螺旋和2个半螺旋;基于同源建模的3D结构预测显示其可以形成同源四聚体。生物信息学预测和在烟草叶片中的亚细胞定位分析显示,HbPIP2;3定位在细胞膜,这同时也得到了双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验的证实。BiFC实验显示,HbPIP2;3可在细胞膜上形成同源四聚体,这进一步得到酵母双杂交结果的验证。本研究结果表明,HbPIP2;3可通过同源四聚体的方式调控乳管的水分平衡,但是否存在异源互作模式还有待进一步研究。

Aquaporins(AQPs),assembled in tetramers at the biological membrane,constitute a class of integral membrane proteins facilitating the passive transport of water.Natural rubber,which is specifically synthesized in the laticifer located in the secondary phloem of the rubber tree(Hevea brasiliensis)trunk,is expelled out in the form of latex that represents the cytoplasmic content of laticiferous cells upon tapping.Water,accounting for approximately 70%of the latex upon each tapping,plays a crucial role in the latex production by regulating the latex viscosity and laticifer turgor.Previous studies suggested that the water balance of laticifers is governed by plasma membrane intrinsic proteins(PIPs)especially HbPIP2;3,an efficient and abundant water transporter.To uncover the regulation mechanism of HbPIP2;3 in the laticifer water balance,its 861 bp full-length coding sequence(CDS)was isolated using RT-PCR.Sequence analysis revealed that HbPIP2;3 putatively encoded 286 amino acids with the theoretical molecular weight(MW)of 30.58 kDa,the isoelectric point(pI)of 8.50,the instability index(II)of 31.68,and the grand average of hydropathicity(GRAVY)of 0.449,implying its basic,hydrophobic,and stable features.The protein was shown to contain one conserved MIP(major intrinsic protein)domain,which including six typical transmembrane helices(i.e.TM1-6)as well as two half helices(i.e.HB and HE).3D prediction via homology modeling suggested that HbPIP2;3 could assemble in homotetramer.In accordance with the bioinformatics prediction,transient over-expression of HbPIP2;3 in Nicotiana benthamiana leaves supported the cell membrane localization,which was also confirmed by bimolecular fluorescence complementation(BiFC).Moreover,BiFC revealed that HbPIP2;3 could interact itself,which was further confirmed by yeast two-hybrid.The results showed that HbPIP2;3 may be involved in the laticifer water balance in the form of homotetramer at the plasma membrane,however,whether it could also function in hereotetramer still needs to be addressed.