橡胶树HbPIP1;1基因的克隆及蛋白的亚细胞定位与多聚化分析

水通道蛋白（aquaporin）是一类广泛存在生物体中高效转运水分子的膜内在蛋白,其在生物膜上以同源或异源四聚体的形式起作用。根据序列相似性及亚细胞定位,植物水通道蛋白可分为PIP （plasma membrane intrinsic protein）、TIP（tonoplast intrinsic protein）、NIP（NOD26-like intrinsic protein）、SIP（small basic intrinsic protein）和XIP（X intrinsic protein）等5大类,其中,PIP定位在细胞膜,是介导细胞间水分跨膜运输的主要通道。橡胶树最早起源于南美亚马逊河流域,是目前天然橡胶的主要商业来源。与其他植物相比,橡胶树除蒸腾耗水外,周期性的割胶活动也会造成水分的大量流失,因此,水分平衡对于橡胶树尤为重要。为揭示橡胶树水分平衡的分子机制,采用RT-PCR技术对1个PIP类水通道蛋白基因HbPIP1;1进行克隆,并在此基础上对其编码蛋白的亚细胞定位和多聚化特征进行分析。结果显示：该基因的编码区长864 bp,预测编码287 aa,其理论分子量为30.80 kDa、等电点8.59、不稳定系数49.27、脂肪族指数22.34、总平均疏水指数为0.639,属于不稳定的疏水型碱性蛋白;蛋白含有水通道蛋白家族特有的MIP（major intrinsic protein）结构域及6个典型的跨膜螺旋,每个螺旋的残基数介于20~23之间。研究构建了基因的亚细胞定位分析载体,并采用农杆菌介导法对烟草叶片进行了瞬时转化。荧光共聚焦显微镜观察显示,HbPIP1;1蛋白定位在细胞膜,与用生物信息学手段预测的结果一致。在烟草叶片中的双分子荧光互补实验显示,HbPIP1;1蛋白自身不能互作,这进一步得到了点对点的酵母双杂交实验的证实。以上结果表明HbPIP1;1蛋白可能通过异源互作的方式参与橡胶树水分的平衡。

Gene Cloning,Subcellular Localization and Multimerization Analysis of HbPIP1;1 from Hevea brasiliensis

Aquaporins (AQPs), a class of integral membrane proteins facilitating the passive transport of water, are widely present in all living organisms. Evidence shows that AQPs function in homotetramers or hereotetramers in biological membranes. On the basis of the sequence similarity and subcellular localization, plant AQPs could be divided into five main subfamilies, i.e. PIP (plasma membrane intrinsic protein), TIP (tonoplast intrinsic protein), NIP (NOD26-like intrinsic protein), SIP (small basic intrinsic protein), and XIP (X intrinsic protein). Among them, PIPs, which are located in the plasma membrane, represent the main channel mediating water transport between cells. Para or Brazilian rubber tree (Hevea brasiliensis Muell. Arg.), a perennial big tree native to the Amazon basin, is the main commercial source of natural rubber currently. Compared with other plants, the water balance is particularly important for rubber tree, because a large amount of water loss could be caused by periodic bark-tapping as well as transpiration. Previous studies showed that the rubber tree genome encodes a high number of 51 AQP genes, which include five PIP1s and ten PIP2s. To uncover the molecular mechanism of PIP-mediated water balance in rubber tree, a key gene named HbPIP1;1 was cloned using the RT-PCR (reverse transcription polymerase chain reaction) technique, followed by investigation of the subcellular localization and multimerization of its coding peptide. Results showed that the CDS (coding sequence) length of HbPIP1;1 is 864 bp (base pairs), putatively encoding 287 aa (amino acids), which includes a MIP (major intrinsic protein) domain specific to the AQP family; it was predicted to be an instable, hydrophobic, and basic protein that harbors the theoretical molecular weight (Mw) of 30.80 kDa, the isoelectric point (pI) of 8.59, the instability index (II) of 49.27, the aliphatic index (AI) of 22.34, and the grand average of hydropathicity (GRAVY) value of 0.639; it was also shown to contain six transmembrane regions that harbor 20-23 residues. Moreover, various expression vectors for subcellular localization, bimolecular fluorescence complementation, and yeast two-hybrid were constructed. Consistent with the bioinformatics analysis, transient overexpression of HbPIP1;1 in tobacco (Nicotiana tabacum) leaves via the Agrobacterium tumefaciens-medicated transformation revealed that the protein is located in the plasma membrane. Further bimolecular fluorescence complementation and yeast two-hybrid experiments showed that HbPIP1;1 could not form a homomultimer. The findings presented in this study suggest that HbPIP1;1 may be involved in water balance in the form of heteromultimer, though detailed mechanisms are to be further studied.