Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2022, Vol. 43 ›› Issue (12): 2405-2412.DOI: 10.3969/j.issn.1000-2561.2022.12.002

• Omics & Biotechnology • Previous Articles     Next Articles

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

QIAO Xueying1,2,3, ZHENG Yujiao1,2,3, YANG Jianghua4, ZENG Changying1,*(), ZOU Zhi2,3,*()   

  1. 1. College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China
    2. Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572024, China
    3. Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions / Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
    4. Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
  • Received:2022-08-01 Revised:2022-09-15 Online:2022-12-25 Published:2023-01-12
  • Contact: ZENG Changying,ZOU Zhi


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.

Key words: Hevea brasiliensis, subcellular localization, multimerization, bimolecular fluorescence complementation, yeast two-hybrid

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