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

doi:10.3969/j.issn.1000-2561.2022.12.002

Abstract

Abstract:

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

CLC Number:

S794.1

QIAO Xueying, ZHENG Yujiao, YANG Jianghua, ZENG Changying, ZOU Zhi. Gene Cloning, Subcellular Localization and Multimerization Analysis of HbPIP1;1 from Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2405-2412.

Figures/Tables 8

References 24

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目的 Use	引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)
基因克隆	HbPIP1;1F	ATGGAGGGCAAGGAAGAAGATG
基因克隆	HbPIP1;1R	TCACTTCTTGAAAGGGATGGCT
亚细胞定位	pCam1304-HbPIP1;1F	AGTGGTCTCTGTCCAGTCCTATGGAGGGCAAGGAAGAAGATG
亚细胞定位	pCam1304-HbPIP1;1F	GGTCTCAGCAGACCACAAGTCTTCTTGAAAGGGATGGCT
酵母双杂交	pGADT7-HbPIP1;1F	TACGACGTACCAGATTACGCTATGGAGGGCAAGGAAGAAGATG
	pGADT7-HbPIP1;1R	CAGTATCTACGATTCATCTGCTCACTTCTTGAAAGGGATGGCT
	pGBKT7-HbPIP1;1F	ATCTCAGAGGAGGACCTGATGGAGGGCAAGGAAGAAGATG
	pGBKT7-HbPIP1;1R	TGCGGCCGCTGCAGGTCGTCACTTCTTGAAAGGGATGGCT
双分子荧光	pNC-BiFC-HbPIP1;1F	AGTGGTCTCTGTCCAGTCCTATGGAGGGCAAGGAAGAAGATG
双分子荧光	pNC-BiFC-HbPIP1;1R	GGTCTCAGCAGACCACAAGTCTTCTTGAAAGGGATGGCT

目的 Use	引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)
基因克隆	HbPIP1;1F	ATGGAGGGCAAGGAAGAAGATG
基因克隆	HbPIP1;1R	TCACTTCTTGAAAGGGATGGCT
亚细胞定位	pCam1304-HbPIP1;1F	AGTGGTCTCTGTCCAGTCCTATGGAGGGCAAGGAAGAAGATG
亚细胞定位	pCam1304-HbPIP1;1F	GGTCTCAGCAGACCACAAGTCTTCTTGAAAGGGATGGCT
酵母双杂交	pGADT7-HbPIP1;1F	TACGACGTACCAGATTACGCTATGGAGGGCAAGGAAGAAGATG
	pGADT7-HbPIP1;1R	CAGTATCTACGATTCATCTGCTCACTTCTTGAAAGGGATGGCT
	pGBKT7-HbPIP1;1F	ATCTCAGAGGAGGACCTGATGGAGGGCAAGGAAGAAGATG
	pGBKT7-HbPIP1;1R	TGCGGCCGCTGCAGGTCGTCACTTCTTGAAAGGGATGGCT
双分子荧光	pNC-BiFC-HbPIP1;1F	AGTGGTCTCTGTCCAGTCCTATGGAGGGCAAGGAAGAAGATG
双分子荧光	pNC-BiFC-HbPIP1;1R	GGTCTCAGCAGACCACAAGTCTTCTTGAAAGGGATGGCT

反应 Reaction	AD质粒 AD plasmid	BD质粒 BD plasmid	涂板种类 Type of coating	备注 Note
1	pGADT7- LargeT	pGBKT7- 53 m	SD-TL、SD-TLHA（SD-TLH）	阳性对照（自激活）
2	pGADT7- LargeT	pGBKT7- LaminC	SD-TL、SD-TLHA（SD-TLH）	阴性对照（自激活）
3	pGADT7	pGBKT7- HbPIP1;1	SD-TL、SD-TLH、SD-TLHA	自激活
4	pGADT7- HbPIP1;1	pGBKT7- HbPIP1;1	SD-TL、SD-TLHA	实验组

反应 Reaction	AD质粒 AD plasmid	BD质粒 BD plasmid	涂板种类 Type of coating	备注 Note
1	pGADT7- LargeT	pGBKT7- 53 m	SD-TL、SD-TLHA（SD-TLH）	阳性对照（自激活）
2	pGADT7- LargeT	pGBKT7- LaminC	SD-TL、SD-TLHA（SD-TLH）	阴性对照（自激活）
3	pGADT7	pGBKT7- HbPIP1;1	SD-TL、SD-TLH、SD-TLHA	自激活
4	pGADT7- HbPIP1;1	pGBKT7- HbPIP1;1	SD-TL、SD-TLHA	实验组