橡胶树HbPIP2;3的亚细胞定位与多聚化分析OA北大核心CSTPCD

Aquaporins(AQPs),assembled in tetramers at the biological membrane,constitute a class of integral mem-brane 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 ac-cordance 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.