Identification and functional characterisation of aquaporins in the grapevine, Vitis vinifera.
Identification and functional characterisation of aquaporins in the grapevine, Vitis vinifera.
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DOI：
10.1071/fp09117
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发表时间：
2009-12
期刊：
影响因子：
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通讯作者：
M. Shelden;S. Howitt;B. Kaiser;S. Tyerman
中科院分区：
文献类型：
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作者：
M. Shelden;S. Howitt;B. Kaiser;S. Tyerman
植物水通道蛋白属于一个保守蛋白超家族，称为主要内在蛋白（MIP）。关于葡萄藤中MIP的多样性及其水分运输能力的信息有限。本研究的目的是确定从葡萄藤和功能性的水转运的一个子集的MIP的MIP。通过对葡萄属酿酒葡萄（vinifera L.）（cv.用基因特异性探针构建了赤霞珠（Cabernet Sauvignon）cDNA文库，用于编码质膜内在蛋白（PIP）和液泡膜内在蛋白（TIP）亚家族成员的水通道蛋白cDNA。筛选鉴定出11个全长和2个部分长度的水通道蛋白cDNA。VvTIP2; 1同种型具有不同的3'UTR，紧邻poly（A）尾的上游，表明存在多聚腺苷酸化的多个切割位点。利用已发表的葡萄藤组序列，我们进行了系统发育分析的MIP与先前表征的MIP从拟南芥。我们从酿酒葡萄近纯合品系（PN 40024）的基因组序列中鉴定了23个全长MIP基因，其聚类到在其他物种中鉴定的四个主要亚科（和亚组内）。然而，基于在赤霞珠中不存在于PN 40024基因组中的PIP 2基因的鉴定，在其他杂合葡萄藤品种中可能存在超过23个MIP基因。通过在非洲爪蟾卵母细胞中表达，测定了几种PIP和TIP的水转运能力。只有VvPIP 2和VvTIP蛋白作为水通道的功能，除了VvPIP 2;5. VvPIP 2; 5与水传导VvPIP 2;1的不同之处在于环B中的两个高度保守的氨基酸（G97 S，G100 W）的取代，这通过同源性建模显示可能形成水孔的疏水块。
Plant aquaporins belong to a large superfamily of conserved proteins called the major intrinsic proteins (MIPs). There is limited information about the diversity of MIPs in grapevine, and their water transport capacity. The aim of the present study was to identify MIPs from grapevine and functionally characterise water transport of a subset of MIPs. Candidate genes were identified, by screening a Vitis vinifera L. (cv. Cabernet Sauvignon) cDNA library with gene specific probes, for aquaporin cDNAs encoding members of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies. The screen resulted in the identification of 11 full-length and two partial length aquaporin cDNAs. VvTIP2;1 isoforms had different 3' UTRs, immediately upstream of the poly(A) tail, suggesting the presence of multiple cleavage sites for polyadenylation. Using published genome sequences of grapevine, we conducted a phylogenetic analysis of the MIPs with previously characterised MIPs from Arabidopsis. We identified 23 full-length MIP genes from the V. vinifera genome sequence of a near homozygous line (PN40024) that cluster into the four main subfamilies (and subgroups within) identified in other species. However, based on the identification of PIP2 genes in Cabernet Sauvignon that were not present in the PN40024 genome, there are likely to be more than 23 MIP genes in other heterozygous grapevine cultivars. Water transport capacity was determined for several PIPs and TIPs, by expression in Xenopus oocytes. Only VvPIP2 and VvTIP proteins function as water channels with the exception of VvPIP2;5. VvPIP2;5 differs from the water conducting VvPIP2;1 by the substitution of two highly conserved amino acids in Loop B (G97S, G100W), which was shown by homology modelling to likely form a hydrophobic block of the water pore.