慈竹细胞壁转化酶BeCWINV1的克隆、亚细胞定位和表达分析

Molecular Cloning,Subcellular Localization and Expression Analysis of a BeCWINV1 Gene in Bamboo(Bambusa emeiensis)

细胞壁转化酶(CWINVs)是糖降解关键酶之一,在植物发育、同化物分配和调节库强等方面具有重要作用。为了探究慈竹中CWINVs基因的亚细胞定位和表达水平,本研究以慈竹转录组数据库为基础,从慈竹笋中克隆到BeCWINV1基因。其开放阅读框序列长度为1758 bp,编码585个氨基酸残基。序列比对和系统进化分析表明,BeCWINV1具有细胞壁转化酶特异的活性功能位点"WECPD",并与绿竹Boβfruct1、水稻CIN2、玉米INCW2亲缘关系较近,聚在一个CWINV分支上。将BeCWINV1-GFP融合载体瞬时转化洋葱表皮细胞的研究也证明了BeCWINV1定位在细胞壁上,表明其可能参与质外体空间蔗糖的水解。实时荧光定量PCR分析表明,BeCWINV1在慈竹茎杆中表达量显著高于其他部位;额外施加蔗糖对BeCWINV1表达没有明显影响,但糖供应受到限制时其表达被显著激活。这些结果表明,BeCWINV1参与慈竹茎杆韧皮部卸载,对逆境胁迫下维持细胞内糖浓度和库活性方面有重要的作用。

Cell wall invertase(CWINV)is one of the key enzymes in sucrose degradation,which plays a key role in assimilate partitioning,the regulation of sink strength,and plant development.In order to explore the subcellular localization and expression level of CWINVs gene in bamboo speices,a BeCWINV1 gene was cloned from the shoots of B.emeiensis based on the transcriptome database.The BeCWINV1 was 1758 bp in the length of open reading frame,encoding a predicted protein of 585 amino acid.Sequence alignment and phylogenetic analysis showed that BeCWINV1 contains a cell-wall invertase active site(WECPD)motif,and shared higher identity with green bamboo Boβfruct1,rice CIN2 and maize INCW2,all of them belonging to a CWINV-specific cluster with other known CWINVs.Transient expression of the BeCWINV1-GFP fusion protein in the onion epidermis cells suggested that BeCWINV1 was located in the cell wall,indicating its role on sucrose hydrolysis in extracellular space.qRT-PCR analysis showed that the expression of BeCWINV1 was significantly higher in the stem than other tissues tested.The extra application of sucrose had no significant effect on the expression of BeCWINV1,but its expression was activated when the sugar supply was limited.These results suggested that BeCWINV1 might be involved in phloem unloading of B.emeiensis stem,and might also have an essential role in maintaining intracellular sugar concentration and sink activity under stress.