摘要
液泡膜内在蛋白(tonoplast intrinsic protein,TIP)是调控植物细胞内水分平衡的主要水通道蛋白类型,其根据进化关系可分为TIP1~TIP5等5个亚类。本研究基于前期获得的油莎豆全长转录组对1个块茎高水平表达的TIP基因(命名为CeTIP4;1)进行克隆和鉴定。序列分析显示,该基因含有2个保守的内含子,编码区全长753 bp,预测编码250个氨基酸,理论分子量为25.57 kDa、等电点(pI)为6.02、总平均疏水指数(GRAVY)为0.884,属于典型的酸性疏水型蛋白;该蛋白含有1个保守的MIP(major intrinsic protein)结构域,且具有已结晶AtTIP2;1类似的结构特征。进化分析显示,CeTIP4;1与AtTIP4;1、OsTIP4;1、OsTIP4;2和OsTIP4;3聚在一起,序列相似性为69.62%~82.94%,支持其划归为TIP4亚类。本研究在基因克隆的基础上进一步构建了其亚细胞定位载体pNC-Cam1304-CeTIP4;1。在烟草叶片中的瞬时过表达和共聚焦显微镜观察显示,CeTIP4;1定位在细胞膜。进一步的表达分析显示,CeTIP4;1具有明显的组织特异性,主要在块茎、根、匍匐茎和芽尖中表达;在起始期、膨大中期、膨大晚期和成熟期等4个典型的块茎发育时期中,CeTIP4;1基因的表达呈现先升后降的趋势,峰值出现在膨大中期,最低的是成熟期,与含水量趋势基本一致。本研究结果为深入解析油莎豆块茎的水分平衡机制奠定坚实的基础。
Tonoplast intrinsic proteins(TIPs),which comprise a subfamily of aquaporin(AQP)mediating the water balance within plant cells,could be divided into five phylogenetic groups on the basis of sequence similarity,i.e.,TIP1–TIP5.Based on the full-length transcriptome previously described in tigernut(Cyperus esculentus),a tuberabundant TIP gene named CeTIP4;1 was cloned and characterized in this study.Sequence analyses revealed that CeTIP4;1 contained two conservative introns with a complete coding sequence of 753 base pairs(bp),putatively encoding 250 amino acids with the theoretical molecular weight of 25.57 kDa,the isoelectric point(pI)value of 6.02,and the grand average of hydropathicity(GRAVY)value of 0.884,implying its acidic and hydrophobic features.The deduced peptide possessed one conservative major intrinsic protein(MIP)domain and exhibited similar structural features with the structure resolved AtTIP2;1.Classification of CeTIP4;1 into the TIP4 group was supported by the phylogenetic analysis,which showed that CeTIP4;1 clusters with AtTIP4;1,OsTIP4;1,OsTIP4;2 and OsTIP4;3 with sequence similarities of 69.62%–82.94%.After gene cloning,a plant expression vector for subcellular localization,i.e.,pNC-Cam1304-CeTIP4;1,was successfully constructed.When transiently overexpressed in tobacco(Nicotiana benthamiana)leaves,confocal laser scanning microscopy imaging showed that CeTIP4;1 was unexpectedly localized to the cell membrane.Further expression analysis indicated that CeTIP4;1 exhibited an apparent tissue-specific expression patter,predominately expressed in tuber,root,rhizome,and shoot apex.Moreover,among the four typical stages of developmental tuber examined in this study,i.e.,initiation,medium swelling,late swelling,and maturation,CeTIP4;1 exhibited a bell-like expression pattern,peaking in medium swelling tubers and lowest in maturation tubers.These results lay a solid foundation for further uncovering the mechanism of water balance in tigernut tubers.
作者
邹智
郑玉皎
ZOU Zhi;ZHENG Yujiao(National Key Laboratory for Tropical Crop Breeding/Institute of Tropical Biosciences and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China)
出处
《热带作物学报》
CSCD
北大核心
2024年第9期1823-1831,共9页
Chinese Journal of Tropical Crops
基金
海南省重点研发计划项目(No.ZDYF2024XDNY171)
国家自然科学基金项目(No.32460342,No.31971688)。
关键词
油料作物
虎坚果
块茎
液泡膜内在蛋白
亚细胞定位
表达模式
oil crop
tigernut
tuber
tonoplast intrinsic protein
subcellular localization
expression profile
