《植物生理学报》 2014, 50(12): 1880-1888

蓖麻质膜水通道蛋白PIP1.3在仓鼠卵巢细胞中的转运功能

董乐¹, 戴聪杰¹, 王芳^1,*, 王云^2,*, 朱国立³, 谢小宾¹, 许珊珊¹, 刘灿阳¹, 林婷¹

¹泉州师范学院化学与生命科学学院, 福建泉州362000; ²内蒙古民族大学农学院, 内蒙古通辽028042; ³通辽市农业科学院, 内蒙古通辽028015

通信作者：王芳；E-mail: dwf320@163.com, txmh@163.com；Tel: 0595-22919563, 0475-8314677

摘　要：

为研究蓖麻质膜水通道蛋白PIP1.3的转运功能, 采用RT-PCR技术扩增基因PIP1.3的全长编码区cDNA序列(861 bp), 将该序列亚克隆至真核表达载体pcDNA3.1/Myc-His A, 酶切、测序分析表明, 重组质粒pcDNA3.1PIP1.3-Myc-His阅读框架正确。将pcDNA3.1PIP1.3-Myc-His和连接氯离子敏感性绿色荧光蛋白突变体(EYFP-H148Q-V163S)的pcDNA3.1Hygro-EYFP-H148Q-V163S重组质粒稳定共转染至中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞中, RT-PCR和Western blot分析表明, PIP1.3的mRNA和蛋白质在选定CHO细胞克隆中高表达。分别通过荧光法和同位素标记的¹⁴C-甘油摄入实验测定该CHO细胞对水分和甘油的通透性, 结果表明, 该CHO细胞表达的PIP1.3对水分子的转运能力很低, 但能选择性转运甘油。

关键词：蓖麻; 质膜水通道蛋白; 真核表达; 转运功能

收稿：2014-11-25   修定：2014-12-04

资助：国家自然科学基金(31270370和31160262)、福建省生物学省级重点学科建设项目、2014年国家级大学生创新创业训练项目(201410399018)和泉州师范学院大学生科研基金项目(2014DKJ05)。

Castor Plasma Membrane Aquaporin (PIP1.3) in Vitro Transprot in Chinese Hamster Ovary

DONG Le¹, DAI Cong-Jie¹, WANG Fang^1,*, WANG Yun^2,*, ZHU Guo-Li³, XIE Xiao-Bin¹, XU Shan-Shan¹, LIU Can-Yang¹, LIN Ting¹

¹College of Chemistry and Life Science, Quanzhou Normal University, Quanzhou, Fujian 362000, China; ²College of Agriculture, Inner Mongolia University for Nationlities, Tongliao, Inner Mongolia 028042, China; ³Institute of Agricultural Science of Tongliao City, Tongliao, Inner Mongolia 028015, China

Corresponding author: WANG Fang; E-mail: dwf320@163.com, txmh@163.com; Tel: 0595-22919563, 0475-8314677

Abstract:

The encoding sequence of plasma membrane aquaporin gene (PIP1.3) in castor (Ricinus communis L.) was amplified with RT-PCR and inserted into pcDNA3.1/Myc-His A vector, double enzyme digestion analysis and DNA sequencing confirmed that recombination plasmid was successfully constructed. pcDNA3.1PIP1.3- Myc-His and pcDNA3.1Hygro-EYFP-H148Q-V163S plasmids linking Cl^--sensitive EYFP mutant (EYFP-H148Q-V163S) were transfected in CHO cells. The stable transfection of CHO cells was analysis with RT-PCR and Western blot. The results showed that PIP1.3 mRNA and protein were overexpressed in cell line screened. Permeabilities of PIP1.3 expressing CHO cells for water and glycerol were measured respectively by fluorescence method and carbon labeling experiment. The results showed that permeability of PIP1.3 expressing CHO cells for water was low, but high for glycerol.

Key words: castor (Ricinus communis L.); plasma membrane aquaporin; mammalian expression; permeability