Abstract:

For a better understanding of the molecular and biochemical processes involved in orthophosphate(Pi) uptake,a phosphate transporter cDNA(OsPT6) from rice was expressed in yeast and HEK293 cell expression systems.The gene OsPT6 codes a protein that belongs to Pht1 family of phosphate transporters with 12 putative transmembrane domains.The yeast mutant complementation analysis suggested that the transporter(OsPT6) was functional Pi transporter in yeast cells mediating Pi uptake at micromolar concentrations.When expressed in a Pi-uptake deficient yeast mutant,the phosphate transporter(OsPT6) protein exhibited an apparent Km of 96 μmol/L and mediated the highest Pi uptake at pH 6.0.The pH experiment suggested that the activity of OsPT6 was dependent on the electrochemical membrane potential mediated by the yeast P-type H+-ATPase.When expressed in HEK293 cells by either OsPT6∶YFP or OsPT6∶CFP proteins,both the YFP and CFP fluorescence were observed at the plasma membrane.This work demonstrated that OsPT6 had a similar function as PHO84 which is a phosphate transporter in yeast Saccharomyces cerevisiae and OsPT6 played an important role in Pi acquisition.

Key words: rice, phosphate transporter, yeast, human embryonic kidney cell, functional analysis

摘要： 为了更好地研究植物在磷素吸收过程中的分子机制以及生物化学过程的变化，将水稻中分离得到的一个磷转运蛋白基因（OsPT6）用于互补实验。互补实验结果表明，OsPT6能够与缺失磷转运功能的酵母突变体实现互补，并在低磷条件下促进酵母突变体对磷的吸收。进一步分析表明OsPT6属于水稻Pht1家族运输蛋白基因，所编码的蛋白对磷酸盐（Pi）的吸收Km值为96 μmol/L，属于高亲和的磷转运蛋白。不同的酵母转化子对不同pH环境的响应实验显示，OsPT6是一个与质子相偶联的磷运输蛋白，其吸收磷素的最佳pH为6.0。对OsPT6在人的胚胎肾细胞（HEK293）中的表达分析表明，该基因能够编码蛋白并定位于细胞膜，证明OsPT6的功能与酵母磷转运子PHO84相似，是一个定位于细胞膜上的具有吸收转运磷素作用的运输蛋白。

关键词: 水稻, 磷转运蛋白, 酵母, 人胚胎肾细胞, 功能分析