酵母异源互补法鉴定MbNramp1基因的功能

【目的】研究MbNramp1基因的功能。【方法】通过异源互补试验鉴定该基因转运铁的功能,并对MbNRAMP1蛋白进行亚细胞定位研究。【结果】MbNramp1基因转化酵母突变株DDY4在缺铁的培养基上恢复生长。在供试BPDS浓度的培养基上,MbNramp1基因转化酵母突变株DDY4生长状况均明显好于空载体转化后的突变株。培养基中BPDS增加到15μmol·L-1时,转化了MbNramp1的酵母细胞生长状况与野生型相差无几。在30μmol·L-1BPDS时,MbNramp1转化的细胞与低浓度BPDS相比生长延缓,但是明显优于空载体转化的DDY4突变株。与较低浓度BPDS相比,野生型酵母(DY1457)的生长量也减少了。亚细胞定位表明,MbNRAMP1主要位于部分质膜上而非整个膜上。【结论】初步证明MbNramp1编码具有功能的铁转运蛋白,能够使酵母吸收铁的突变株恢复突变,MbNRAMP1主要位于部分细胞膜上行使铁营养转运功能。

Function Analysis of MbNramp1 Gene from Malus baccata(L.)Borkh Through Yeast Complementation Experiments

【Objective】 The objective of the study is to investigate the function of MbNramp1 gene. 【Method】 Heterogenous complementation experiments were employed to confirm the iron- transporting ability of MbNramp1,and study subcellular localization of the MbNRAMP1 protein 【Result】 MbNramp1 was expressed in yeast mutant DDY4, which could restore growth in the media without iron. At tested concentrations of BPDS, the yeast cells DDY4 mutant after MbNramp1 being transformed grew better than mutant strain transformed with empty vector. When the concentration of BPDS in the media reached 15 μmol?L-1, the growth of yeast cells with MbNramp1 was comparable with that of wild type. Although the growth of MbNramp1 transformed cells in the media with 30 μmol?L-1 BPDS was slower than those in the media with low concentration of BPDS, they were significantly faster that DDY4 mutant transformed with empty vector. In addition, wild type grew slower with 30 μmol?L-1 BPDS. The result of subcellular localization showed that MbNRAMP1 proteins were mainly positioned in the part of plasma membrane not in the whole membrane. 【Conclusion】 Initial results suggested that MbNRAMP1 protein has the ability of transferring Fe and also help yeast mutant to grow. In addition, MbNRAMP1 proteins were mainly positioned in the part of plasma membrane