水稻磁性蛋白OsMR的真核表达及活性分析

Eukaryotic expression and activity analysis of aicemagnetic protein OsMR

MagR蛋白是生物体内发现的第一个具有磁感应功能的蛋白质,通过氨基酸序列比对发现在水稻中含有一个与MagR蛋白相似性高达82%的未知蛋白。利用生物信息学分析对水稻OsMR进行蛋白质功能预测,构建pFastBac1表达质粒转染SF9细胞。通过昆虫细胞真核表达带有Fc标签的重组蛋白OsMR后,Western blot被用于检测样本中蛋白质的表达量。再对目的蛋白进行Ni亲和层析,利用瞬态荧光光谱法检测纯化蛋白OsMR的磁感应能力。结果表明,水稻OsMR蛋白与前人研究的MagR磁蛋白在各物种中相似性高达80%;在MagR磁蛋白的关键功能位点Fe-S簇序列完全一致;在进化上始于动植物分化之前且高度保守;跨膜结构域分析表明该蛋白是一个线粒体内膜结合蛋白;信号肽分析表明该蛋白可能是一个核内合成蛋白,并且最终被运送到细胞膜。通过SDS-PAGE与Western blot实验分析,表明OsMR蛋白的相对分子质量为42 000,与预测大小相符。在黑暗条件下,OsMR蛋白的瞬态荧光光谱随磁场的变化而变化,说明OsMR蛋白在体外具有一定的磁场感应能力。

MagR protein is the first protein with magnetic induction function found in organisms. Through amino acid sequence comparison, it is found that rice contains an unknown protein that is 82% similar to MagR protein. Using bioinformatics analysis to predict the protein function of rice OsMR, construct pFastBac1 expression plasmid to transfect SF9 cells. After eukaryotic expression of the Fc-tagged recombinant protein OsMR by insect cells, Western blot was used to detect the protein expression in the sample. Then the target protein was subjected to Ni affinity chromatography, and transient fluorescence spectroscopy was used to detect the magnetic induction ability of the purified protein OsMR. The results show that the rice OsMR protein is as much as 80% similar to the previously studied MagR magnetin in each species;the sequence of the Fe-S cluster at the key functional site of the MagR magnetin is exactly the same;the evolution began before the differentiation of animals and plants and highly conserved;transmembrane domain analysis shows that the protein is a mitochondrial inner membrane binding protein;signal peptide analysis shows that the protein may be a nuclear synthetic protein and is eventually transported to the cell membrane. Analysis by SDS-PAGE and Western blot experiments showed that the relative molecular mass of the OsMR protein was 42 kDa, which was consistent with the predicted size. Under dark conditions, the transient fluorescence spectrum of the OsMR protein changes with the change of the magnetic field, indicating that the OsMR protein has a certain magnetic field sensing ability in vitro.