马铃薯钾离子通道蛋白SKT1的克隆与原核表达

发布时间：2018-11-05 12:53

【摘要】：钾是植物生长发育所必需的大量元素之一,参与植物体内许多重要的生理功能。马铃薯属喜钾作物,钾营养能够提高马铃薯的光合效率,还可影响光合产物的合成和运输,影响马铃薯的品质和产量高低。因此,钾元素在植物体内的积累对植物的生长发育起着至关重要的作用。以马铃薯组培幼苗反转录出的cDNA为模板,PCR扩增了 SKT1蛋白的胞内区的1758 bp的基因片段。构建原核表达质粒pET30-SKT1,转化E.coli BL21(DE3)表达株菌,用IPTG诱导表达,SDS-PAGE检查发现SKT1蛋白主要是以包涵体的形式存在。本文利用6 M的盐酸胍对SKT1包涵体进行溶解,利用Ni柱亲和纯化,在复性缓冲液中复性。目的蛋白SKT1复性得率大于40%,纯化后纯度达到95%以上,Western Blotting鉴定结果正确。本论文克隆了马铃薯费乌瑞它钾离子通道蛋白SKT1基因片段,原核表达并纯化SKT1蛋白,为下一步研究蛋白SKT1的晶体结构与功能,以及与相关蛋白的相互作用奠定基础。
[Abstract]:Potassium is one of the essential elements for plant growth and development, and participates in many important physiological functions in plants. Potassium nutrition can improve the photosynthetic efficiency of potato, affect the synthesis and transportation of photosynthetic products, and affect the quality and yield of potato. Therefore, the accumulation of potassium in plants plays an important role in the growth and development of plants. The 1758 bp gene fragment in the intracellular region of SKT1 protein was amplified by PCR using cDNA reverse transcribed from potato tissue culture seedlings. The prokaryotic expression plasmid pET30-SKT1, was constructed and transformed into E.coli BL21 (DE3) expression strain, and the expression was induced by IPTG. SDS-PAGE analysis showed that SKT1 protein mainly existed in the form of inclusion body. In this paper, SKT1 inclusion bodies were dissolved with 6 M guanidine hydrochloride and purified by Ni column. Objective: the SKT1 renaturation rate of protein was more than 40%, and the purity of purified protein was over 95%, Western Blotting. In this paper, the SKT1 gene fragment of Potato Fiurita potassium channel protein was cloned, and the prokaryotic expression and purification of SKT1 protein, which laid a foundation for the further study of the crystal structure and function of the protein SKT1 and its interaction with related proteins.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S532

【参考文献】