苦荞FtPCS基因克

发布时间：2019-05-27 02:35

【摘要】：该研究以Pb~(2+)诱导的苦荞(Fagopyrum tataricum)叶片转录组数据为基础,通过RT-PCR克隆,获得苦荞植物络合素合酶(Phytochelatins,PCs)基因(FtPCS);采用无缝克隆构建原核表达载体pET28a-PCS,并通过反向-HPLC结合DTNB[5,5′-二硫代双(2-硝基苯甲酸)]柱后衍生的方法,对纯化的FtPCS重组蛋白在Pb~(2+)存在条件下的催化活性进行初步分析,为进一步揭示FtPCS基因在苦荞重金属富集和解毒过程中的机制奠定基础。结果表明:苦荞FtPCS基因组序列全长5 456bp,包括8个外显子和7个内含子;FtPCS基因ORF序列全长1 485bp,编码494个氨基酸,预测分子量为55.10kDa。可溶性分析表明,苦荞FtPCS基因在E.coli BL21Star(DE3)中以包涵体的形式表达,采用梯度透析复性并结合钴离子螯合层析的方法获得纯化的FtPCS重组蛋白,复性的FtPCS蛋白具有催化GSH生成PC化合物的活性,而且低浓度的Pb~(2+)对其催化活性具有激活作用。
[Abstract]:Based on the data of Tartary buckwheat (Fagopyrum tataricum) leaf transcriptional group induced by Pb~ (2), the (FtPCS); gene of Tartary buckwheat plant complex enzyme (Phytochelatins,PCs) was obtained by RT-PCR cloning. The prokaryotic expression vector pET28a-PCS, was constructed by seamless cloning and derivatization was carried out by reverse-HPLC binding DTNB [5, 5] dithiobis (2-nitrobenzoic acid). The catalytic activity of purified FtPCS recombinant protein in the presence of Pb~ (2) was analyzed, which laid a foundation for further revealing the mechanism of FtPCS gene in the process of heavy metal enrichment and detoxification in Tartary buckwheat. The results showed that the full length of Tartary buckwheat FtPCS genome sequence was 5456 BP, including 8 exons and 7 introns, and the ORF sequence of FtPCS gene was 1485 BP, encoding 494 amino acids and predicting molecular weight of 55. 10 KD. Soluble analysis showed that Tartary buckwheat FtPCS gene was expressed in the form of inclusion body in E.coli BL21Star (DE3). The purified FtPCS recombinant protein was obtained by gradient dialysis renaturation and cobalt ion chelating chromatography. The renatured FtPCS protein has the activity of catalytic GSH to PC compound, and low concentration of Pb~ (2) can activate its catalytic activity.
【作者单位】：西北农林科技大学生命科学学院;
【基金】：国家自然科学基金(31171606) 西北农林科技大学基本科研业务费(24520152214)
【分类号】：S517;Q943.2

【相似文献】