苦荞FtPCS基因克隆、表达及酶活性初步分析

Cloning,Expression and Activity Analysis of a Phytochelatin Synthase Gene from Fagopyrum tataricum

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。

Based on the RNA-seq data of Pb-（2＋）-induced tartary buckwheat leaves,we cloned a phytochelatin synthase gene（FtPCS）from Fagopyrum tataricum using the RT-PCR method.The FtPCS gene was sub-cloned into pET28a（＋）using In-fusion technology,which was expressed in Escherichia coli BL21Star（DE3）.Being applied with Pb-（2＋）,the catalytic activity of the purified FtPCS protein was detected by reverse-HPLC involving DTNB（5,5′-dithiobis-2-nitrobenzoic acid）postcolumn derivatization method.The results indicated that the full length of genomic sequence of FtPCS was 5 456 bp,containing 8exons and 7introns;the CDs sequence of FtPCSwas 1 485 bp in length,which encoded a 55.10 kDa protein consisted of 494 amino acids.The FtPCSwas highly expressed in E.coli in the form of inclusion body,which was refolded by dialysis and purified by Co-（2＋）chelation chromatography.The purified FtPCS recombinant proteins still kept the biological activity for transforming GSH to PC compounds,and the low concentration of Pb-（2＋）could remarkably increase its catalytic activity.