苦荞FtPCS基因克隆、表达及酶活性初步分析
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国家自然科学基金(31171606);


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

    该研究以Pb2+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Pb2+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5 456 bp,包括8个外显子和7个内含子;FtPCS基因ORF序列全长1 485 bp,编码494个氨基酸,预测分子量为55.10 kDa。可溶性分析表明,苦荞FtPCS基因在E.coli BL21 Star(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Pb2+对其催化活性具有激活作用。

    Abstract:

    Based on the RNAseq data of Pb2+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 BL21 Star(DE3). Being applied with Pb2+, 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 8 exons and 7 introns; the CDs sequence of FtPCS was 1 485 bp in length, which encoded a 55.10 kDa protein consisted of 494 amino acids. The FtPCS was highly expressed in E.coli in the form of inclusion body, which was refolded by dialysis and purified by Co2+ chelation chromatography. The purified FtPCS recombinant proteins still kept the biological activity for transforming GSH to PC compounds, and the low concentration of Pb2+ could remarkably increase its catalytic activity.

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袁 勇,陈 鹏.苦荞FtPCS基因克隆、表达及酶活性初步分析[J].西北植物学报,2017,37(3):419-427

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  • 在线发布日期: 2017-04-18
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