蛇足石杉内生真菌Shiraia sp.Slf14中赖氨酸脱羧酶基因的克

发布时间：2018-10-19 19:13

【摘要】：【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~( 3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。
[Abstract]:[objective] the biosynthesis pathway of Huperzine A Hup A was initiated by lysine decarboxylase (Lysine decarboxylase,LDC) in the treatment of Alzheimer's disease. In this study, the LDC gene of endophytic fungi derived from Hup A was cloned and expressed, and its function was studied. [methods] LDC gene was obtained from an endophytic fungus of Hup A, Shiraia sp.Slf14, by RT-PCR amplification. Expression plasmids p ET-22b-LDC and p ET-32a-LDC, transformant E.coli BL21, were added to IPTG at the final concentration of 1 脳 10 ~ (3) mol/L, at 24 掳C ~ (2) r/min for 8 h to induce the expression of LDC protein. The recombinant LDC was purified by Ni~ (2) metal affinity chromatography and the enzymatic reaction system was established. The catalytic activity of LDC was determined by TLC. The physicochemical properties of LDC and the spatial structure of the protein were analyzed by bioinformatics software. [results] the recombinant protein LDC and Trx-LDC, were successfully cloned and heterogeneously expressed. The molecular weights of the recombinant protein LDC and Trx-LDC, were 24.4 k Da and 42.7 k Da, respectively, identified by SDS-PAGE electrophoresis. TLC results showed that both LDC and Trx-LDC had Lysine decarboxylase activity. [conclusion] the LDC gene was cloned and expressed heterologous from the endophytic fungus Shiraia sp.Slf14, which produced Hup A. The catalytic activity was detected, which provided reference data for enriching LDC molecular information and elucidating the biosynthesis mechanism of Hup A in endophytic fungi.
【作者单位】：江西师范大学生命科学学院江西省亚热带植物资源保护与利用重点实验室;江西科技师范大学生命科学学院江西省生物加工过程重点实验室;
【基金】：国家自然科学基金(31460021) 国家“十二五”重大科技支撑项目(2011BAC13B04) 江西省自然科学基金(20142BAB214008,20151BAB204003,20151BA204002)~~
【分类号】：Q78

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