两种不同植物来源白藜芦醇生物合成关键酶——芪合酶催化效率比较  

Comparison of stilbene synthase from different plant sources for resveratrol biosynthesis

作　　者：郭辉力[1,2] 罗在柒[1,2] 杨亚东[3] 杨明峰[2] 吕鹤书[2] 柳春梅[2] 杨静[4] 王有年[2] 马兰青[2] 

机构地区：[1]北京林业大学林学院,北京100083 [2]北京农学院农业部都市农业(北方)重点实验室,北京102206 [3]北京农学院植物科学技术学院,北京102206 [4]中北大学化工与环境学院,山西太原030051

出　　处：《生物工程学报》2014年第10期1622-1633,共12页Chinese Journal of Biotechnology

基　　金：北京市自然科学基金重点项目(No.5111001);国家自然科学基金(Nos.31300620;31370674);北京市教委面上项目(Nos.KM201310020002;KM201310020015;KM201110020001;KM201210020009);北京市属高校人才强教深化计划项目(No.PHR201108279)资助~~

摘　　要：白藜芦醇是一种天然植保素,且具有特殊的药理和保健功能,芪合酶(Stilbene synthase,STS)是该化合物生物合成的关键酶和限速酶。白藜芦醇存在于有限几种植物且含量差异很大,虎杖中白藜芦醇含量比葡萄、花生高1 000倍以上,推测不同STS的催化能力有可能是白藜芦醇含量差异的原因之一。为验证上述推测,文中通过overlap PCR技术从葡萄叶片基因组DNA中克隆得到葡萄STS基因,连同前期工作中获得的虎杖STS基因(PcPKS5),进行了原核表达分析。诱导表达产物经过Ni2+亲和柱纯化和PD-10柱脱盐后,均得到分子量约43 kDa的可溶性纯化蛋白。酶促产物分析结果表明,两种酶催化产物均为白藜芦醇。酶动力学分析表明,虎杖STS催化效率(Kcat/Km)是葡萄STS的2.4倍。文中从植物类型Ⅲ聚酮化合物合酶(Polyketide synthase,PKS)超家族催化活性位点和保守位点角度分析了造成上述两种酶活性产生差异可能存在的原因。Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase （STS） is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1 000 times higher than grapes and peanuts. We speculate that the catalytic abilily of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene （VvSTS） was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS （PcPKS5） were analyzed through heterologous expression in Escherichia coil The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency （Kcat/Kra） of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.

关 键 词：葡萄 虎杖 芪合酶 基因克隆 原核表达 功能分析 

分 类 号：Q946.5[生物学—植物学]