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中国科学院大学学报 ›› 2013, Vol. 30 ›› Issue (2): 206-212.DOI: 10.7523/j.issn.1002-1175.2013.02.010

• 化学与生命科学 • 上一篇    下一篇

虎杖查尔酮合酶PcCHS1基因的克隆与功能分析

李星, 王红   

  1. 中国科学院研究生院, 北京 100049
  • 收稿日期:2012-03-29 修回日期:2012-04-13 发布日期:2013-03-15
  • 通讯作者: 王红
  • 基金资助:

    国家自然科学基金(61173098)和中国科学院"十二五"基础前沿专项(KSCX2-EW-J-29)资助

Cloning and characterization of PcCHS1 from Polygonum cuspidatum

LI Xing, WANG Hong   

  1. College of life science, Graduate University, Chinese Academy of Sciences, Beijing 100049, China
  • Received:2012-03-29 Revised:2012-04-13 Published:2013-03-15

摘要:

从中药虎杖中通过RACE等方法克隆到一个查尔酮合酶基因的全长cDNA,命名为PcCHS1.该cDNA全长1182 bp,编码一个含393个氨基酸的蛋白质.体外酶促活性研究表明,重组PcCHS1在pH 7~8时催化形成查尔酮为其单一产物,在pH 9时除催化形成查尔酮外,还产生一定量的苯亚甲基丙酮.对PcCHS1第216位和第333位氨基酸进行了定点突变研究,结果表明这些位点对PcCHS1的体外酶促活性影响较大.

关键词: 虎杖, 查尔酮合酶, 定点突变, 酶活性

Abstract:

A full length cDNA encoding a type Ⅲ PKS (PcCHS1) was isolated from Polygonum cuspidatum Sieb. et Zucc. Functional and enzymatic assays biochemically confirmed that PcCHS1 was a chalcone synthase. When incubated with p-coumaroyl-CoA and malonyl-CoA at pH 7-8, PcCHS1 catalyzed the formation of chalcone as the major product. However, at pH 9 both p-hydroxybenzalacetone and chalcone were detected. Site-directed mutagenesis of Phe216 into Leu caused reduction of enzyme activity. Another mutation N333K performed activity of releasing some common derailment products, CTAL and BNY, of many CHSs in vitro.

Key words: Polygonum cuspidatum, chalcone synthase, site-directed mutagenesis, enzyme activity

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