多花黄精查尔酮合酶PcCHS的原核表达、亚细胞定位及表达分析  

Prokaryotic Expression,Subcellular Localization and Expression Analysis of PcCHS Gene from Polygonatum cyrtonema Hua

作　　者：潘萍萍 徐志浩 张怡雯 李青 王忠华[1] PAN Ping-ping;XU Zhi-hao;ZHANG Yi-wen;LI Qing;WANG Zhong-hua(College of Biology and Environment,Zhejiang Wanli University,Ningbo 315100)

机构地区：[1]浙江万里学院,宁波315100

出　　处：《生物技术通报》2024年第5期280-289,共10页Biotechnology Bulletin

基　　金：浙江省生物工程一流学科学生创新项目(CX2022018);浙江省中药材新品种选育重大科技专项(2021C02074);浙江省一流学科开放基金项目(KF2021009)。

摘　　要：【目的】探究查尔酮合酶(chalcone synthase,PcCHS)基因在多花黄精类黄酮合成中的作用,为后续解析PcCHS功能以及多花黄精新品种选育提供可靠的理论依据。【方法】以多花黄精为cDNA模板,克隆多花黄精PcCHS基因的编码序列,对该基因进行生物信息学分析。通过构建PcCHS的原核表达载体,纯化目的重组蛋白,验证该酶的体外表达活性。利用瞬时超表达体系探究该基因过表达后总黄酮的含量变化。利用Gateway技术构建亚细胞定位载体35S::PcCHS-GFP,通过本氏烟草表达系统确定目的蛋白亚细胞定位情况。【结果】PcCHS基因的开放阅读框为1 251 bp,理论分子量为44.63 kD,等电点为5.89,属于亲水蛋白,与石刁柏(Asparagus officinalis)CHS亲缘关系较近。原核表达实验表明,pET28a-PcCHS经IPTG(异丙基-β-D-硫代半乳糖苷)诱导表达可溶性重组蛋白,Western-blot显示大小约为45 kD,与预期大小一致,且纯化的目的蛋白具有一定的酶活性,能催化对香豆酰辅酶A和丙二酰辅酶A转化为柚皮素查尔酮。此外,PcCHS瞬时超表达中,PcCHS组的表达量显著高于空载K组,总黄酮含量也显著高于空载K组,最高可达1.83倍。亚细胞定位结果显示,该基因在细胞膜和细胞核中发挥作用。【结论】PcCHS基因原核表达的酶具有体外酶活性,其亚细胞定位于细胞膜和细胞核,且瞬时超表达能够显著提高多花黄精叶片总黄酮含量。【Objective】This work aims to explore the role of chalcone synthase(chalcone synthase,PcCHS)gene in the synthesis of flavonoids in Polygonatum cyrtonema Hua,which may provide a reliable theoretical basis for the subsequent analysis of the function of PcCHS and the breeding of new varieties of P.cyrtonema Hua.【Method】Using P.cyrtonema Hua as cDNA template,the coding sequence of PcCHS gene was cloned,and the gene was analyzed bioinformatically.The prokaryotic expression vector of PcCHS was constructed and the recombinant protein was purified to verify the expression activity of PcCHS in vitro.Transient overexpression system was used to investigate the changes of total flavonoids content after overexpression of this gene.Gateway technology was used to construct the subcellular localization vector 35S::PcCHS-GFP,and the subcellular localization of target protein was determined by the tobacco expression system.【Result】The results showed that PcCHS was a hydrophilic protein with an open reading frame of 1251 bp,a theoretical molecular weight of 44.63 kD and an isoelectric point of 5.89,and was closely related to AoCHS(Asparagus officinalis).Prokaryotic expression experiment showed that pET28a-PcCHS was induced to express soluble recombinant protein by IPTG(isopropyl-β-d-thiogalactoside).Western-blot showed that the size of pET28a-PcCHS was about 45 kD,which was consistent with the expected size.The purified protein had certain enzymatic activity and catalyzed the conversion of p-coumaryl-CoA and malonyl-CoA into naringin chalcone.In addition,in the transient overexpression of PcCHS,the expression level of PcCHS group was significantly higher than that of no-load K group,and the total flavonol content was also significantly higher than that of no-load K group,up to 1.83 times.Subcellular localization results showed that the gene plays a role in the cell membrane and nucleus.【Conclusion】Prokaryotic expression of PcCHS gene has enzyme activity in vitro,and its subcellular location is in cell membrane and n

关 键 词：多花黄精 查尔酮合酶基因(CHS) 原核表达 瞬时超表达 蛋白纯化 体外酶活 亚细胞定位 

分 类 号：R28[医药卫生—中药学]