摘要
2-酮戊二酸依赖性双加氧酶(2-oxoglutarate-dependent dioxygenase,2-ODD)是芳基萘类木脂素-鬼臼毒素合成途径的关键酶。为深入解析Sc2-ODD基因的表达及其功能,本研究克隆了五味子Sc2-ODD基因,并进行生物信息学分析、果实发育期基因表达特征分析、原核表达载体构建、蛋白诱导与纯化研究。五味子Sc2-ODD基因ORF全长1077 bp,编码358个氨基酸,蛋白分子质量40.16 kD,具2OG-FeII_Oxy结构域;系统进化显示Sc2-ODD与长蒴黄麻2-ODD亲缘关系较近;qRT-PCR结果显示Sc2-ODD表达量呈现果实膨大期快速增加而进入着色期迅速降低的变化趋势;将Sc2-ODD基因插入到原核表达载体,并构建pGS21T-Sc2-ODD重组表达载体,转化Rosetta(DE3),经诱导表达获得大量融合蛋白,利用GST融合蛋白纯化技术对融合蛋白进行纯化,获得高纯度可溶性重组蛋白。研究结果为进一步研究Sc2-ODD基因在五味子芳基萘类木脂素代谢中的分子调控机制提供依据,从而为利用基因工程提高五味子芳基萘类木脂素含量并最终提升五味子药材品质奠定基础。
The 2-oxoglutarate-dependent dioxygenase(2-ODD)gene is regarded as the key enzyme gene involved with aryl naphthalene lignan-podophyllotoxin synthesis.To study the expression pattern and function of the Sc2-ODD gene,a full-length cDNA of the gene was cloned.Bioinformatic analysis,the expression pattern,and prokaryotic expression and purification were implemented.The open reading frame of Sc2-ODD gene was 1077 bp and encoded 358 amino acids with a molecular weight of 40.16 kD.The Sc2-ODD protein contained the conserved2 OG-FeII-oxy sequence of the 2-ODD protein.The results of phylogenetic analysis revealed that Sc2-ODD is most closely related to Corchorus olitorius 2-ODD.qRT-PCR results showed that Sc2-ODD expression displayed obvious up-regulation at the fruit-swelling stage,then down-regulation in the fruit-coloring period.The Sc2-ODD gene was cloned into the bacterial expression vector pGS21 T,the recombinant Sc2-ODD protein was expressed in Escherichia coli Rosetta(DE3)cells and the fusion protein was obtained and purified by GST fusion protein purification technology.This study will lay a foundation for further research on the function and expressional regulation of the Sc2-ODD gene in the aryl naphthalene lignans biosynthesis pathway,and also provides a scientific basis for improving the lignan content and the medicinal quality of Schisandra chinensis using plant genetic engineering.
作者
李海燕
刘久石
王婷
刘宇阳
王熙昂
李宏博
LI Hai-yan;LIU Jiu-shi;WANG Ting;LIU Yu-yang;WANG Xi-ang;LI Hong-bo(College of Horticulture,Shenyang Agricultural University,Shenyang 110866,China;Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China)
出处
《药学学报》
CAS
CSCD
北大核心
2020年第9期2226-2233,共8页
Acta Pharmaceutica Sinica
基金
2018年全国中药资源普查项目(1102-01042918001)。
