摘要
查尔酮异构酶是高等植物黄酮类化合物生物合成中的关键限速酶,决定了植物体内黄酮类化合物的产生。该研究从菘蓝的不同部位中提取RNA,反转录为cDNA,并设计带有酶切位点的特异性引物,克隆得到1个菘蓝查尔酮异构酶基因,命名为IiCHI。该基因全长756 bp,含有1个完整的开放阅读框,编码251个氨基酸。通过同源比对显示IiCHI与拟南芥Arabidopsis thaliana的CHI蛋白亲缘关系较近,并且具有查尔酮异构酶典型的活性位点,系统发育树分析显示IiCHI归属于Ⅰ型CHI。构建原核表达载体pET28a-IiCHI并进行诱导表达,纯化后获得IiCHI的重组蛋白。体外酶活性检测实验显示IiCHI重组蛋白可以把柚皮素查尔酮催化生成柚皮素,而不能把异甘草素催化生成甘草素。实时荧光定量PCR结果显示菘蓝中IiCHI在地上部分的表达量明显高于地下部分,其中地上部分在花中表达量最高,叶与茎次之,在地下部分的须根、根的皮部与木质部几乎不表达。该研究为进一步探讨菘蓝中查尔酮异构酶的功能及其在黄酮类化合物的生物合成研究中提供了参考。
Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants,which determines the production of flavonoids in plants.In this study,RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA.Specific primers with enzyme restriction sites were designed,and a chalcone isomerase gene was cloned from I.indigotica,named IiCHI.IiCHI was 756 bp in length,containing a complete open reading frame and encoding 251 amino acids.Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase.Phylogenetic tree analysis showed that IiCHI was classified into typeⅠCHI clade.Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein.In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin,but could not catalyze the production of liquiritigenin by isoliquiritigenin.The results of real-time quantitative polymerase chain reaction(qPCR)showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts,followed by leaves and stems,and no expression was observed in the roots and rhizomes of the underground parts.This study has confirmed the function of chalcone isomerase in I.indigotica and provided references for the biosynthesis of flavonoid components.
作者
张苛苛
孙术富
谭宇萍
徐朝阳
姜银银
杨健
李大勇
唐金富
ZHANG Ke-ke;SUN Shu-fu;TAN Yu-ping;XU Zhao-yang;JIANG Yin-yin;YANG Jian;LI Da-yong;TANG Jin-fu(State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;College of Life Sciences,Shandong Normal University,Ji'nan 250014,China;Beijing Key Laboratory of Vegetable Germplasm Improvement,Beijing Vegetable Research Center,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第6期1510-1517,共8页
China Journal of Chinese Materia Medica
基金
中国中医科学院科技创新工程项目(CI2021A04114)
国家自然科学基金项目(81974517)。
关键词
菘蓝
查尔酮异构酶
黄酮类化合物
表达分析
Isatis indigotica
chalcone isomerase
flavonoid
expression analysis
