title: Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.
abstract: Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China. zhxn0609@163.com.
authors.forename: Xianan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China. 13716402100@139.com.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Guan
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. dai_zb@tib.cas.cn.
authors.forename: Zhubo
authors.initials: Z
authors.lastname: Dai
authors.affiliation: Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing 100700, China. guojuanzy@163.com.
authors.forename: Juan
authors.initials: J
authors.lastname: Guo
authors.affiliation: Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing 100700, China. shenyezy@sina.com.
authors.forename: Ye
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing 100700, China. guanghongcui@126.com.
authors.forename: Guanghong
authors.initials: G
authors.lastname: Cui
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China. weigao@ccmu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing 100700, China. huangluqi68@163.com.
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D045782:Hemiterpenes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D030821:Plants, Genetically Modified; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D027561:Salvia miltiorrhiza / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013729:Terpenes / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D013729:Terpenes; C004809:isopentenyl pyrophosphate; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase
keywords: RNA interference; Salvia miltiorrhiza; color complementation; functional identification; isopentenyl diphosphate isomerase