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.
Highlights
Salvia miltiorrhiza Bunge is a traditional Chinese medicine that is frequently used for curing cardiovascular diseases, and its preparations are currently in phase II clinical trials [1]
RNA interference (RNAi) SmMIPoleIc1ulesh2a01i5r,y20,rpoagoet–spaglienes were established to evaluate the effect of Salvia miltiorrhiza IPI1 gene (SmIPI1) on tanshinone accumulatitoanns.hTinhoneesseinstSu. mdiiletisorrhhaizva,ewtehceonpdoutcetendtitahel ftoolloiwnicnrgeaexspeeroimuerntksn: foirwstllye,dthgeeSmofIPIt1hgeenme ewtaasbolism of terpenes fotrrmanesfderrtehdrionutoglhycothpeenMe-eEngPinpeaertehdwEsacyh.erichia coli to determine whether expression of the gene would influence metabolic flow and promote the yield of lycopene
Lycopene engineering bacteria has been used to discover the function of SmIPI1 in E. coli
Summary
Salvia miltiorrhiza Bunge is a traditional Chinese medicine that is frequently used for curing cardiovascular diseases, and its preparations are currently in phase II clinical trials [1]. We selected multiple candidate genes involved in tanshinone biosynthesis in the hairy roots of S. miltiorrhiza, as revealed by a cD20N2A07microarray [17] using ~4400 expressed sequence tags, including SmIPI1. We selected multiple candidate genes involved in tanshinone biosynthesis in the hairy roots of S. miltiorrhiza, as revealed by a cDNA microarray [17] using ~4400 expressed sequence tags, including SmIPI1. RNA interference (RNAi) SmMIPoleIc1ulesh2a01i5r,y20,rpoagoet–spaglienes were established to evaluate the effect of SmIPI1 on tanshinone accumulatitoanns.hTinhoneesseinstSu. mdiiletisorrhhaizva,ewtehceonpdoutcetendtitahel ftoolloiwnicnrgeaexspeeroimuerntksn: foirwstllye,dthgeeSmofIPIt1hgeenme ewtaasbolism of terpenes fotrrmanesfderrtehdrionutoglhycothpeenMe-eEngPinpeaertehdwEsacyh.erichia coli to determine whether expression of the gene would influence metabolic flow and promote the yield of lycopene.
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