| Prunella vulgaris L is one of the widly used Traditional Chinese Medicine in ancient China and Asia which presents various medicinal activities. Pharmacopoeia of People’s Republic of China(2015 edition, first volumn) stipulates that rosmarinic acid(RA) is the indext component of P. vulgaris that can be used to determine the medicinal quality of this TCM. RA itself, at the meanwhile, presents multiple biological activities and belongs to an important classification of phenolic acids, while the medicinal effects and biosynthetic pathway of RA have been investigated broadly.This study isolated genes of two enzymes playing significant roles in RA biosynthesis in P. vulgaris using homology based cloning, i.e., rosmarinic acid synthase(PvRAS) and cytochrome P450 monooxygenase(PvCYP). And we analyzed the amino acid sequences encoded by the two OFRs using bioinformatics to determine the evolutionary classification of these two proteins. Besides, our study established the hairy root cultivation system of P. vulgaris and further explored the effects of elicitors on biomass accumulation, RA synthesis and gene expression in P. vulgaris hairy root. The results are as the following:1. The cDNA of PvRAS(KM053280) contains a 1305 bp ORF, encoding a 435 amino acids polypeptide; the cDNA of PvCYP(KM053281) contains a 1530 bp ORF, encoding a 510 amino acids polypeptide. Gene expression profiling analysis suggests that the two genes can be expressed in various tissues while the expression levels are highest in root for both of the two genes, and the expression preference for PvRAS is: root>leaf>stem>spike; for PvCYP is: root>stem>leaf>spike.2. The PvRAS protein has a calculated molecular mass of 48.1 kDa and an isoelectric point(pI) of 5.97; the PvCYP protein has a calculated molecular mass of 57.8 kDa and an isoelectric point(pI) of 8.62. Their amino acid sequences share high identities with RASs and CYPs of other species and both of them possess four types of secondary structures. Alignment of multiple sequences and construction of phylogenetic trees indicate that PvRAS protein belongs to BAHD acyltransferase family while PvCYP belongs to CYP98 A subfamily of cytochrome P450 superfamily.3. Hairy root cultivation system was established for P. vulgaris by inducing the leaves of aseptic seedlings using Agmbacterium rhizogenes ATCC 15834. And the positive hairy root lines were identified by amplifying rolB and rolC genes which were originally located in the Ri plasmid of Agmbacterium. To select the optimal elicitation time point, we characterized the growth curve of hairy root and found the 15 d- old hairy root presented the best conditions. Therefore, we selected 15 d-old hairy root to conduct the elicitation experiment in the following study.4. We treated the hairy root system using two elicitors: ethylene(Eth) and salicylic acid(SA) and screened the optimal concentrations: 200 μg/L for Eth and 6.9 mg/L for SA. We added the elicitors in selected concentrations at the appropriate time point into the hairy root culture and selected samples at different time points. Our results showed that both of the elicitors could increase the biomass accumulation and RA biosynthesis while the effect of Eth was more significant than that of SA: the highest RA content after Eth elicitation was 63.5 mg/g at 5 d while the highest RA content after SA elicitation was 58.3 mg/g at 2 d. At some selected time points, neither of these elicitors could upregulate the expression of PvRAS and PvCYP except 0.5 d after the addition of SA and 5 d after the addition of Eth, when the expression level of PvRAS and PvCYP increased significantly, and the expression of PvCYP alone presented a dramatic increase, respectively. |
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