Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages.

The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.

Analysis of the Prunellae Spica transcriptome under salt stress.

Prunella vulgaris L. is a moderately salt tolerant plant commonly found in China and Europe, whose spica (Prunellae Spica) has been used as a traditional medicine. The scant transcriptomic and genomic resources of Prunellae Spica have greatly hindered further exploration of the underlying salt tolerance mechanism of this species. To clarify the genetic basis of its salt tolerance, high-throughput sequencing of mRNAs was employed for de novo transcriptome assembly differential expression analysis of Prunellae Spica under salt stress. 118,664 unigenes were obtained by assembling pooled reads from all libraries with 68,119 sequences annotated. A total of 3857 unigenes were differentially expressed under low, medium and high salt stress, including 2456 up-regulated and 1401 down-regulated DEGs, respectively. Gene ontology analysis revealed that salt stress-related categories involving 'catalytic activity', 'binding', 'metabolic process' and 'cellular process' were highly enriched. KEGG pathway annotation showed that the DEGs from different salt stress treatment groups were mainly enriched in the pathways of translation, signal transduction, carbohydrate metabolism, energy metabolism, lipid metabolism and amino acid metabolism, accounting for over 60% of all DEGs. Finally, it showed that the results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis for 10 unigenes that randomly selected were significantly consistent with RNA-seq data, which further assisted in the selection of salt stress-responsive candidate genes in Prunellae Spica. This study represents a significant step forward in understanding the salt tolerance mechanism of Prunellae Spica, and also provides a significant transcriptomic resource for future work.

Promiscuous terpene synthases from Prunella vulgaris highlight the importance of substrate and compartment switching in terpene synthase evolution.

The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these. We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS-a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS-a enzymes from Lamiaceae were cytosolic and reported to act on the 15-carbon farnesyl diphosphate. Plastidial TPS-a enzymes using the 20-carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family. All four new enzymes were found to be active on multiple prenyl-diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11-hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins. We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification.

Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

[Genetic variation, correlation and principle component analysis on morphological characteristics of various germplasm from Prunella vulgaris].

OBJECTIVE: To study the correlation among the morphological characteristics and the spica yield of various germplasm of Prunella vulgaris. METHOD: The various P. vulgaris germplasm from all over the country were investigated by analysis of correlation analysis, path analysis and principle component analysis in a randomized block experiment. RESULT: The 6 morphological characteristics were greatly different from each other in various germplasm. The spica yield per plant had a very significantly positive correlation with the number of spica per plant and fresh leaves weight per plant, meanwhile the correlation between the spica yield per plant and spica length was very significantly. Three principal components which accounted for 87.533% of total variance were extracted from the principal component analysis. CONCLUSION: The strong growth potential, the number of spica per plant and spica length were main factors for the selection of high yield breeding of P. vulgaris.

[AFLP analysis of genetic diversity of Prunella].

OBJECTIVE: To explore the variety of the genetic polymorphism of eight Prunella germplasm resources by AFLP analysis. METHOD: The amplified fragment length polymorphism (AFLP) tags were applied to screen out 32 selective amplification primer pairs, the amplified bands as original matrix were analyzed with NTSYS-PC software for the similarity between the Prunella germplasm and the construction of genetic phylogenetic tree. RESULT: SDS extraction of genomic Prunella DNA showed a good quality, could meet the requirements of AFLP analysis. From 32 selective amplification primer pairs, 10 pairs with strong polymorphism, better band and higher resolution were used for the construction of the AFLP Prunella fingerprint, all eight Prunella germplasms were separated, they were divided into 3 categories. CONCLUSION: Prunella germplasm resources are rich in genetic diversity, certain morphological characteristics and differences are associate with genotype.

[Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.