[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

[Genome-wide identification of bZIP family genes and screening of candidate AarbZIPs involved in terpenoid biosynthesis in Artemisia argyi].

Artemisia argyi is an important medicinal and economic plant in China, with the effects of warming channels, dispersing cold, and relieving pain, inflammation, and allergy. The essential oil of this plant is rich in volatile terpenoids and widely used in moxi-bustion and healthcare products, with huge market potential. The bZIP transcription factors compose a large family in plants and are involved in the regulation of plant growth and development, stress response, and biosynthesis of secondary metabolites such as terpenoids. However, little is known about the bZIPs and their roles in A. argyi. In this study, the bZIP transcription factors in the genome of A. argyi were systematically identified, and their physicochemical properties, phylogenetic relationship, conserved motifs, and promoter-binding elements were analyzed. Candidate AarbZIP genes involved in terpenoid biosynthesis were screened out. The results showed that a total of 156 AarbZIP transcription factors were identified at the genomic level, with the lengths of 99-618 aa, the molecular weights of 11.7-67.8 kDa, and the theoretical isoelectric points of 4.56-10.16. According to the classification of bZIPs in Arabidopsis thaliana, the 156 AarbZIPs were classified into 12 subfamilies, and the members in the same subfamily had similar conserved motifs. The cis-acting elements of promoters showed that AarbZIP genes were possibly involved in light and hormonal pathways. Five AarbZIP genes that may be involved in the regulation of terpenoid biosynthesis were screened out by homologous alignment and phylogenetic analysis. The qRT-PCR results showed that the expression levels of the five AarbZIP genes varied significantly in different tissues of A. argyi. Specifically, AarbZIP29 and AarbZIP55 were highly expressed in the leaves and AarbZIP81, AarbZIP130, and AarbZIP150 in the flower buds. This study lays a foundation for the functional study of bZIP genes and their regulatory roles in the terpenoid biosynthesis in A. argyi.

[Chloroplast genome structure characteristics and phylogenetic analysis of Artemisia indica].

Artemisia indica is an important medicinal plant in the Asteraceae family, but its molecular genetic information has been rarely reported. In this study, the chloroplast genome of A. indica was sequenced, assembled, and annotated by the high-throughput sequencing technology, and its sequence characteristics, repeat sequences, codon usage bias, and phylogeny were analyzed. The results showed that the length of the chloroplast genome for A. indica was 151 161 bp, which was a typical circular four-segment structure, including two inverted repeat regions(IRs), a large single-copy(LSC) region, and a small single-copy(SSC) region, with a GC content of 37.47%. A total of 132 genes were annotated, and 114 were obtained after de-duplication, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Fifty long repeat sequences and 191 SSRs were detected in the chloroplast genome of A. indica, and SSRs were mainly single nucleotides. Codon usage bias analysis showed that leucine was the most frequently used amino acid(10.77%) in the chloroplast genome, and there were 30 codons with relative synonymous codon usage(RSCU)>1 and all ended with A/U. The phylogenetic tree constructed based on the chloroplast genomes of the 19 species from the Asteraceae family showed that A. indica and A. argyi were closest in the genetic relationship, and Artemisia species clustered into separate evolutionary branches. The results of this study are expected to provide a theoretical basis for the genetic diversity and resource conservation of Artemisia medicinal plants.

[Molecular genetics of medicinal plants, past achievement, and new era].

Unraveling the genetic basis of medicinal plant metabolism and developmental traits is a long-standing goal for pharmacologists and plant biologists. This paper discusses the definition of molecular genetics of medicinal plants, which is an integrative discipline with medicinal plants as the research object. This discipline focuses on the heredity and variation of medicinal plants, and elucidates the relationship between the key traits of medicinal plants(active compounds, yield, resistance, etc.) and genotype, studies the structure and function, heredity and variation of medicinal plant genes mainly at molecular level, so as to reveal the molecular mechanisms of transmission, expression and regulation of genetic information of medicinal plants. Specifically, we emphasize on three major aspects of this discipline.(1)Individual and population genetics of medicinal plants, this part mainly highlights the genetic mechanism of the domestication, the individual genomics at the species level, and the formation of genetic diversity of medicinal plants.(2)Elucidation of biosynthetic pathways of active compounds and their evolutionary significance. This part summarizes the biosynthesis, diversity and molecular evolution of active compounds in medicinal plants.(3) Molecular mechanisms that shaping the key agronomic traits by internal and external factors. This part focuses on the accumulation and distribution of active compounds within plants and the regulation of metabolic network by environmental factors. Finally, we prospect the future direction of molecular genetics of medicinal plants based on the rapid development of multi-omics technology, as well as the application of molecular genetics in the future strategies to achieve conservation and breeding of medicinal plants and efficient biosynthesis of active compounds.

[Synthesis of triterpenoid saponins from Aesculus chinensis based on transcriptome data].

Aesculus chinensis belongs to Hippocastanaceae family,bears medicinal and ornamental values. The oleanane type triterpenoid saponin aescin is regarded as active ingredient and accumulated in seed. In order to understand its molecular basis of the triterpenoid biosynthesis,we used high-throughput sequencing under Illumina Hi Seq 2000 platform to obtain the transcriptome data of seed and flower from A. chinensis to further mine the genes involved in its metabolic pathway. Unigene's de novo splicing was performed using Trinity software; the transcriptome results were annotated with KEGG database to predict the specific pathways of the aescin triterpenoid metabolism. Terpenoid and triterpenoid pathways were found from transcriptome data,and forty seven and twenty seven corresponding genes were uncovered respectively. It was found that there are eight kinds of enzymes related to the terpenoid metabolism pathway precursors and three kinds of enzymes related to the triterpenoid metabolism pathway. In this study,five genes corresponding to triterpene cyclase were analyzed in A. chinensis for the first time,which may participate in the synthesis of triterpenoid. It' s revealed that there were thirty three differential genes associated with the ko00900 and ko00909 pathways by analysis on the difference in transcriptome expression between seeds and flowers; seventeen unigenes were up-regulated and sixteen unigenes were down-regulated in the seeds relative to flowers. In this study, qRT-PCR experiments were used to verify the expression of three key enzyme genes of SQE( Unigene25806),HMGS( Unigene36710),and ß-AS( Unigene33291). The results of qRT-PCR were consistent with the transcriptome data. The candidate genes related to triterpenoid saponin aescin synthesis in A. chinensis found in this study can provide theoretical basis for the metabolism synthesis and regulation of aescin.