Transcriptome analysis of three medicinal plants of the genus Polygonatum: identification of genes involved in polysaccharide and steroidal saponins biosynthesis.

Polysaccharides and saponins are the main active components of Polygonati Rhizoma. Studying the molecular mechanism of their synthesis pathway is helpful in improving the content of active components at the molecular level. At present, transcriptome analysis of three Polygonatum species (Polygonatum sibiricum Red., Polygonatum cyrtonema Hua, Polygonatum kingianum Coll. et Hemsl.) has been reported, but no comparative study has been found on the transcriptome data of the three species. Transcriptome sequencing was performed on the rhizomes of three Polygonatum species based on high-throughput sequencing technology, and all transcripts were assembled. A total of 168,108 unigenes were generated after the removal of redundancy, of which 121,642 were annotated in seven databases. Through differential analysis and expression analysis of key enzyme genes in the synthesis pathway of three Polygonatum polysaccharides and steroidal saponins, 135 differentially expressed genes encoding 18 enzymes and 128 differentially expressed genes encoding 28 enzymes were identified, respectively. Numerous transcription factors are involved in the carbohydrate synthesis pathway. Quantitative real-time PCR was used to further verify the gene expression level. In this paper, we present a public transcriptome dataset of three medicinal plants of the genus Polygonatum, and analyze the key enzyme genes of polysaccharide and steroidal saponins synthesis pathway, which lays a foundation for improving the active component content of Polygonati Rhizoma by molecular means.

Isolation, characterisation, and expression profiling of DXS and DXR genes in Atractylodes lancea.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

[Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province].

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Full-length transcriptome analysis of two chemotype and functional characterization of genes related to sesquiterpene biosynthesis in Atractylodes lancea.

Atractylodes lancea (Thunb.) DC. is an important medicinal plant mainly distributed in China. A. lancea is rich in volatile oils and has a significant effect on various diseases, including coronavirus disease 2019 (COVID-19). Based on the signature constituents of volatile oils, A. lancea is divided into two chemotypes: the Dabieshan and Maoshan chemotype. Gas chromatography-mass spectrometry (GC-MS) results revealed that the hinesol and ß-eudesmol contents in the Dabieshan chemotype were higher than those in the Maoshan chemotype. Next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technologies were combined to investigate the molecular mechanisms of sesquiterpenoid biosynthesis in A. lancea. A total of 42 differentially expressed genes (DEGs) for terpenoid biosynthesis were identified in the two chemotype groups, and nine full-length terpene synthase (TPS) genes were identified. Subcellular localization revealed that AlTPS1 and AlTPS2 proteins were localized in the nucleus and endoplasmic reticulum. They use FPP as a substrate to generate sesquiterpenoids. AlTPS1 catalyzes biosynthesis of elemol while AlTPS2 is observed to perform ß-farnesene synthase activity. This study provides information for understanding the differences in the accumulation of terpenoids in two chemotypes of A. lancea and lays a foundation for further elucidation of the molecular mechanism of sesquiterpenoid biosynthesis.