Full-length transcriptome profiling of Acanthopanax gracilistylus provides new insight into the kaurenoic acid biosynthesis pathway.

Acanthopanax gracilistylus is a deciduous plant in the family Araliaceae, which is commonly used in Chinese herbal medicine, as the root bark has functions of nourishing the liver and kidneys, removing dampness and expelling wind, and strengthening the bones and tendons. Kaurenoic acid (KA) is the main effective substance in the root bark of A. gracilistylus with strong anti-inflammatory effects. To elucidate the KA biosynthesis pathway, second-generation (DNA nanoball) and third-generation (Pacific Biosciences) sequencing were performed to analyze the transcriptomes of the A. gracilistylus leaves, roots, and stems. Among the total 505,880 isoforms, 408,954 were annotated by seven major databases. Sixty isoforms with complete open reading frames encoding 11 key enzymes involved in the KA biosynthesis pathway were identified. Correlation analysis between isoform expression and KA content identified a total of eight key genes. Six key enzyme genes involved in KA biosynthesis were validated by real-time quantitative polymerase chain reaction. Based on the sequence analysis, the spatial structure of ent-kaurene oxidase was modeled, which plays roles in the three continuous oxidations steps of KA biosynthesis. This study greatly enriches the transcriptome data of A. gracilistylus and facilitates further analysis of the function and regulation mechanism of key enzymes in the KA biosynthesis pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01436-7.

Comparative transcriptome analysis of different tissues of Solanum khasianum reveals candidate genes involved in steroidal glycoalkaloid biosynthesis.

Fruits and leaves of Solanum khasianum C. B. Clarke have long been used as a common Chinese herbal medicine. Steroidal glycoalkaloids (SGAs), the main active ingredient in S. khasianum, exhibit various pharmacological effects. However, genes involved in the SGA biosynthetic pathway in S. khasianum have not yet been identified. Genes encoding potential key SGA biosynthesis enzymes were identified through comprehensive RNA sequencing analysis (RNA-seq) of S. khasianum leaves, stems, and fruits. A total of 123,704 unigenes were obtained, of which 109,775 (88.74%) were annotated in seven public databases. Among these, 54 unigenes potentially involved in SGA biosynthesis were identified. Additionally, 23,636 differentially expressed genes were identified by comparing gene expression levels among the fruits, stems, and leaves of S. khasianum. The structural characteristics and phylogenetic relationship of cycloartenol synthase involved in SGA biosynthesis were further analyzed. Solasodine constituent was detected by high-performance liquid chromatography. This is the first study to report the comparative transcriptome analysis of different tissues of S. khasianum that identifies valuable genes potentially involved in SGA biosynthesis in this species.

Comparative transcriptomic analysis reveals the regulatory mechanisms of catechins synthesis in different cultivars of Camellia sinensis.

Camellia sinensis (L.) O. Kuntze is used to produce tea, a beverage consumed worldwide. Catechins are major medically active components of C. sinensis and can be used clinically to treat hyperglycaemia, hypertension, and cancer. In this study, we aimed to identify the genes involved in catechins biosynthesis. To this end, we analysed transcriptome data from two different cultivars of C. sinensis using DNBSEQ technology. In total,47,717 unigenes were obtained from two cultivars of C. sinensis, of which 9429 were predicted as new unigenes. In our analyses of the Kyoto Encyclopedia of Genes and Genomes database, 212 unigenes encoding 13 key enzymes involved in catechins biosynthesis were identified; the structures of leucoanthocyanidin reductase and anthocyanidin reductase were spatially modelled. Some of these key enzymes were verified by real-time quantitative polymerase chain reaction, and multiple genes encoding plant resistance proteins or transcription factors were identified and analysed. Furthermore, two microRNAs involved in the regulation of catechins biosynthesis were explored. Differentially expressed genes involved in the flavonoid biosynthesis pathway were identified from pairwise comparisons of genes from different cultivars of tea plants. Overall, our findings expanded the number of publicly available transcript datasets for this valuable plant species and identified candidate genes related to the biosynthesis of C. sinensis catechins, thereby establishing a foundation for further in-depth studies of catechins biosynthesis in varieties or cultivars of C. sinensis.

Probing the transcriptome of Boehmeria nivea reveals candidate genes associated with the biosynthesis of chlorogenic acid.

Boehmeria nivea (L.) Gaudich is used in traditional Chinese medicine. Chlorogenic acids are major medically active components of Boehmeria nivea, which can be used clinically to treat hyperglycemia, pneumonia, and cancer. To identify the genes involved in chlorogenic acid biosynthesis, we analyzed transcriptome data from leaf, root, and stem tissues of Boehmeria nivea using the Illumina Hi-Seq 4000 platform. A total of 146,790 unigenes were obtained from Boehmeria nivea, of which 106,786 were annotated in public databases. In analyses of the KEGG (Kyoto Encyclopedia of Genes and Genome) database, 484 unigenes that encode the five key enzymes involved in chlorogenic acid biosynthesis were identified, and shikimate O-hydroxycinnamoyl transferase was spatially simulated. Some of these key enzyme unigenes expression levels were verified by RT-qPCR (real-time quantitative Polymerase Chain Reaction). Furthermore, multiple genes encoding plant resistance proteins or transcription factors were identified and analyzed. Differentially expressed genes were identified by performing pairwise comparison of genes between tissues. This study increases the number of public transcript datasets of this species and identifies candidate genes related to the biosynthesis of chlorogenic acid, laying a foundation for the further exploration of this pathway in Boehmeria nivea.

[Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea].

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid ß-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid ß-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid ß-oxidation in A. lancea.

Genetic basis of maize kernel oil-related traits revealed by high-density SNP markers in a recombinant inbred line population.

BACKGROUND: Maize (Zea mays ssp. mays) is the most abundantly cultivated and highly valued food commodity in the world. Oil from maize kernels is highly nutritious and important for the diet and health of humans, and it can be used as a source of bioenergy. A better understanding of genetic basis for maize kernel oil can help improve the oil content and quality when applied in breeding. RESULTS: In this study, a KUI3/SC55 recombinant inbred line (RIL) population, consisting of 180 individuals was constructed from a cross between inbred lines KUI3 and SC55. We phenotyped 19 oil-related traits and subsequently dissected the genetic architecture of oil-related traits in maize kernels based on a high-density genetic map. In total, 62 quantitative trait loci (QTLs), with 2 to 5 QTLs per trait, were detected in the KUI3/SC55 RIL population. Each QTL accounted for 6.7% (qSTOL1) to 31.02% (qBELI6) of phenotypic variation and the total phenotypic variation explained (PVE) of all detected QTLs for each trait ranged from 12.5% (OIL) to 52.5% (C16:0/C16:1). Of all these identified QTLs, only 5 were major QTLs located in three genomic regions on chromosome 6 and 9. In addition, two pairs of epistatic QTLs with additive effects were detected and they explained 3.3 and 2.4% of the phenotypic variation, respectively. Colocalization with a previous GWAS on oil-related traits, identified 19 genes. Of these genes, two important candidate genes, GRMZM2G101515 and GRMZM2G022558, were further verified to be associated with C20:0/C22:0 and C18:0/C20:0, respectively, according to a gene-based association analysis. The first gene encodes a kinase-related protein with unknown function, while the second gene encodes fatty acid elongase 2 (fae2) and directly participates in the biosynthesis of very long chain fatty acids in Arabidopsis. CONCLUSIONS: Our results provide insights on the genetic basis of oil-related traits and a theoretical basis for improving maize quality by marker-assisted selection.

Transcriptome analysis identifies putative genes involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of ß-amyrin synthase, the key synthase of ß-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

Comparative transcriptome analysis of tubers, stems, and flowers of Gastrodia elata Blume reveals potential genes involved in the biosynthesis of phenolics.

Orchidaceae, well known for its fascinating flowers, is one of the largest and most diverse families of flowering plants. There are many kinds of plants in this family; these are distributed practically globally and have high ornamental and medicinal values. Gastrodia elata Blume, a traditional Chinese medicinal herb, is a rootless and leafless achlorophyllous orchid. Phenolic compounds are considered to be the major bioactive constituents in G. elata, with antioxidant, antiangiogenic, neuroprotective, antidepressant, anxiolytic, and sedative activities. In this study, we determined the contents of six main phenolic components in tubers, stems and flowers from G. elata. Meanwhile, the transcriptomes of the tuber, stem and flower tissues of G. elata were obtained using the BGISEQ-500 platform. A total of 58.29 Gb of data and 113,067 unigenes were obtained, of which 74,820 unigenes were functionally annotated against seven public databases. Differentially expressed genes between tuber, stem and flower tissues were identified. A total of 76 DEGs encoding eight key enzymes were identified as candidate genes involved in the biosynthesis of phenolics in G. elata. For further validation, the expression levels of unigenes were measured using quantitative real-time PCR. Our results greatly enrich the transcriptomic data of G. elata and provide valuable information for the identification of candidate genes involved in the biosynthesis of secondary metabolites.

Full-length transcriptome sequences by a combination of sequencing platforms applied to isoflavonoid and triterpenoid saponin biosynthesis of Astragalus mongholicus Bunge.

BACKGROUND: Astragalus mongholicus Bunge is an important medicinal plant used in traditional Chinese medicine. It is rich in isoflavonoids and triterpenoid saponins. Although these active constituents of A. mongholicus have been discovered for a long time, the genetic basis of isoflavonoid and triterpenoid saponin biosynthesis in this plant is virtually unknown because of the lack of a reference genome. Here, we used a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to identify genes involved in the biosynthetic pathway of secondary metabolites in A. mongholicus. RESULTS: In this study, NGS, SMRT sequencing, and targeted compound analysis were combined to investigate the association between isoflavonoid and triterpenoid saponin content, and specific gene expression in the root, stem, and leaves of A. mongholicus. Overall, 643,812 CCS reads were generated, yielding 121,107 non-redundant transcript isoforms with an N50 value of 2124 bp. Based on these highly accurate transcripts, 104,756 (86.50%) transcripts were successfully annotated by any of the seven databases (NR, NT, Swissprot, KEGG, KOG, Pfam and GO). Levels of four isoflavonoids and four astragalosides (triterpenoid saponins) were determined. Forty-four differentially expressed genes (DEGs) involved in isoflavonoid biosynthesis and 44 DEGs from 16 gene families that encode enzymes involved in triterpenoid saponin biosynthesis were identified. Transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs, and 11 bZIPs, were also identified. The above transcripts showed different expression trends in different plant organs. CONCLUSIONS: This study provides important genetic information on the A. mongholicus genes that are essential for isoflavonoid and triterpenoid saponin biosynthesis, and provides a basis for developing the medicinal value of this plant.

Tissue-specific metabolite profiling of Fallopia multiflora (Heshouwu) and Fallopia multiflora var. angulata by mass spectrometry imaging and laser microdissection combined with UPLC-Q/TOF-MS.

Heshouwu, derived from root tubers of Fallopia multiflora (Thunb.) Harald., is a well-known herb used for millennia in traditional Chinese medicine. However, different forms of root tubers of Heshouwu have occurred in current Chinese herbal market and used in clinic, although it is still unknown whether their quality is consistent. In the present study, a mass spectrometry imaging and laser microdissection combined with UPLC-Q/TOF-MS were therefore used for the metabolite profiling on the whole and different parts of root tubers of F. multiflora and F. multiflora var. angulata. Our results suggested that the character of "woody heart" root tubers of F. multiflora was similar to that of F. multiflora var. angulata, but the latter had more phloem fibers and larger diameter vessel in the normal vascular bundle. Moreover, 140 compounds including stilbenes, anthraquinones, phenolic acids, naphthalenes, and other compounds were identified or putatively characterized from F. multiflora and F. multiflora var. angulata. Both unsupervised principal component analysis (PCA) and supervised Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) multivariate statistics allowed discriminating F. multiflora and F. multiflora var. angulata. And a total of 32 potential markers were identified. The tissue-specific study indicated that the compounds in the phelloderm of F. multiflora and F. multiflora var. angulata were the most abundant. This is the first study on metabolite profiling and comparison of root tubers between F. multiflora and F. multiflora var. angulata, which would provide reasonable basis for further quality evaluation and safe medication of F. multiflora.

Genome survey sequencing of Atractylodes lancea and identification of its SSR markers.

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine rich in sesquiterpenes that has been widely used in China and Japan for the treatment of viral infections. Despite its important pharmacological value, genomic information regarding A. lancea is currently unavailable. In the present study, the whole genome sequence of A. lancea was obtained using an Illumina sequencing platform. The results revealed an estimated genome size for A. lancea of 4,159.24 Mb, with 2.28% heterozygosity, and a repeat rate of 89.2%, all of which indicate a highly heterozygous genome. Based on the genomic data of A. lancea, 27,582 simple sequence repeat (SSR) markers were identified. The differences in representation among nucleotide repeat types were large, e.g., the mononucleotide repeat type was the most abundant (54.74%) while the pentanucleotide repeats were the least abundant (0.10%), and sequence motifs GA/TC (31.17%) and TTC/GAA (7.23%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. A total of 93,434 genes matched known genes in common databases including 48,493 genes in the Gene Ontology (GO) database and 34,929 genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This is the first report to sequence and characterize the whole genome of A. lancea and will provide a theoretical basis and reference for further genome-wide deep sequencing and SSR molecular marker development of A. lancea.

[Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida].

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.