[Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa].

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

Molecular Regulation of Catalpol and Acteoside Accumulation in Radial Striation and non-Radial Striation of Rehmannia glutinosa Tuberous Root.

Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

[Comparison of chemical quality characteristics between radial striations and non-radial striations in tuberous root of Rehmannia glutinosa].

An HPLC method was established to determine the contents of catalpol, acteoside, rehmaionoside A, rehmaionoside D, leonuride in three part of Rehmanni glutinosa in Beijing No.1 variety R. glutinosa during the growth period, This method, in combination with its HPLC fingerprint was used to evaluate its overall quality characteristics.The results showed that：① the content of main components of R. glutinosa varied in different growth stages ;② there was a great difference of the content of main components between theradial striations and the non-radial striations; ③ the two sections almost have the same content distribution of catalpol, acteoside and rehmaionoside D; ④the content of rehmaionoside A in non-radial striations was higher than that in radial striations,while the content of leonuride in radial striations was higher than that in non-radial striations.; ⑤the HPLC fingerprint of radial striations, non-radial striations and whole root tuber were basically identical, except for the big difference in the content of chemical components. The result of clustering displayed that the radial striations, non-radial striations, and whole root were divided into two groups. In conclusion, there was a significant difference in the quality characteristics of radial striations and non-radial striations of R. glutinosa. This research provides a reference for quality evaluation and geoherbalism of R. glutinosa.

Endophytic Fungi and Secondary Metabolites of Rehmannia Glutinosa Based on Traditional Chinese Medicine Fingerprints.

Research on the active components of medicinal plants has always been the focus of research, and research on the active components of medicinal plant endophytic fungi and their secondary metabolites has also attracted widespread attention. Endophytic fungi of medicinal plants are widely distributed and are ubiquitous in various biological groups in nature. Rehmannia glutinosa contains a variety of active ingredients, which are regarded as the top grade of Chinese medicinal materials. It is of certain significance to study endophytic fungi and their metabolites of Rehmannia glutinosa. In this paper, endophytic fungi and their secondary metabolites of Rehmannia glutinosa were studied using fingerprint technology, which initially understands the diversity of endophytic fungi in Rehmannia glutinosa. In this paper, the roots and leaves of Rehmannia glutinosa were used as experimental materials. The fungi were cultured in the medium, the fungi were isolated and purified by the tissue block method, the fungal growth of Rehmannia glutinosa in different parts was determined, and the types of endophytic fungi were identified by microscopic identification and fingerprinting. The isolated strains were tested for biological activity using oryza oryzae spores, and highly active strains were screened. Fermentation products of endophytic fungi were separated and purified by chromatography, and the structure of the compounds was identified by nuclear magnetic resonance spectroscopy. Through the above studies, the population structure of endophytic fungi of Rehmannia glutinosa was determined, 3 highly active strains were found, and the structures of 7 endophytic fungi metabolites were identified, of which 3 were newly discovered compounds.