Root rot was occurred widely in the production area of Rehmannia glutinosa, and which result in serious influence on the yield and quality of R. glutinosa. In the present work, a new phytopathogen was isolated from roots with root rot symptom in the production area of R. glutinosa. The colony of the pathogen growing on PDA medium was gray-black, the structure of hyphae was compact, the aerial hyphae was less developed, and the back of the colony was black. The hyphae of the pathogen were uneven in size, about 2 to 3 μm in diameter and twined with each other, the conidia of the pathogen were small, nearly round and about 1 μm in diameter. The healthy roots of R. glutinosa were inoculated with the pathogen in vitro, black-brown rot was observed at the inoculate sites after a few days' incubation. The rhizosphere soil of healthy R. glutinosa seedlings were inoculated in vivo, the leaves were wilted and the roots were black-brown rotted after several days' normal culture, the symptoms were consistent with those observed in the field. The genomic DNA of the pathogen was amplified by fungus rDNA-ITS universal primer ITS1/ITS4 and homologous analyzed, the pathogen was in a branch with Heterophoma sp., Phoma sp., P. novae-verbascicola and P. herbarum with the nuclear acid homology of 99.21% to 99.43%. The pathogen shown 97.00% to 98.02% nuclear acid homology with H. verbascicola, H. novae-verbascicola, H. poolensis, P. herbarum, H. sylvatica, H. verbascicola and H. verbasci-densiflori when amplified by the tub2 gene special primer Btub2 fd/Btub4 rd, and H. novae-verbascicola was the highest. The pathogen was in a branch with H. novae-verbascicola when amplified by the lsu gene special primer LR0 R/LR7. Based on the morphological characteristics, nucleotide sequence analysis and Koch's test results, the isolated pathogen causing root rot of R. glutinosa was identified as H. novae-verbascicola. This study is of great significance for the further theoretical research on root rot of R. glutinosa and root rot control in field.
DNA, Ribosomal ; Fungi/genetics* ; Plant Leaves ; Rehmannia/genetics* ; Seedlings

NRT1 family proteins play an important roles for absorbing and transporting of nitrate in different plants. In order to identify the NRT1 family genes of Rehmannia glutinosa, this study used 11 NRT1 homologous proteins of Arabidopsis as probe sequences and aligned with the transcriptome data of R. glutinosa by using NCBI BLASTN software. Resulting there were 18 NRT1 proteins were identified in R. glutinosa. On basis of this, a series of the molecular characteristics of R. glutinosa NRT1 proteins including the conserved domains, the transmembrane structure, the subcellular location and phylogenetic features were in detail analyzed. At same time, it were systematically analyzed that the temporal and spatial expression patterns and characteristics of R. glutinosa NRT1 family genes in response to different stress factors. The results indicated that 18 R. glutinosa NRT1 family genes with the length of coding region from 1 260 bp to 1 806 bp, encoded proteins ranging from 419 to 601 amino acids, and all of they owned the domains of typical peptide transporter with 7 to 12 transmembrane domains. These R. glutinosa NRT1 family proteins mostly were found to locate on cellular plasma membrane, and belonged to the hydrophobic proteins. Furthermore, the evolutionary analysis found that the 18 R. glutinosa NRT1 protein family could be divided into two subfamilies, of which 14 NRT1 family genes might occur the positive selection, and 4 genes occur the passivation selection during the evolution process of R. glutinosa. In addition the expression analysis showed that 18 R. glutinosa NRT1 family genes have the distinct expression patterns in different tissues of R. glutinosa, and their expression levels were also obvious difference in response to various stress. These findings infield that 18 R. glutinosa NRT1 family proteins might have obviously different functional roles in nitrate transport of R. glutinosa. In conclusion, this study lays a solid theoretical foundation for clarifying the absorption and transport molecular mechanism of N element during R. glutinosa growth and development, and at same time for deeply studying the molecular function of R. glutinosa NRT1 proteins in absorption and transport of nitrate.
Anion Transport Proteins ; Membrane Transport Proteins ; Nitrates ; Phylogeny ; Plant Proteins/metabolism* ; Rehmannia/genetics* ; Transcriptome

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.
Gene Expression Profiling ; Plant Leaves/genetics* ; Plant Roots/genetics* ; Rehmannia/genetics* ; Transcription, Genetic

This study investigated the differential mechanisms of Rehmanniae Radix and Rehmanniae Radix Praeparata in improving diabetes in mice through AMPK-mediated NF-κB/NLRP3 signaling pathway. The diabetic mouse model was established with high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days), after which the mice were randomly divided into model group, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, catalpol group(250 mg·kg~(-1)), 5-hydroxymethylfurfural(5-HMF) group(250 mg·kg~(-1)), metformin group(250 mg·kg~(-1)), with the normal group also set. The organ indexes of heart,liver, spleen, lung, kidney and pancreas were calculated after four weeks of administration. The pathological changes and fibrosis of pancreas, kidney and liver in mice were observed by hematoxylin-eosin(HE) staining and Masson staining. Western blot was used to determine the expression levels of Toll-like receptor-4(TLR4), nuclear factor-κB(NF-κB), Nod-like receptor protein 3(NLRP3),interleukin-1β(IL-1β), adenosine monophosphate-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK) in the pancreas, kidney and liver of mice. Compared with the model group, the administration groups witnessed significant decrease in the liver,spleen, kidney, pancreas and fat indexes of diabetic mice, and there was no significant difference in heart and lung indexes. The pathological states and fibrosis of pancreatic, kidney and liver tissues were significantly improved after administration. Additionally, the expression levels of TLR4, NF-κB and NLRP3 in pancreas, kidney and liver of diabetic mice were significantly lowered. The expression levels of p-AMPK/AMPK were enhanced significantly in kidney and liver of mice in Rehmanniae Radix group while in pancreas, kidney and liver in Rehmanniae Radix Praeparata group. This suggests that Rehmanniae Radix and Rehmanniae Radix Praeparata differ in the mechanism of regulating energy metabolism of multiple organs and thereby exerting anti-inflammatory effects to alleviate symptoms of diabetic mice.
AMP-Activated Protein Kinases/genetics* ; Animals ; Diabetes Mellitus, Experimental/drug therapy* ; Diet, High-Fat/adverse effects* ; Mice ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Plant Extracts ; Rehmannia ; Signal Transduction ; Streptozocin

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.
Cloning, Molecular ; Genes, Plant ; Iridoids ; metabolism ; Ligases ; genetics ; Phylogeny ; Rehmannia ; enzymology ; genetics

Catalpol, a major bioactive component from Rehmannia glutinosa, which has been used to treat diabetes. The present study was designed to elucidate the anti-diabetic effect and mechanism of action for catalpol in db/db mice. The db/db mice were randomly divided into six groups (10/group) according to their blood glucose levels: db/db control, metformin (positive control), and four dose levels of catalpol treatment (25, 50, 100, and 200 mg·kg), and 10 db/m mice were used as the normal control. All the groups were administered orally for 8 weeks. The levels of fasting blood glucose (FBG), random blood glucose (RBG), glucose tolerance, insulin tolerance, and glycated serum protein (GSP) and the globe gene expression in liver tissues were analyzed. Our results showed that catalpol treatment obviously reduced water intake and food intake in a dose-dependent manner. Catalpol treatment also remarkably reduce fasting blood glucose (FBG) and random blood glucose (RBG) in a dose-dependent manner. The RBG-lowering effect of catalpol was better than that of metformin. Furthermore, catalpol significantly improved glucose tolerance and insulin tolerance via increasing insulin sensitivity. Catalpol treatment significantly decreased GSP level. The comparisons of gene expression in liver tissues among normal control mice, db/db mice and catalpol treated mice (200 and 100 mg·kg) indicated that there were significant increases in the expressions of 287 genes, whichwere mainly involved in lipid metabolism, response to stress, energy metabolism, and cellular processes, and significant decreases in the expressions of 520 genes, which were mainly involved in cell growth, death, immune system, and response to stress. Four genes expressed differentially were linked to glucose metabolism or insulin signaling pathways, including Irs1 (insulin receptor substrate 1), Idh2 (isocitrate dehydrogenase 2 (NADP), mitochondrial), G6pd2 (glucose-6-phosphate dehydrogenase 2), and SOCS3 (suppressor of cytokine signaling 3). In conclusion, catalpol ecerted significant hypoglycemic effect and remarkable therapeutic effect in db/db mice via modulating various gene expressions.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; Gene Expression ; drug effects ; Glucosephosphate Dehydrogenase ; genetics ; metabolism ; Humans ; Hypoglycemic Agents ; administration & dosage ; Insulin ; metabolism ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Iridoid Glucosides ; administration & dosage ; analysis ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Rehmannia ; chemistry ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism

The study aims at evaluating genetic diversity and medicinal quality of cultivated germplasm in Rehmannia glutinosa, and providing theoretical guidance for screening excellent germplasm. The genetic diversity of 21 species of R. glutinosa were analyzed by SRAP molecular markers, and the catalpol and verbascoside was determined by HPLC. The mass fraction of catalpol and verbascoside in R. glutinosa germplasm were respectively in the range of 2.393%-6.519% and 0.063%-0.478%, the germplasm 14, 16, 15 and 20 germplasm, witch catalpol and verbascoside content was higher. A total of 57 bands were produced by 10 primer, among which 40 polymorphic bands were polymorphic bands, and the percentage of polymorphic loci was 8.77%-54.39%, the Nei's genetic diversity index (H) was 0.374 1, Shannon's polymorphism information index (I) was 0.546 6. Gst and gene flow Nm were 0.608 8 and 0.321 3, respectively. Based on the genetic uniformity, 21 species of germplasm were grouped into 2 categories. The genetic diversity level of R. glutinosa was medium low. The comprehensive consideration of the genetic diversity and the content inculde catalpol and verbascoside, germplasm 7 and germplasm 18 could be used as the preferred materials for the cultivation of reticulum. Germplasm 15 and 16 can be used as the preservation and breeding object of rhubarb germplasm.
Animals ; Gene Flow ; Genetic Variation ; Phylogeny ; Plant Breeding ; Plants, Medicinal ; genetics ; Rehmannia ; genetics

The history of Rehmannia glutinosa breeding has already beyond 100 years. There are rich cultivated varieties and wild germplasm resources in R. glutinosa. However, there also exist a lot of problems, such as, the pedigree of the existing varieties is not clear, the genetic basis is narrow, backward method of germplasm enhancement and breeding. Breeding of new varieties has been unable to meet the demand of R. glutinosa production in the new era. This paper summarizes the species of Rehmannia and their distribution, the diversity of plant morphology and the quality of R. glutinosa germplasm resources, as well as the progress of R. glutinosa breeding in recent 100 years. For ensuring the orderly, effective and safe production of R. glutinosa, the authors suggest to establish the wild resources protection area and germplasm resources garden, deeply study the genetic base of quality, strengthen application of new breeding method such as mutation breeding, haploid breeding and gene editing.
Plant Breeding ; Plants, Medicinal ; genetics ; Rehmannia ; genetics

Using cDNA from Rehmannia glutinosa leaf as template, a 972 bp fragment of expansin gene which containing a 762 bp ORF that encoded 253 amino acids, was cloned, named RgEXPA10, which GenBank accession number for this gene is KF011918. A 1 207 bp genomic sequence of RgEXPA10 was amplified by PCR with leaf DNA as template, sequencing analysis revealed that three exons and two introns in RgEXPA10 genomic sequence, and which GenBank accession number is KF011919. Molecular and bioinformatic analyses indicated that RgEXPA10 protein have DPBB_1 and Pollen_allerg_1 domain, also including a 26 aa nuclear localization signal and a 19 aa transmembrane region. Phylogenetic analysis revealed that RgEXPA10 showed the highest homology with AtEXPA8 among the 26 α-expansins in Arabidopsis thaliana. However, the RgEXPA10 indicated the highest homology with the expansin from Solanum lycopersicum among 22 plant species. Expression patterns using qRT-PCR analysis showed that RgEXPA10 mainly expressed in unfolded leaf, followed by the tuberous root at stage of expanding period, and rarely expressed in senescing leaf. And RgEXPA10 showed higher expression level in tuberous root at 60 and 90 days after emergence. The transcription level of RgEXPA10 significantly reduced under all the three stresses including continuous cropping conditions, salinity and waterlogging. This study will lay foundations for molecular function in development and regulation of different stresses for R. glutinosa.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Gene Expression Regulation, Plant ; Genes, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Leaves ; Plant Proteins ; genetics ; Plant Roots ; Rehmannia ; genetics

In order to study the development characteristics of Rehmannia glutinosa tuberous root expansion and reveal the regulation mechanism of the genes related to hormones in this process, R. glutinosa "wen-85" was used as the experimental material in this study. R. glutinosa tuberous roots of different developmental stages were collected to observe phenotype and tissue morphology using resin semi-thin sections method. The genes related to hormone biosynthesis and response were chosen from the transcriptome of R. glutinosa, which was previously constructed by our laboratory, their expression levels at different development stages were measured by real-time quantitative PCR. The results showed that the root development could be divided into six stages: seeding, elongation, pre-expanding, mid-expanding, late-expanding and maturity stage. The anatomic characteristics indicated that the fission of secondary cambium initiated the tuberous root expansion, and the continuous and rapid division of secondary cambium and accessory cambium kept the sustained and rapid expansion of tuberous root. In addition, a large number oleoplasts were observed in root on the semi-thin and ultra-thin section. The quantitative analysis suggested that the genes related to biosynthesis and response of the IAA, CK, ABA,ethylene, JA and EB were up-regulated expressed, meanwhile, GA synthesis and response genes were down-regulated expressed and the genes of GA negative regulation factors were up-regulated expressed. The maximum levels of most genes expression occurred in the elongation and pre-expansion stage, indicating these two stages were the key periods to the formation and development of tuberous roots. Oleoplasts might be the essential cytological basis for the formation and storage of the unique medicinal components in R. glutinosa. The results of the study are helpful for explanation of development and the molecular regulation mechanism of the tuberous root in R. glutinosa.
Gene Expression Regulation, Developmental ; drug effects ; genetics ; Gene Expression Regulation, Plant ; drug effects ; genetics ; Lipid Droplets ; metabolism ; ultrastructure ; Microscopy, Electron, Transmission ; Plant Growth Regulators ; biosynthesis ; pharmacology ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; genetics ; growth & development ; metabolism ; Rehmannia ; genetics ; growth & development ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors