[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.

[Cloning and expression analysis of a tyrosine decarboxylase gene from Rehmannia glutinosa].

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.

[Responses of physiological ecology and quality evaluation of Rehmannia gltinosa in continuous cropping].

OBJECTIVE: To study responses of physiological ecology and quality evaluation of Rehmannia glutinosa in continuous cropping. METHOD: The potted plant R. glutinosa which consists of first cropping, 1 year continuous cropping and 2 year continuous cropping were used as experimental materials. The photosynthetic activity, descending axis vitality, the protective enzymes system and MDA content were measured, the quality was evaluated by FTIR and HPLC. RESULT: Continuous cropping reduced the content of chlorophyll in the non-first cropping R. glutinos, the photosynthetic activity and descending axis vitality were weakened. Because of the increase of the free radical in the R. glutinos due to the continuous cropping, the activity of protective enzymes including POD, SOD and CAT were enhanced and MDA content were increased, more importantly the medical potency declined . And along with the increasing years of the continuous cropping, this effect becomes even stronger. CONCLUSION: Continuous cropping affects the descending axis ability of absorbing water and nutrition and photosynthesis are inhibited R. glutinosa, at the same time, it also causes the disorders of antioxidation systems in R. glutinos, resulting in continuous cropping obstacle and decline of the medicinal materials quality.

[Effects of continuous cropping obstacle on growth of Rehmannia glutinosa].

OBJECTIVE: To study the effects of continuous cropping obstacles on growth of Rehmannia glutinosa. METHOD: The growth indexes, activity of root ATPase, root activity and mineral nutritional absorption were determined. RESULT: Continuous copping decreased growth rate and declined the size of leaves. Activity of root ATPase and root activity were also inhibited. CONCLUSION: The deficiency of source capacity is an important factor to restrain the root development of R. glutinosa with continuous cropping, the decrease of root activity and ATPase activity as well as nutritional stress of potassium and nitrogen are the reasons for the effects of continuous cropping on the growth and development of R. glutinosa.

Induction of phenylalanine ammonia-lyase gene expression by paraquat and stress-related hormones in Rehmannia glutinosa.

Rehmannia glutinosa is a medicinal herb that is tolerant to the non-selective herbicide paraquat. Acteoside, a phenolic compound present in the plant, has been shown to inhibit paraquat. To understand regulation of the phenylpropanoid pathway that produces the acteoside moiety, we isolated a phenylalanine ammonia-lyase (PAL) cDNA clone (RgPAL1) and used it to examine PAL expression. The deduced 712 amino acid sequence of the open reading frame contains the conserved active site and potential phosphorylation sites of other plant PALs. RgPAL1 mRNA was detected in the leaves, flowers and roots of healthy plants, and the level of the mRNA was higher in leaves than in flowers and roots. RgPAL1 mRNA was induced in leaves by paraquat, H2O2, UV light, wounding, yeast extract, jasmonic acid and ethephon. The transcript level and enzyme activity increased gradually from 6 to 24 h after exposure to paraquat or jasmonic acid. Induction of RgPAL1 by paraquat and stress-related phytohormones suggests that it is involved in the regulation of the phenylpropanoid pathway under oxidative stress.

Isolation and Characterization of Basic Exochitinase from Leaf Extract of Rehmannia glutinosa.

Rehmannia chitinases were extracted from the leaves of Rehmannia glutinosa under acidic conditions (pH 2.9). We purified a 28.6-kDa chitinase, designated as P2, from crude extract to homogeneity by (NH4)2SO4 precipitation, chromatography with regenerated chitin affinity and hydrophobic interaction column, and preparative native PAGE. Isolated P2 showed maximum chitinase activity at pH 5.0 and 60°C, and had a isoelectric point of 8.46. P2 produced only (GlcNAc)2 from (GlcNAc)4-6 and regenerated chitin. Based on these results, we arrived at the conclusion that P2 was a basic exochitinase.