Structural and interactions analysis of a transcription factor PnMYB2 in Panax notoginseng.

The main active ingredients of the traditional Chinese medicinal plant, Panax notoginseng, are the Panax notoginseng saponins (PNS). They can be synthesized via the mevalonate pathway; PnSS and PnSE1 are the key rate-limiting enzymes in this pathway. In this study, an interaction between PnMYB2 and the key enzymes was identified and characterized from the P. notoginseng cDNA library using the Y1H technique. Subsequently, X-α-gal color reaction confirmed the interaction between PnMYB2 and the upstream sequences of PnSS and PnSE1 promoters. Full-length cDNA sequence of PnMYB2 was isolated and characterized. PnMYB2 has an open reading frame of 864 bp, encoding 287 amino acids. 3D structural analysis of PnMYB2 indicated that its structure was similar to that of the template. Phylogenetic analysis revealed that PnMYB2 and PgMYB2 are highly homologous and belong to the R2R3 MYB transcription factor (TF). Subcellular localization analysis showed that PnMYB2 was localized in the nucleus. The recombinant protein PnMYB2 was successfully obtained through prokaryotic expression and was confirmed to be an inclusion body protein. Furthermore, electrophoretic mobility shift assay (EMSA) experiments demonstrated that PnMYB2 specifically binds to MYB core and AC-rich elements. This study provides a theoretical basis for transcriptional regulation of saponin biosynthesis in P. notoginseng.

Molecular cloning and structural analysis of key enzymes in Tetrastigma hemsleyanum for resveratrol biosynthesis.

Resveratrol (RES), a plant antitoxin, has antioxidant, anti-inflammatory, anti-cancer and cardiovascular protection effects. It has been reported that RES can be stably detected in a Chinese herbal medicinal plant Tetrastigma hemsleyanum. At present, the research of T. hemsleyanum mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites in T. hemsleyanum. In this experiment, four key enzymes (ThPAL/ThC4H/Th4CL/ThRS) involved in the RES biosynthesis pathway were cloned and obtained. They contained an open reading frame (ORF) of 2139 bp, 1518 bp, 1716 bp and 1035 bp, encoding 712, 505, 571 and 344 amino acids, separately. Various bioinformatics tools were used to analyze these deduced protein domains, secondary structures, three-dimensional (3D) structures and phylogenetic trees. Subsequently, quantitative primers were designed to conduct the tissue-specific expression. Quantitative results displayed that the four genes were expressed in all tested tissues, and their expression in root tubers was more stable. Moreover, the subcellular localization of the four genes was studied by constructed recombinant green fluorescent expression vectors. Herein, by digging out the key enzyme genes in the biosynthesis of RES in T. hemsleyanum, this experiment tried to reveal the expression patterns of these key enzyme genes. It also provided the basis for the research on the molecular level, which will help people further illuminate and clarify the biosynthesis and regulation mechanism of secondary metabolites in T. hemsleyanum.

The research progresses and future prospects of Tetrastigma hemsleyanum Diels et Gilg: A valuable Chinese herbal medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetrastigma hemsleyanum Diels et Gilg, known as Sanyeqing in China, was an extensively used folk Chinese herbal medicine. This plant had been validated to be highly effective for treating high fever, pneumonia, hepatitis, gastritis, cervicitis, lymphatic tuberculosis, septicemia, and viral meningitis. AIM OF THE REVIEW: As a review in T. hemsleyanum, this article aims to provide a critical and comprehensive evaluation for future research as well as the development of new drugs. The possible uses and future research directions of this plant were also discussed. MATERIALS AND METHODS: A literature search was conducted on different scientific search engines, including Google Scholar, Science Direct, PubMed, Web of Science, and CNKI. Additional information was obtained from classic books about Chinese herbal medicine and scientific databases. RESULTS: T. hemsleyanum was a perennial herb climbing vine, which was mainly based on field cultivation. About 150 chemical compounds have been isolated from T. hemsleyanum, including flavonoids, phenolic acids, polysaccharides, triterpenoids, steroids, and organic acids. Studies on the physiological aspects of T. hemsleyanum have been focused on the effects of light and fertilizer on their growth, and few other studies have been conducted. The plant had widespread pharmacological effects on the immune system, as well as anti-tumor, anti-inflammatory, analgesic, and antipyretic. CONCLUSIONS: T. hemsleyanum was a valuable traditional Chinese medical herb with pharmacological activities that mainly affected the immune system. This review summarized its botanical description, cultivation techniques, physiology, ethnopharmacology, chemical components, and pharmacological functions. This information suggested that we should focus on the development of new drugs related to T. hemsleyanum. Meanwhile, it was important to emphasize the traditional use of T. hemsleyanum, avoiding over-harvesting that exerted a great impact on resource scarcity. And developing its new clinical usage and comprehensive utilize would augment the therapeutic potentials of T. hemsleyanum.

An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

[Clinical observation of the curative effect of Qiangxin Mixture on congestive heart failure].

OBJECTIVE: To investigate the efficacy of Qiangxin Mixture in patients with congestive heart failure (CHF). METHODS: Sixty cases of CHF were randomly divided into Qiangxin Mixture group (treatment group, n=30) and digoxin group (control group, n=30). The total clinical effective rate, integra of the symptoms of kidney deficiency, classification of functional capacity of the New York Heart Association (NYHA), and echocardiogram [ejection fraction (EF), cardiac output (CO), interventricular septal thickness (IVST), posterior wall thickness (PWT), left ventricular mass index (LVMI)] as well as the measurement of plasma endothelin, angiotensin II and atrial natriuretic peptide were observed in both groups. RESULTS: The total clinical effective rate of Qiangxin Mixture group was 87%, and improvement was significantly observed in the Lee CHF score, classification of functional capacity of the NYHA, EF and CO (P<0.05, vs before treatment), but no significant improvement in digoxin group (P>0.05). The integra of the symptoms of kidney deficiency, the levels of plasma ET, Ang II and ANP decreased significantly (P<0.01, vs before treatment and digoxin group respectively). IVST, PWT and LVMI were also reduced significantly (P<0.01, vs before treatment; P<0.05 vs digoxin group). CONCLUSION: The Qiangxin Mixture is effective in enhancing cardiac contraction, improving hemodynamics in the short-term and rectifying some indexes in the long-term, so it could postpone the processes of CHF. This mechanism may be related to decreasing the stimulating factors (angiotensin, endothelin) which trigger the cardiac remodeling, delaying or reversing the cardiac remodeling.