[Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata].

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified in vitro. The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its in vivo function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

[Overexpression and isolation of CYP76AH1 in Salvia miltiorrhiza hairy roots].

Protein complexes are involved in the synthesis of multiple secondary metabolites in plants, and their separation is essential to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0.5% Triton X-100 was selected as the best extraction buffer, and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.

[Effect of Chemical Constituents from Glycyrrhiza uralensis Induced by Exogenous 6-Benzylaminopurine in Different Growth Periods].

Objective: To study the effect of chemical constituents from Glycyrrhiza uralensis induced by exogenous 6-benzylaminopurine( 6-BA) in different growth periods, aim to explore the regulatory network of Glycyrrhiza uralensis. Methods: The transplants of two-year-old Glycyrrhiza uralensis were subjected to four concentrations of 6-BA( 15,20,50,100 mg / L) at June and July. The content of seven kinds of chemical constituents including glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, liquiritin apioside,isoliquiritin apioside were determined by HPLC. Then long-term dynamic changes of the content of seven chemical compositions were analyzed. Results: In June, the content of seven compositions increased remarkably after 6-BA stimulating than that in July. Seven kinds of chemical constituents increased remarkably after 100 mg / L 6-BA stimulating for 3 months, and the increase rates were59. 34%,71. 14%,57. 31%,16. 36%,30. 17%,80. 26%,91. 90%,respectively. The greatest impact was glycyrrhizic acid with different concentration stimulating. 6-BA stress had a protection ability against the decrease of flavonoids in licorice under natural growth condition. The treatment of 6-BA had effect to the content of Glycyrrhiza uralensis,but hardly altered the ratio of their chemical composition. Conclusion: A regulatory network is existed among medicinal ingredients of Glycyrrhiza uralensis. Glycyrrhizic acid and flavonoids accumulation of Glycyrrhiza uralensis can be affected by exogenous 6-BA stimulation in certain case.

[Study on chemical constituents in Lysinotus wilsonii by UPLC-Q-TOF-MS].

The Ultra-high Performance Liquid Chromatography Quadrupole Time-of-flight Mass Spectrometry（UPLC-Q-TOF-MS）was applied to analyze the chemical components in Lysinotus wilsonii. A Waters ACQUITY UPLC-BEH-C18 S column（2.1 mm×100 mm,1.7 µm）was used with a gradient elution of acetonitrile-water containing 0.1％ formic acid. The mass spectrometry equipped with ionization source was used and the data was collected in negative ion mode. Results showed that 57 components were identified as 42 phenylethanoid glycosides, 5 benzyl alcohol glycosides, 6 flavonoids and 4 other components. Among them, 43 compounds were firstly identified in Gensneriaceae and one benzyl alcohol glycoside may be a new compound. We have quite completely identified the components in L. wilsonii for the first time, which may lay the foundation for further study and utilization of the medicinal plant.

[Rapid analysis of compounds in leaves of Chinese seabuckthorn and Tibetan seabuckthorn by UPLC/Q-TOF-MS].

The study is aimed to analyze the chemical components in leaves of Chinese seabuckthorn and Tibetan seabuckthorn qualitatively and compare the differences between them by using ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS).The chromatographic separation of the components was achieved ona Waters ACQUITY UPLC-T3 column (2.1 mm×100 mm, 1.7 µm）using gradient mobile phase consisting of acetonitrile (A) and aqueous solution (B). The identification of the separated compounds was performed on atandem mass spectrometry (MS/MS)by fragmentation patterns under the negative electrospray ionization. The parameters of ion source were as follows：capillary voltage, 2 000 V; Cone voltage, 40 V. The ion source temperature, 100 ℃; collision gas argon; sheath gas flow rate, 900 L•h⁻¹; sheath gas temperature, 450 ℃. Through the analysis of mass spectrometry data and with the help of literature data, a total of 35 compounds were detected and most of them were flavonoids. Among these compounds, 29 were common components for the two species, two components were unique to Chinese seabuckthorn and 4 were characteristic components of Tibetan seabuckthorn. The results indicated that the compositions of the two kinds of seabuckthorn leaves were quite similar. It is also demonstrated that UPLC/Q-TOF-MS method could be applied to rapidly and effectively analyze and speculate the compounds in leaves of Chinese seabuckthorn and Tibetan seabuckthorn.