[Directing construction of CRISPR/Cas9 vector of SmPAL1 in Salvia miltiorrhiza by target efficiency detection in vitro].

To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences (SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity.

AIM: To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS: Primary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations (1, 3, 10 and 30 micromol/L) of berberine or 1 micromol/L Glibenclamide (GB) for 24 h. Glucose-stimulated insulin secretion (GSIS) assay was conducted and insulin was determined by radioimmunoassay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha (HNF4alpha) was determined by reverse transcription polymerase chain reaction (RT-PCR). Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4alpha in the islets. Glucokinase (GK) activity was measured by spectrophotometric method. RESULTS: Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 mumol/L. Both mRNA and protein expressions of HNF4alpha were up-regulated by berberine in a dose-dependent manner, and GK activity was also increased accordingly. However, GB demonstrated no regulatory effects on HNF4alpha expression or GK activity. CONCLUSION: Berberine can enhance GSIS in rat islets, and probably exerts the insulinotropic effect via a pathway involving HNF4alpha and GK, which is distinct from sulphonylureas (SUs).

[Relationship between photosynthetic characteristics and environmental factors in leaves of Pueraria lobata].

OBJECTIVE: To study the relationship between photosynthetic characteristics and environmental factors in leaves of P. lobata. METHOD: Photosynthetic characteristics and environmental factors were measured by using CIRAS-2 portable photosynthesis system. RESULT: The apparent quantum yield in leaves was 0.0173 micromol CO2 x micromol(-1) photon. The dark respiration rate was 2.9333 micromol x m(-2) x s(-1). The light compensation point of photosynthesis was 180 micromol x m(-2) x s(-1). The light saturation point was 1600 micromol x m(-2) x s(-1). The carboxylation efficiency was 0.0338 micromol x m(-2) x s(-1). The light respiration rate was 2.5 micromol x m(-2) x s(-1). The CO2 compensation point was 100 micromol x mol(-1), The CO2 saturation point was 1 600 micromol x mol(-1). CONCLUSION: Photo flux density and air temperature are major environmental factors influencing diumal changes of net photosynthetic rate.

[The qualitative analysis and quantitative analysis of purification of salvianolic acids by macroreticular resin].

OBJECTIVE: To purify salvianolic acids by macroreticular resin,then mensurate the contents of salvianolic acids and analyse the chromatogram with HPLC. METHOD: Make salvianolic acids with macroreticular resin; mensurate the content of Salvianolic acids with UV spestrophotometry: the control compound is protocaechuic aldehyde, and the wavelength is 281 nm. Analysis the chromatogram with HPLC, and compare the chromatogram in different technics: zorbax ODS column (4.6 mm x 250 mm, 5 microm), mobilephase: 1% aceticacid-water and methanol in different proportions, the wavelength is 281 nm. RESULT: The contents of salvianolic acids is 53.8%; HPLC chromatogram indicate that the method is reasonable to make salvianolic acids. CONCLUSION: Determination of contents and HPLC chromatogram can control the quality of Salvianolic acids more accurately.