Cryopreservation Conditions of Carthamus tinctorius Seeds / 中国实验方剂学杂志

ObjectiveTo study on the suitable cryopreservation conditions of Carthamus tinctorius seeds. MethodThe germination rate，relative conductivity，soluble sugar，soluble protein， and related enzyme activities of C. tinctorius seeds， as well as the hydroxysafflor yellow A （HSYA） content in Carthami Flos after storage and breeding for four months were detected under different temperature conditions （long-term storage，medium-term storage，short-term storage，room temperature，and ultra-low temperature refrigerator），different water content （8.1%，6.6%，5.2%，and 3.9%），and different storage time （2，4，6，8， 10 months）. SPSS 20.0 was used for statistical analysis. ResultDuring the storage for 10 months，the changing trend of the germination rate of C. tinctorius seeds revealed that it was more suitable to store seeds with low water content at a lower temperature. The differences in germination rate of seeds caused by storage temperature，seeds water content， and storage time were statistically significant. After storage for 10 months，the germination rate was significantly correlated with other detection indexes. ConclusionThe proper water content of C. tinctorius seeds in long-term and medium-term storage is 5.2% or 6.6%，and that in short-term and ultra-low temperature refrigerator is 3.9% or 5.2%. As revealed by the comparison results， the optimal storage conditions for C. tinctorius seeds were long-term storage and water content of 5.2%， which resulted in the highest germination rate and content of soluble sugar and soluble protein and the lowest relative conductivity after storage for 10 months. Additionally， the content of hydroxy safflor yellow A （HSYA） in Carthami Flos obtained after breeding and regeneration for four months was higher than that obtained after room temperature storage.

Analysis of Equivalence Between Traditional and Nontraditional Medicinal Varieties of Citri Reticulatae Semen from Sichuan Province / 中国实验方剂学杂志

Objective:To study the differences in genetic relationship， shape， size， and flavonoid content between traditional and nontraditional medicinal varieties of Citri Reticulatae Semen produced in Sichuan province as well as their equivalence. Method:Six batches of traditional medicinal Citri Reticulatae Semen （<italic>Citrus reticulata</italic> 'Dahongpao'） and 23 batches of nontraditional medicinal varieties were collected， and their genetic relationship was explored using sequence-related amplified polymorphism （SRAP） markers. Following the observation of their shapes and sizes under a stereomicroscope， the contents of naringin， hesperidin， and neohesperidin were measured by high performance liquid chromatography （HPLC）. SIMCA 14.1 software was used for cluster analysis of their shapes， sizes， and flavonoid contents， thus figuring out the similarities between the traditional and nontraditional medicinal varieties in character， size， and chemical components. Result:SRAP markers-based genetic relationship analysis effectively distinguished different Citri Reticulatae Semen varieties from each other. Some samples collected from the same or adjacent places exhibited a close genetic relationship and they shared high similarities in shape， size， and flavonoid content. However， the traditional medicinal Citri Reticulatae Semen was still quite different from most nontraditional medicinal varieties. Conclusion:The analysis of differences in genetic materials， appearance， character， and active ingredient content between the traditional and nontraditional medicinal varieties revealed that the equivalence<italic> </italic>of <italic>C.</italic> <italic>reticulata</italic> 'Ponkan' samples from some regions with the traditional medicinal variety was the largest， enabling them to be considered as the emerging medicinal variety.

Correlation between accumulation of flavoneand expression of functional genes in Carthamus tinctorius / 中国中药杂志

The average yield of safflower blooming from 1 to 7 day was recorded and calculated, HPLC was used to detect the percentage composition of HYSA,quercetin,naringenin and kaempferol, and the real-time PCR was used to analyze the expression of chs and chi. The average yield,percentage composition of HYSA and naringenin as well as functional genes' expression presented similar trends. The average yield reached the highest peak at the third day, showing highpositive correlation with the contents of HYSA (r=0.756,P<0.05), and significant correlation with the expression of chi (r=0.892,P<0.01). The contents of naringenin showed a high positive correlation with the expression of chs(r=0.766,P<0.05). The study provides a theory basis for the composition and regulation mechanism of the flavonoid constituents and lays foundation for molecular mechanisms which lead to the difference of quality in C. tinctorius.

Cloning of ureI gene from Helicobacter pylori and its expression in E. coli / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the urea membrane channel gene (ureI) from Helicobacter pylori (Hp) for its expression in E. coli, and evaluate the expression conditions and immunological features of the fusion protein.</p><p><b>METHODS</b>ureI gene cloned by PCR from Hp was inserted into the plasmid pET32a (+) to construct the recombinant plasmid pET32a/ureI, followed by identification by BglII and HindIII digestion and sequencing. E. coli BL-21+(DE3) was transformed with pET32a/ureI to obtain the engineered bacterium BL21+/UreI, which was cultured at different temperatures and induced with 1.0 mmol/L IPTG for expression of the recombinant protein. The expressed proteins were identified by SDS-PAGE and analyzed by Pro-gel analyzer 4.0. Western blotting was performed to evaluate the immunogenicity of the expressed protein.</p><p><b>RESULTS</b>The cloned gene fragment was about 650 bp in length, and BglII and HindIII digestion of pET32a/ureI yielded a 650-bp band. Sequence analysis revealed that the cloned ureI gene contained 646 bp without reading frame alterations. Comparison against GenBank indicated a homology of 100% of the cloned gene with ureI gene of the corresponding Hp strains, and also one no less than 98.5% with ureI gene from other strains. The engineered E. coli BL21+/UreI could express recombinant UreI (rUreI) with His tag, and the target protein accounted for 20.2% of the total bacterial proteins after 1.0 mmol/L IPTG induction of the bacterium at 37 degrees C for 14 h. SDS-PAGE and Western blotting showed that the recombinant UreI protein was produced mainly in the inclusion bodies and fused with his-tag (rUreI/his), which could react with human anti-Hp and mAb to his tag but not with mAb to Hp UreB.</p><p><b>CONCLUSIONS</b>We have successfully cloned ureI gene and constructed the prokaryotic expression plasmid for efficient rUreI expression, and the fusion protein rUreI/his expressed in the inclusion bodies can react specifically with both Hp antibody and his-tag antibody.</p>