The characteristics and functions of CtXTH1: the gene booststhe corolla elongation in safflower / 药学学报

In this study, 13 xyloglucan endotransglycosylases/hydrolases (XTHs) and 8 expansin (EXPs) were screened from safflower floret transcriptome database. Through correlation analysis between the safflower gene expression profile chip and the corolla development, only 4 XTHs (CtXTH1-4) and 1 EXP (CtEXP1) have positive relevance with corolla elongation (r≥0.60) and were therefore validated by qRT-PCR. The full length of these genes were cloned by RACE. According to the bioinformatic analysis, CtXTH1 correlated with the development of the floret, and the expression pattern analysis indicated that CtXTH1 had accumulated in the floret. The recombinant vector (pMT39-CtXTH1) was constructed for gene transformation. Overexpression of CtXTH1 significantly increased the corolla length (about 5.34% to 10.25%) and corolla weight (about 30.00% to 36.02%) in transgenic safflower. The overexpression lines also showed an increasing tendency in the weight of seeds, average number of corollas per cone and average number of seeds in each cone. Meanwhile, overexpression of CtXTH1 had no significant effect on flavonoids. According to the corolla microstructure, the OVX-line tubular part of floret exhibited a looser and irregular character. These data suggested that CtXTH1 can potentially increase relaxation of the tissues and boost corolla elongation. Our study provides a valuable clue for plant breeding in the future.

CtCHS4 induces the accumulation of safflower quinone chalcones in response to methyl jasmonate induction / 药学学报

Flavonoids, especially chalcones such as hydroxysafflor yellow A and carthamin are the main active ingredients of safflower. To study the biosynthesis pathway of safflower flavonoids is of great significance for the quality control of safflower. Chalcone synthase (CHS) is an enzyme that plays an important role in regulation of the synthesis of flavonoids. However, for the time being, the role of CHS is not yet clear in the biosynthesis of safflower flavonoids. As a plant signaling regulator, JA/MeJA can activate CHS gene expression in plants. CtCHS1, one of the CHS genes in safflower, was elucidated in our previous work. In our continuous search for CtCHSs functions from this plant, other CHS genes CtCHS2 and CtCHS4 in safflower were examined. The floret was stimulated with methyl jasmonate (MeJA) and the transcriptome expression of CtCHS2 and CtCHS4 was analyzed by qRT-PCR at different time points of 0, 3, 6, and 12 h after stimulation. Further metabolites under stimulation by MeJA were analyzed by UHPLC/Q-TOF-MS. The results showed that the expression of CtCHS4 in response to MeJA significantly increased at 3 and 6 h, while the expression of CtCHS2 showed a trend of decrease after induction. Meanwhile, the accumulation of rutin, hydroxysafflor yellow A, D-phenylalanine, kaempferol-3-O-β-rutinoside and carthamin increased obviously. Especially, accumulation of hydroxysafflor yellow A was increased significantly at 3, 6 and 12 h after induction (P ≥ 0.05 or 0.01), but the change in kaempferol, kaempferol-3-O-β-D-glucoside, luteolin, quercetin-3-β-D-glucoside was not significant. The accumulation of hydroxysafflor yellow A and carthamin was positively correlated with the expression abundance of CtCHS4 with Pearson correlation analysis method (r ≥ 0.8). The data suggest that CtCHS4 may be a key gene for forming hydroxysafflor yellow A and carthamin and plays an important role in the accumulation of safflower chalcones. The CtCHS4-pMAL-C5X recombinant vector was successfully expressed in BL21 (DE3) Plys to express the product naringenin in vitro under the catalytic substrates p-coumaryol-COA and malonyl-CoA. The results of this study provide a new insight into synthetic genes involved in flavonoids biosynthetic pathway to elucidate the biosynthesis pathway of safflower chalcones.

Regulation of anthocyanin biosynthesis by circadian clock / 药学学报

As a secondary metabolite in plant, anthocyanins plays an important role in many aspects of plant life, and also exhibits various activities including the anti-oxidation, anti-inflammatory, antibacterial, antitumor and cardio-cerebral vascular protective in animals. They are a group of important natural drug candiadtes in the prevention of cardiovascular and cerebrovascular diseases and metabolic diseases. Therefore, exploration of the biosynthetic pathway and regulatory mechanism of anthocyanins is of great interest for improvement of anthocyanin production and development of low-cost production methods. Circadian clock, as a ubiquitous regulatory system in organisms, affects plant physiological and molecular processes, and also regulate the anthocyanin biosynthesis. To provide new ideas on anthocyanin biosynthesis, we provide a review of the recent progress in circadian rhythm clock with regard on regulation of anthocyanin biosynthesis in this paper.

Surgical strategies for treatment of T1b gallbladder cancers diagnosed intraoperatively or postoperatively / 北京大学学报(医学版)

Objective:To explore the surgical strategies for the treatment of T1 b gallbladder cancer patients diagnosed intraoperatively or postoperatively.Methods:A retrospective analysis of 42 patients with T1 b gallbladder cancers was performed.There were 14 patients diagnosed intraoperatively and 28 patients diagnosed postoperatively.The reevaluations of T stages were conducted in the 28 T1 b gallbladder cancer patients diagnosed postoperatively by the professional pathologist.After T stage reevaluation,25 confirmed T1 b patients with complete follow-up data were divided into simple cholecystectomy group and radical resection group,and the clinicopathologic characteristics between the two groups were analyzed.Results:Only 2 of the 14 T1 b gallbladder cancer patients diagnosed by the intraoperative frozen specimen proved to be T1b on postoperative paraffin pathology,and for the remaining 13 patients,T2 was in 11 patients,and T3 in one patient.The rate of misdiagnosis was 85.7％ by the intraoperative frozen specimens,postoperative T stages were equal or higher than intraoperative T stages.Two of the 28 postoperatively diagnosed T1b patients were proved to be T2 after reevaluation,the rate of misdiagnosis was 7.1％,the reevaluated T stages were equal to or higher than the previous stages.Twenty-five confirmed T1 b gallbladder cancer patients had complete follow-up data,11 of whom underwent simple cholecystectomy and the remaining 14 radical resections.No patient had vessel or perineural invasion on pathology in the 25 confirmed T1b patients.Metastasis was absent in all the 30 lymph nodes examined,which achieved from 14 patients with radical resection.The survival rate after simple cholecystectomy was comparable to that after radical resection (P =0.361).Only one patient with radical resection had abdominal cavity implantation relapse,who received gallbladder compression during operation and 2 years later died from metastasis.Conclusion:Intraoperatively diagnosed T1 b gallbladder cancer should receive radical resection.Reevaluation of the T stage is necessary and the initial step for postoperative diagnosed T1b gallbladder cancer patients.The pros and cons of radical surgery for definitive T1b patients should be carefully evaluated,and systemic chemotherapy is recommended for those with bile spillover.

A new anti-hypoxia sesquiterpene from the rhizome of Petasites japonicus / 药学学报

A new sesquiterpene, bakkenolide-Ⅵa (1), was isolated from the rhizome of Petasites japonicas (Sieb. et Zucc.) Maxim. The structure was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, HMBC and NOESY) and mass spectrometry data. Bakkenolide-Ⅵa showed potent cerebral hypoxia-ischemia protective activity in mice subjected to decapitation through prolonging the survival time and gasping time. It also exhibited a protective activity against hypoxia injury in PC12 cells in anaerobic culture by inhibition of lactate dehydrogenase (LDH) release.

Expression of ATP synthase CF1 alpha subunit gene (CTL-spn) as screened by the cDNA-SRAP approach is correlated with spininess in Carthamus tinctorius L / 药学学报

The safflower floret is a traditional Chinese medicine used to promote blood circulation and remove obstruction in the channels. The spines on its bracts are considered a handicap when manual harvest is involved. In this study, cDNA-SRAP was used to systematically investigate which genes are associated with the spines. Sixty pairs of possible primer combinations were used on two cDNA pools representing spininess and spinelessness. Six transcript-derived fragments were identified, of which two with low recombination were sequenced successfully and named as GPY-1 and GPY-2. By using the RACE method, the full-length cDNA of GPY-2 is cloned and named as CTL-spn. The full-length cDNA of CTL-spn was 1 679 bp long with a 1 524 bp ORF encoding a 508 aminoacid protein. The deduced amino acid sequence of the CTL-spn gene shared a high homology (97%) with other known ATP synthase CF1 alpha subunits. Semiquantitative RT-PCR analysis revealed that the mRNA of GPY-1 and GPY-2 accumulated in only spiny lines. Considering the important role of ATP synthase CF1 alpha subunit in plants, it may directly take part in the formation process of spininess and enhancing resistance reaction of spiny safflower. Also, our results provide the important insights for breeding spineless cultivars of safflower.

Determination of ellagic acid, flavonoids and goshonoside-F5 in Rubi Fructus by HPLC / 中国中药杂志

High-performance liquid chromatographic coupled with variable wavelength detection (HPLC-VWD) has been developed for simultaneous determination of 5 analytes including ellagic acid, quercetin, kaempferol-3-O-beta-D-rutinoside, tiliroside and kaempferol, and high-performance liquid chromatographic with an evaporative light scattering detector (HPLC-ELSD) has been established to determine goshonoside-F5 in extract of Rubi Fructus. Chromatographic separations were carried out on an Agilent ZORBAX SB-C18 column (4.6 mm x 250 mm, 5.0 microm). All calibration curves of reference standards revealed good linearity (R2 > 0.999 5) within the concentration ranges tested. The method limits of detection ranged 0.297-90.144 ng and the method limits ofquantitation ranged 0.990-300.480 ng, respectively. Recoveries of 6 analytes were from 97.11% to 101.7%, with RSD less than 2.1%. The result shows that amounts of the 6 analytes in the samples from 16 localities were found to be different. The higher latitude of growing environment, the more ellagic acid in herb. The content of total flavonoids in sample from east localities were higher than that in middle and west localities, and the content of goshonoside-F5 in Bozhou, Anhui province was higher than others. This method was found to be simple, accurate, sensitive with good repeatability. Those results might serve as a sound foundation for further study, quality control and application of Rubi Fructus.

HPLC fingerprints in seed of Celosia argentea / 中国中药杂志

For preferable authentication and regulation of material quality of Celosia argentea, HPLC fingerprints of different habitats were studied. Analysis was carried out on a Hypersil ODS2 column (4.6 mm x 250 mm, 5 microm) with acetonitrile-0.1% glacial acetic acid as the mobile phase, and eluates were detected by an evaporative light scattering detector (ELSD). The similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine ( Version 2004 A) was applied to analyses the similarity of the fingerprint of diverse habitats. The similarity results were verified by SPSS. The chromatographic profiles of the samples from different regions were very similar. HPLC fingerprints of Semen C. argentea 12 common peaks and each peak in the fingerprint was well separated under the chromatographic condition above. The different habitats of C. argentea can be grouped to two types: the middle region and the south region. The chemical constituents of C. argentea vary with different habitats so selection of material habitat is very important for quality control of C. argentea. The fingerprint with high individuality and specificity could be applied in the identification and quality control of the material of C. argentea.

SRAP fragments linked to spines of Carthamus tinctorius / 药学学报

Sequence related amplified polymorphism (SRAP) technique was used to identify SRAP fragment linked to Carthamus tinctorius L. spines of outer involucral bract (OIB) , experimental evidence for molecular marker assistant breeding of Carthamus tinctorius L. has been provided. Based on the strategy of bulk segregate analysis (BSA), two gene pools were separately constructed according to the extreme trait of OIB with many long spines and no spines from Carthamus tinctorius L. Forty-five pairs of SRAP primers were selected and screened from two parents and two gene pools, and one SRAP marker M3E3 was found to be linked to the spines in segregating F2 population confirmation. M3E3 SRAP band was excised, cloned and sequenced. In 20 spininess individuals, this marker was present in 16 spininess individuals and absent in 4 individuals. This band was absent in the 15 spineless F, segregating individuals, which accounted for 11.4% recombination. The M3E3 extract length was 349bp, of which the base components of A + T accounted for 41. 08%. One SRAP marker M3E3 linked to the spines in Carthamus tinctorius L. will be of good use for breeding spineless cultivars at the molecular level in the future.

Determination of safflor yellow A by RP-HPLC and resources quality comparison in Carthamus tinctorius / 中国中药杂志

<p><b>OBJECTIVE</b>To provide the basis for establishing evaluation criterion, selecting good strains and carring out good agricultural practice of the crude drug.</p><p><b>METHOD</b>Representative 22 varieties of Carthamus tinctorius were selected and cultivated in different ecological localities and different years. And the content of safflor yellow A in their corollas were measured by RP-HPLC to compare the differences and their genetic stabilities among varieties.</p><p><b>RESULT</b>The range of of safflor yellow A content was 0.70%-1.85% which were varied among varieties (P < 0.01). The content of safflor yellow A in varieties Yutai Honghua, Hefei Honghua, Rucheng Honghua were higher than in others.</p><p><b>CONCLUSION</b>The effective compound safflor yellow A in C. tinctorius was one of the main quality evaluation criterions. Varieties Yutai Honghua, Hefei Honghua and Rucheng Honghua were good resources.</p>

Analysis of intraspecific variation of Chinese Carthamus tinctorius L. using AFLP markers / 药学学报

<p><b>AIM</b>To investigate the intraspecific variation of Carthamus tinctorius L. (safflower) and establish foundation for further breeding of safflower germplasm resource and screening the quality correlation genes.</p><p><b>METHODS</b>Amplified fragment length polymorphism (AFLP) was carried out to analyze genetic variation of 28 safflower populations collected in China. Unweighed pair-group method of with arithmetical averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among the populations.</p><p><b>RESULTS</b>All populations could be uniquely distinguished using 12 selected primer combinations. Similarity coefficients ranged from 0.48 to 0.96 among the populations. Dendrogram revealed distinct segregation of all the cultivars into three main groups and one midst group.</p><p><b>CONCLUSION</b>Limited genetic diversity exists within the tested 28 collections at intra specific level and AFLP-based phyiogeny was not absolutely consistent with that based on morphological characters may be due to the interaction effect between genotype and environment.</p>