[Cloning and expression analysis of U6 promoters in Panax quinquefolius].

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Antioxidative 2-(2-phenylethyl)chromones in Chinese eaglewood from Aquilaria sinensis.

Two new compounds 6,7-dimethoxy-2-[2-(2'-hydroxyphenyl)ethyl]chromone (1) and 6,7-dimethoxy-2-[2-(4'-hydroxyphenyl)ethenyl]chromone (2), together with ten known 2-(2-phenylethyl)chromones (3-12) were isolated from the resinous wood of Aquilaria sinensis (Lour.) Gilg. Their structures were elucidated by detailed IR, MS, NMR spectroscopic analyses, and comparison with reported. The absolute configuration of 3 was confirmed by Cu Kα X-ray crystallographic experiment, and the X-ray crystallographic data of 3 were firstly reported. Compounds 2, 8, 10, and 11 exhibited strong ABTS•+ radical scavenging activity, with IC50 values of 12.3, 16.5, 12.1, and 34.7 µM, respectively.[Formula: see text].

Three new sulphur glycosides from the seeds of Descurainia sophia.

Three new sulphur glycosides, raphanuside B-D (1-3), together with a known sulphur glycoside, raphanuside (4) were isolated from the decoction of the seeds of Descurainia sophia (L.) Webb ex Prantl, and the compound 4 was reported for the first time from this plant. Their structures were identified by means of UV, IR, 1D, 2D NMR (HSQC, HMBC and NOESY) and HR-ESI-MS spectroscopic data.

Extractions of oil from Descurainia sophia seed using supercritical CO2, chemical compositions by GC-MS and evaluation of the anti-tussive, expectorant and anti-asthmatic activities.

Descurainia sophia is widely distributed in China and is one of the most troublesome annual weeds. It has diverse medicinal usage. D. sophia has abundant oil, making it an important oil plant in China. The main goal of this study was to obtain the maximum yield of the oil by an optimal selection of supercritical fluid extraction parameters. According to the central composite design and response surface methodology for supercritical fluid extraction method, a quadratic polynomial model was used to predict the yield of D. sophia seed oil. A series of runs was performed to assess the optimal extraction conditions. The results indicated that the extraction pressure had the greatest impact on oil yield within the range of the operating conditions studied. A total of approximately 67 compounds were separated in D. sophia seed oil by GC-MS, of which 51 compounds represented 98.21% of the total oils, for the first time. This study was also aimed at evaluating the anti-asthmatic, anti-tussive and expectorant activities in vivo of D. sophia seed oil which supplied for further research on bioactive constituents and pharmacological mechanisms.

Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHß and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species.

A new kaempferol trioside from Silphium perfoliatum.

A new apiose-containing kaempferol trioside, kaempferol-3-O-α-L-rhamnosyl-(1‴ → 6″)-O-ß-D-galactopyranosyl-7-O-ß-D-apiofuranoside, along with 16 known compounds, were isolated from 50% acetone extract of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic techniques including UV, IR, MS, ¹H, ¹³C, and 2D-NMR. In addition, the pharmacological activity of compound 1 was tested with HepG2 and Balb/c mice (splenic lymphocytes and thymic lymphocytes) in vitro, and it exhibited inhibitory effect on the proliferation of HepG2 cells and showed the immunosuppressive activity.