Objective To develop a convenient, rapid and specific method using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) for detection of facioscapulohumeral muscular dystrophy(FSHD). Methods Genomic DNA was extracted and digested by restricted endonuclease EcoR Ⅰ , followed by agarose electrophoresis. The DNA (< 38 kb) was retrieved from agarose electrophoretic gels. The primers and probe were designed in D4ZA gene in chromosome 4. One hundred and fifteen subjects were examined by FQ-PCR using the retrieved DNA (<38 kb) as a template and the result was analyzed by fluorescent curve comparing with positive control. Results The results by FQ-PCR showed that 13 cases were positive in 16 FSHD cases whose EcoR Ⅰ fragment sizes were known, 75 cases were negative in 78 cases of normal controls, 15 cases were positive in 16 FSHD cases diagnosed clinically whose EcoR Ⅰ fragment sizes were unknown, and 3 cases were positive in 5 cases of relatives of FSHD patients. Consistency was checked using Kappa index between the 2 gene diagnostic tests for FSHD (FQ-PCR test and the traditional Southern blotting test), and between the 2 diagnostic criterions (gene diagnosis by FQ-PCR and clinical diagnosis). The results were statistically significant (κ = 0. 765, P = 0. 002 ; κ = 0. 844, P = 0. 000). Conclusions A new genetic diagnostic method of FSHD by FQ-PCR was developed, which was more simplified and reliable compared to the time-consuming, radioactive Southern blotting. It could also detect the D4Z4 arrays in cases having deletion of p13E-11 as well as the interchromosomal exchange between 4q35 and 10q26. The new method of FQ-PCR for FSHD may be extended to utilize clinically in future.

Saussurea medusa is a rare traditional Chinese medicinal herb. Besides anti-inflammatory and analgesic activities, it has effects of disinhibiting cold, dispelling dampness and promoting blood circulation. Flavonoids are the main medicinal compounds in S. medusa. Contents of flavonoids and expression of flavonoids biosynthesis related genes in white and red (induced by low temperature, high sucrose and high light) callus were analyzed. The results showed that the total flavone in red line was 3.60 times higher compared to white line. The accumulation of rutin in red line (0.25% of dry weight) was 2.40 times higher compared to white line. Anthocyanins were abundant in red line, with the contents of cyanidin 3-O-glucosidechloride and cyanidin 3-O-succinyl glycoside 0.12% and 0.19% of dry weight respectively. CHS, F3'H, FNS, FLS, DFR and ANS genes were highly expressed in red line compared to white line. Expression of three transcription factors (MYB, bHLH and WD40) in red line was significantly higher than that in white line, especially the expression of MYB (19.70 times higher compared to white line). These results indicated that high expression levels of transcription factors induced high expression of structural genes in red line, thereby enhancing the flavonoids biosynthesis. The expression of bHLH and WD40 was similar, whereas it was significantly different from that of MYB, indicating that bHLH and WD40 could form a binary complex to regulate expression of structural genes and flavonoids biosynthesis.
Anthocyanins ; chemistry ; Cell Line ; Flavonoids ; chemistry ; Glucosides ; chemistry ; Plant Proteins ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Saussurea ; chemistry ; genetics ; Transcription Factors ; genetics ; metabolism