Cloning and expression analysis of cycloartenol synthase gene in Fritillaria cirrhosa / 中草药

Objective To obtain the key enzyme gene involved in alkaloids biosynthesis pathway of Fritillaria cirrhosa, cycloartenol synthase (CAS) gene was cloned, and its bioinformatics analysis and gene expression pattern were also performed. Methods The open reading frame (ORF) of F. cirrhosa CAS (FcCAS) was amplified by PCR based on transcriptome sequencing. The bioinformatics analysis of FcCAS cDNA sequences was carried out by some online tools. Meanwhile, using real-time quantitative PCR (qRT-PCR) to analyze the gene expression patterns between wild and regeneration bulbs. Moreover, the content of total alkaloids in wild and regenerated bulbs were also investigated. Results The results showed that CAS had a length of 2 271 bp ORF, which encoding 756 amino acids. The phylogenetic tree analysis showed that FcCAS were highly similar to the corresponding proteins in Asparagus officinalis, Musa acuminate, and Elaeis gunineensis from the NCBI website, and the similarities were more than 80%. The results of qRT-PCR and total alkaloids assay showed that the changing trend of the expression level of FcCAS was consistent with that of the content of total alkaloids, and higher alkaloid accumulation was in regeneration bulbs than wild bulbs. Conclusion The expression of FcCAS gene varied widely in different tissues. These findings suggested that FcCAS was a biological functional protein induced by hormone combination, which laid a theoretical foundation for the improvement of the alkaloid content by using the genetic engineering.

Cloning and identification of a specific standard DNA fingerprint fragment from Chinese herbal medicine Fritillaria cirrhosa D. Don / 中国药学杂志

OBJECTIVE: To establish a standard method for preserving the Fritillaria cirrhosa D. Don DNA fingerprint fragments. METHODS: Independently developed F. cirrhosa test kit was used to extract genomic DNA. Specific F. cirrhosa DNA fragments were cloned in vitro, and ligated into a carrier vector then transformed into a self replicating host cells to produce a large amount of specific F. Cirrhosa DNA. Plasmid DNA was extracted using Plasmid Extraction Kit and confirmed by PCR or restriction digestion of the insert. Bacterial colonies carrying authentic Sichuan F. cirrhosa specific DNA sequences were stored at -80℃. Stability was monitored at intervals of 1, 2, 3 and 6 months after DNA recovery using the boiling method of genomic DNA extraction. RESULTS: The amplified F. cirrhosa DNA clones showed clear bands in the electrophoresis, and had stable results in the repeated verification test. The amplified clones from resuscitated bacterial colonies which had been stored for 1, 2, 3, 6 months at -80℃ still displayed bright and clear bands after electrophoresis. CONCLUSION: It is feasible and effective to preserve DNA fingerprint of F. Cirrhosa by the established method, and this simple and reliable method can be used as the basis of establishing the new genes database of traditional Chinese medicine.

Development and evaluation of Fritillaria cirrhosa D. Don DNA detection kit / 中国药学杂志

OBJECTIVE: To develop a kit for detection of DNA of Fritillaria cirrhosa D. Don and optimize its components as well as process protocols, in order to set up a simple, rapid molecular biology method for identification of Fritillaria cirrhosa D. Don. METHODS: All genomic DNA of Fritillaria cirrhosa D. Don was extracted by kit assay and pharmacopoeia method recorded in the expanded supplement of China Pharmacopoeia 2010, respectively; ultraviolet spectrophotometer was used to measure the quantity of extracted DNA; PCR amplification and restriction fragment length polymorphism analysis (RFLP) were carried out to identify the authentication of Fritillaria cirrhosa D. Don. RESULTS: The maximum value of genomic DNA extracted by pharmacopoeia method was (1.57±0.05) (OD260/OD280) and (1.73±0.10) by kit assay. The PCR amplification showed a single band over 300 bp, while the RFLP showed two distinct bands between 100 and 250 bp in agarose electrophoresis. CONCLUSION: The data demonstrated that the kit assay was better than the pharmacopoeia method, especially in the extraction quantity and DNA purity of Fritillaria cirrhosa D. Don nicleic acid; the PCR and RFLP results showed that the kit assay was consistent with pharmacopoeia method. The detection kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fritillaria cirrhosa D. Don.

Direct identification of Fritillaria cirrhosa, F. ia thunbergii and F. thunbergii var. chekiangensis by FTIR / 中草药

Object To identify Fritillaria cirrhosa D. Don., Fritillaria thunbergii Miq. and Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia. with FTIR.Methods Their IR spectra were obtained by direct FTIR.Results The infrared spectra of F. cirrhosa, F. thunbergii, F. thunbergii var. chekiangensis were different.Conclusion F. cirrhosa, F. thunbergii, and F. thunbergii var. chekiangensis were identified by FTIR directly, fastly and accurately.