RÉSUMÉ
Based on the ITS2 and psbA-trnHsequences, molecular biological identification and genetic relationship of Fritillaria cirrhosa with its relative species were carried out. In this paper, the PCR-RFLP method specified by the Chinese Pharmacopoeia was performed on all samples at first. Secondly, the ITS2 and psbA-trnH sequences of all samples were amplified. Then, the amplified products were used to analyze the genetic distance, construct the phylogenetic tree, assess the identification efficiency, and evaluate the genetic relationship as well. The result showed that all the samples were divided into two groups by PCR-RFLP method. The samples in the first group, including Fritillaria ussuriensis, Fritillaria thunbergii and Fritillaria pallidiflora, could not be digested by SmaI, while the other samples in the second group, including Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa, could be digested by SmaI. Then, ITS2 and psbA-trnH sequences of all samples were obtained. The length of various ITS2 sequences were distributed from 235 to 239 bp, and the average intra- and inter-specific genetic distance were 0.001 and 0.022, respectively. NJ tree showed that all samples were separated into "Northern Fritillaria" group (Fritillaria ussuriensis and Fritillaria pallidiflora) and "Southern Fritillaria" group (Fritillaria thunbergii, Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa). The latter group could be further divided into Fritillaria thunbergii and Fritillaria cirrhosa subgroup, and the species in Fritillaria cirrhosa subgroup had close phylogenetic relationships. The length of psbA-trnH sequences was distributed from 337 to 373 bp, and the intra- and inter-specific genetic distance were 0.263 and 0.329, respectively. The samples in this paper could not be clustered effectively by NJ tree. This indicated that the ITS2 sequences were not only able to identify Fritillaria cirrhosa with its partial relative species quickly and accurately, but also clarify the relationship between different Fritillaria species. Therefore, it provided an important theoretical foundation for the development of molecular markers, effective protection, and rational development and utilization of Fritillaria resources.
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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.
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OBJECTIVE:To investigate antitussive,antiasthmatic,phlegm-resolving and anti-inflammatory effects of Fritillaria cirrhosa and Fritillaria unibracteata. METHODS:Mice and guinea pig were randomly divided into blank control group (distilled water),ethanol extract groups,water extraction groups and powder groups of F. cirrhosa and F. unibracteata(1.13 g/kg for mice, 0.42 g/kg for guinea pig,calculated by crude drug),positive control group,with 10 mice(guinea pig)in each group. Each group was given relevant medicine intragastrically once a day for consecutive 5 d. Relative indicators were detected 30 min after last medication. The ammonia water induced cough method was used to investigate antitussive effect (dextromethorphan hydrobromide as positive drug,0.013 g/kg)by determining coughing latent period and coughing times within 3 min. After inducing asthma with 2％ histamine phosphate for 15 s, antiasthmatic effect (aminophylline as positive drug, 0.033 g/kg) was investigated by determining coughing latent period and the number of guinea pig with convulsive fall. The phenol red injection method was used to investigate phlegm-resolving effect (ambroxol as positive drug,21.304 g/kg) by determining the content of phenol red in the trachea of mice in phenol red expectoration test. The anti-inflammatory effect(dexamethasone acetate as positive drug,9.225×10-4 g/kg)was investigated by determining ear swelling degree and inhibition rate of ear swelling mice in xylene-induced inflammation test. RESULTS:Compared with blank control group, coughing latent period and cough induction latent period prolonged significantly in ethanol extract of F. cirrhosa and F. unibracteata groups,F. cirrhosa and F. unibracteata powder groups,positive control group, and coughing time decreased significantly within 3 min. The number of mice with convulsive fall decreased significantly in F. cirrhosa powder group, F. unibracteata ethanol group,F. unibracteata powder group and positive control group. The content of phenol red in the trachea of mice increased significantly,while ear swelling and inhibitory rate of ear swelling decreased significantly,with statistical significance (P<0.05 or P<0.01). There was no statistical significance in each index of mice between powder group and positive control group (P>0.05). Compared with corresponding groups of F. cirrhosa,coughing latent period of mice prolonged significantly in F. unibracteata ethanol extract group (P<0.05), other indexes had no statistical significance(P>0.05). CONCLUSIONS:The antitussive,antiasthmatic,phlegm-resolving and anti-inflammatory effects of F. cirrhosa are similar to or worse than those of F. unibracteata.
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ABSTRACT The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in Mevalonate (MVA) pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR from Fritillaria cirrhosa (FcHMGR), a bulbous medicinal plant. The full-length cDNA of FcHMGR was 2072 base pair (bp), containing a 1680-bp open reading frame. Bioinformatical analyses revealed that FcHMGR had HMG CoA-binding domains and two NADPH binding domains, which are required for HMGR activity. Quantitative real-time PCR (qRT-PCR) analysis revealed that FcHMGR expressed high in mature bulbs. A truncated version of FcHMGR protein lacking the N-terminal 249-bp GC rich area was expressed in Escherichia coli. The crude cell lysate containing the recombinant protein showed a better HMGR activity than the control and the relative enzyme activity was calculated to be 1.62 U/mg. The cloning, characterization and functional analysis of FcHMGR gene allowed us to further understand the role of FcHMGR involved in steroidal alkaloid biosynthetic pathway in F. cirrhosa at the molecular level.
Sujet(s)
Clonage moléculaire , Fritillaria , Méglutol , Biologie informatiqueRÉSUMÉ
Understanding of codon usage bias of Fritillaria cirrhosa can provide theoretical basis for heterologous biosynthesis of F. cirrhosa alkaloids by genetic engineering technology. A total of 9 843 full length coding sequences (CDS) from the F. cirrhosa transcriptome data were used for the analysis of codon usage bias. The GC and GC3s contents, effective number of codons(ENC) and relative synonymous codon usage (RSCU) were calculated using the CodonW software. The results show that the codon usage bias value is low in the CDS of F. cirrhosa. A total of 15 codons, including UUG, CUU, AUU, GUU, UCA, CCU, CCA, ACU, ACA, GCA, UAU, CAU, AAU, AGA and GGA, were identified as optimal codons in F. cirrhosa. The optimal codons generally end with A/T at the third codon position. By the transcriptome annotation, we found 26 CDSs possibly involved in the biosynthesis of alkaloids in the F. cirrhosa. The proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae are low in these CDSs. We also proposed a method for the codonoptimization in these target genes. Our work lays the foundation for further study on the biosynthesis of alkaloids of the F. cirrhosa in heterologous species.
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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.
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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.
RÉSUMÉ
This study was aimed to find the optimal conditions for seed propagation of Fritillaria cirrhosa in the plateau region in order to summarize the best sowing time and method of artificial propagation. Seeds were stored in appropriate environment. After the seeds matured, the seedbed of F. cirrhosa was treated with cattle dung humus and small shed. The results showed that early March is the best sowing time and cattle dung humus with a thickness about 1 cm is the best coving for propagation of F. cirrhosa; the growing period of film propagation (FP) is about 160 days each year compared with that of control about 50-60 days, respectively. It was concluded that the propagating seeds in plastic greenhouses by using cattle dung humus as planting substrates, using sunshade nets for shading and keeping humidity by spraying can effectively prolong the growing period, improve the retention rate of annual bulbs and the production of F. cirrhosa.
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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.