Cloning of FPS gene and its role in regulation of alkaloid synthesis in Fritillaria thunbergii / 中草药

Objective: Farnesyl pyrophosphate synthase (FPS) gene of Fritillaria thunbergii was cloned and the correlation between the expression content of gene and alkaloid accumulation was analyzed to explore the role of FPS gene in the regulation of alkaloid synthesis and metabolism. Methods: In this study, the new bulbs and different tissues of Fritillaria thunbergii varieties "Narrow leaf" "Broad leaf" and "New Meiyuan" were used to clone the full-length sequence of FPS gene via RACE technology. RT-qPCR and HPLC-ELSD technology was used to determine the expression of FPS in 10 different developmental stages and the content of alkaloid (Peimine and Verticinone), respectively. Finally, the correlation was analyzed. Results: The full-length cDNA sequence of FPS gene was 1 629 bp. Open Reading Frame (ORF) was 1 056 bp and encoded 351 amino acids. Among them, the tissue specific expression trends of FPS gene were same, flowers had the highest expression level, followed by the new bulb. The results of correlation analysis showed thatthe content Peimine and Verticinone had significant positive correlation with the expression level of FPS gene in "Broad leaf" and "New Meiyuan" (The correlation coefficients were 0.289 and 0.613, 0.427 and 0.622, respectively);FPS gene expression and alkaloid content in different tissues had a positive correlation (The correlation coefficients were 0.057 and 0.476, 0.085 and 0.495, 0.375 and 0.432, respectively). Conclusion: The full-length sequence of FPS gene was successfully cloned in this study. The FPS gene involved in the regulation of alkaloid synthesis and metabolism.

Cloning and bioinformatics analysis of Actin gene in Fritillaria thunbergii / 中成药

AIM To clone the Actin gene in Fritillaria thunbergii Miq.and to make bioinformatics analysis.METHODS The total mRNA in roots,stems,leaves,flowers and bulbs of F.thunbergii was extracted,and the degenerate primer was designed and synthesized.With total mRNA in leaves as a template,the conserved fragments of Actin gene was cloned by RT-PCR and Ta cloning technology.Using this gene as a reference gene,tissue specificity expression analysis was adopted in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene.RESULTS One gene sequence (463 bp) was obtained by RT-PCR amplification and Ta cloning.The Actin gene in F.thunbergii showed high similarities to those in Lilium regale Wilson,Tulipa gesneriana,Ornithogalum caudatum Jacq.,Dendrobium officinale Kimura et Migo,Diospyros kaki Thunb.,Betula luminifera H.Winkl.and Zea mays L.(84％-98％),the homologies of its amino acid sequence to Drosera adelae F.Muell,Brassica napus L.,Vanilla peanigoeia Ancer,L.regale,Jatropha carcas L.,Lycium barbarum L.and Rhizophora stylosa amino acid sequences were all more than 89％,and the Actin protein had close genetic relationships with Lotus corniculatus L.,L.regale and T.gesneriana.The expressions of HMGR gene in various parts of this plant showed obvious differences,which was in sequence of bulbs ＞ flowers ＞ leaves ＞ stems ＞ roots.CONCLUSION It is the first time that Actin gene (named as FtActin) is coloned in F.thunbergii,which can lay the basis for its effective application.