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

ABSTRACT

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.