Determination Of Alkaloids In Fritillaria Cirrhosa D.Don And Cloning And Expression Of Key Enzyme Genes For Its Synthesis

Objective Fritillaria cirrhosa D.Don,as a medicinal homologous plant,can be used not only as food,but also for medicinal purposes.In order to ensure the edible and medicinal safety of Fritillaria cirrhosa D.Don,the quality control of Fritillaria cirrhosa D.Don was studied to lay a foundation for strict control of the quality of Fritillaria cirrhosa D.Don medicinal materials,so as to ensure the edible safety.Methods HPLC-ELSD was used to determine the content of Imperialine,Peimine and Peiminine in 1-2 years and 3-4 years of wild Fritillaria cirrhosa.which provided the basis for material selection for Fritillaria cirrhosa alkaloid metabolic engineering.The open reading frame(ORF)sequence of CAS gene(FcCAS)and the full-length fragment of FcHMGR gene(FcHMGR)were cloned from the bulbs of Fritillaria cirrhosa collected from the wild tending base of Zheduoshan,Kangding,China.The FcHMGR and FcCAS sequences were analyzed by Bioinformatics Based on online tools.The relative content of FcCAS and FcHMGR in wild bulb cDNA samples of 1-2 years and 3-4 years was detected by fluorescence quantitative analysis(qRT-PCR).In addition,the activity of HMGR was determined by prokaryotic expression system.Results1 Analysis of the alkaloid content data of wild Fritillaria cirrhosa in different years(1-2 years and 3-4 years)found that the accumulation of alkaloids in wild 3-4 years was higher,which might be related to the level of gene expression synthesized by Fritillaria cirrhosa.2 In response to the increase in alkaloid accumulation over time,two genes associated with alkaloid synthesis were cloned in this study: HMGR and CAS.(1)The full length of FcHMGR gene is 2072 bp.It contains a 1680 bp complete open reading frame and encodes 559 amino acids.The results of homology and amino acid conserved domains showed that FcHMGR had the typical polypeptide sites necessary for HMGR activity.Real-time fluorescence quantitative PCR showed that FcHMGR was more expressed in wild Fritillaria cirrhosa bulbs in 3-4 years.SDS-PAGE analysis showed that FcHMGR gene could be expressed in the expressing competent cell BL21,and it is a fusion protein.FcHMGR enzyme activity assay indicated that FcHMGR is a functional protein;(2)The FcCAS coding region ORF is 2 271 bp and encodes 756 amino acids.The similarity between FcCAS and CAS proteins of asparagus,banana,oil palm and other plants published on NCBI is more than 80%.QRT-PCR experiments show that the expression level of FcCAS in bulbs of wild Fritillaria cirrhosa in 3-4 years is higher than that in bulbs of 1-2 years old.Conclusion The higher the content of Fritillaria cirrhosa alkaloids with the increase of years may be positively correlated with the high expression of genes related to its synthesis.The cloning and function of these genes are further analyzed,which lays a foundation for the study of the content and expression regulation of Fritillaria alkaloids alkaloids.