| Objective The Fritillaria Cirrhosa D.Don is an expensive plant with high medicinal value,and its dried bulb is its main medicinal part.The steroidal alkaloids in the alkaloids,together with the saponins and terpenoids in the non-alkaloids,make F.cirrhosa effective in clearing heat and moistening the lungs,resolving phlegm and relieving cough.Recent studies have found that F.cirrhosa was effective against lung,stomach,ovarian,esophageal,liver and breast cancers.However,the current scarcity of wild F.cirrhosa resources and low alkaloid content make it difficult to meet the market demand.Studies have shown that transcription factors have a regulatory role on plant secondary metabolites.Therefore,this thesis aims at improving the alkaloid content of F.cirrhosa and obtaining excellent F.cirrhosa gene resources through molecular biology,in order to improve the current situation of the scarcity of fritillaria cirrhosa resources.Methods First,based on the wild F.cirrhosa and regenerated F.cirrhosa transcriptome data obtained by Illumina double-end sequencing technology in our laboratory,this paper used bioinformatics tools and database analysis to screen and predict the association of F.cirrhosa MYB-type transcription factors with genes related to regulating the alkaloid synthesis pathway of F.cirrhosa.In addition,the transcription factors that potentially regulate genes related to the alkaloid synthesis pathway were verified and identified by q-PCR method on predicted related genes and transcription factors that can regulate these related genes.Second,to further validate the transcription factor,the gene was cloned and characterized using molecular biology and bioinformatics.Finally,the gene was transferred into the model plant tobacco using Agrobacterium-mediated transfer,and the transgenic plants were identified using PCR techniques.Results By bioinformatic means,this study analyzed a MYB-type transcription factor Fc R2R3-MYB(Unigene0052072)that could potentially enhance the alkaloid content of fritillaria cirrhosa.The results of q-PCR experiments showed that the expression of this transcription factor was higher in regenerated bulbs than in wild bulbs,which was consistent with the trend of higher expression of genes related to alkaloid synthesis in regenerated bulbs than in wild bulbs,and thus was considered to have a potential function in regulating alkaloid synthesis.In addition,the full length of the gene was successfully obtained and cloned using molecular biology,totaling 849 bp,which was analyzed by bioinformatics software and found to encode a total of 283 amino acids with a protein size of 31.50 KDa.After homology matching analysis by NCBI,this paper found that this transcription factor protein showed high homology with lily R2R3-MYB type protein,reaching 92.98%.Through the analysis of the predicted secondary and tertiary structures of Fc R2R3-MYB protein,the results of this paper showed that Fc R2R3-MYB protein mainly consisted of irregular coiled and α-helix,and the homology model was PDB: 6kks.1.A,which had 62.83% identity with this amino acid sequence.The Fc R2R3-MYB gene was transferred into tobacco by Agrobacterium-mediated method,and the success of the transgene was verified by using kanamycin(Kan)resistance screening method and PCR and RT-PCR,and the results showed that positive tobacco plants were successfully obtainedConclusion Based on wild F.cirrhosa and regenerated F.cirrhosa transcriptomic data,this study screened and validated a potential transcription factor regulating the alkaloid content of F.cirrhosa.In this paper,the transcription factor was successfully cloned using molecular biology and bioinformatics methods,and positive transgenic plants were successfully obtained.Further studies on the transcriptional regulation and metabolite detection of this gene in transgenic tobacco will be carried out later,laying the foundation for molecular studies on improving the alkaloid content of F.cirrhosa. |
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