Studies On The Key Enzyme Genes For The Synthesis Of Sanguinarine And Chelerythrine In Chelidonium Majus L.

Chelidonium majus L.is the herb medicine,for the poppy celandine(Papaveraceae)genera Chelidonium L.perennial herbaceous plants.It is not strict with the growing environment and hardy to cold.It has a weak smell,bitter taste,cool,a small poison.The alkaloids in the secondary metabolites of chelandine,such as berberine,coptidine,chelandine,apigenine,sanguine and protropine,are bioactive compounds with extensive pharmacological functions and have great value in scientific research and clinical application.Based on the transcriptome sequencing of pre-flowering and post-flowering tissue materials,this study combined the existing mechanisms of plant secondary metabolites biosynthesis with genetic engineering and molecular biology methods.We preliminarily analyzed seven genes involved in the synthesis pathway of isoquinoline alkaloids in celandine.The correlation between their gene expression level and the dynamic change of alkaloid content was explored.Two key enzyme genes related to the biosynthesis of isoquinoline alkaloids were found.The two genes were cloned and the plant overexpression vectors were constructed.This study laid a foundation for elucidating the roles of these two genes in the synthesis of chelandine and sanguine.The main research aspects are as follows.1.Key genes to the biosynthesis of chelerythrine and sanguinarine were screened.Based on Illumina Hi Seq TM 2500 sequencing platform,transcriptome sequencing was performed on6 samples from the preanthesis and reproductive stage of celandine.261 genes related to alkaloid synthesis were identified,among which 141 genes were involved in the isoquinoline alkaloid synthesis pathway.Combined with the study of KEGG metabolic pathway,115 unigene annotation information were mapped in the isoquinoline alkaloid biosynthesis pathway of chelandine(KO00950).Encoding 9 key enzymes in the biosynthesis pathway of isoquinoline alkaloids,including 5 transaminases,2 oxidases,1 decarboxylase and 1 methyltransferase.Seven genes are important in the synthesis pathway,including canadine ynthase,cheilanthifolinesynthase,methyltetrahydroprotoberberine 14-monooxygenase,Tetrahydroprotoberberine-cis-N-methyltransferase,scoulerine-9-O-methyltransferase,3′-hydroxy-N-methyl-(S)-coclaurine 4′-O-methyltransferase,stylopine synthase.They will be further studied in subsequent studies.2.Studies on the correlation between the genes related to the synthesis pathway of isoquinoline alkaloids and the dynamic accumulation of active components in celandine.The relative expression levels of 7 related enzyme genes in five growth stages of celandine were obtained by real-time fluorescence quantitative PCR.The contents of chelidine,chelerythrine,coptisine,proopioid,berberine and sanguinarine were determined by HPLC.And the correlation between them was studied.The results showed that the gene expression in the materials of different periods differed greatly.CYP719A13/CAS is a very important enzyme gene,which is significantly related to the content of various alkaloids.CYP719A14/CFS is a relatively complex gene in the pathway.It may be involved in the negative regulation of sanguinarine and the positive regulation of chelandine,and may be in competition with several other negatively correlated alkaloids.3.Cloning and construction of plant transformation vector of C_mCAS Gene.The specific primers were designed and the Cm CAS gene was cloned from the leaves of in Chelidonium majus L.by PCR.The length of open reading frame(ORF)of Cm CAS gene was 1494bp,which could encode 497 amino acids in total.The molecular weight was predicted to be 56451.78k D,and the allelic point showed PI7.00.The cloned vector p MD19 was selected.The target sequence was confirmed to be correct by double enzyme digestion and sequencing after transformation of Escherichia coli DH5α.The cloned vector p MD19T-CMCAS was successfully constructed.Plant overexpression vector of PRI-201-AN-CMCAS was constructed and transformed into agrobacterium tumefaciens LBA4404 by electric shock method.4.Cloning and construction of plant transformation vector of Cm CFS Gene.The specific primers were designed and the Cm CFS gene was cloned from the leaves of in Chelidonium majus L.by PCR.The length of open reading frame(ORF)of Cm CFS gene was 1473bp,which could encode 490 amino acids in total.The molecular weight was predicted to be 55035.08k D,and the allelic point showed PI8.96.The cloned vector p MD19 was selected.The target sequence was confirmed to be correct by double enzyme digestion and sequencing after transformation of Escherichia coli DH5α.The cloned vector p MD19T-CMCFS was successfully constructed.Plant overexpression vector of PRI-201-AN-CMCFS was constructed and transformed into agrobacterium tumefaciens LBA4404 by electric shock method.