The Effect Of Overexpression Of Protopine 6-hydroxylase(P6H) On Alkaloid Macleaya Cordata

Macleaya cordata is an important medicinal plants.Some studies have found that some benzylisoquinoline alkaloids(BIAs)have significant biological activity.Such as Sanguinarine(SAN),chelerythrine(CHE),berberine(BBR),protopine(PRO)and other cryptoline(ALL).At present,molecular biology,genetic engineering and other means can be used to over-express key genes in the pathogenesis of hematorrhizin to improve the alkaloid content in M.cordata,the protopine-6’-hydroxylase(P6H)catalyzes the formation of dihydrosanguinarine base by the original opioid to form Sanguinarine.The results of this paper were as follws:1.Designing specific primers based on M.cordata to the protopine-6’-hydroxylase(P6H)gene sequence,using molecular biology techniques to clone the P6 H gene2.The cloned P6 H gene was integrated into the plant expression vector pCAMBIA2301 D for transgenic experiments.3.The plant expression vector 2301-P6 H was successfully introduced into Agrobacterium GV3101,and identified by PCR and GUS identification,and positive plants were obtained.4.The content of benzylisoquinoline alkaloids in three different parts of roots,stems and leaves of overexpressed P6 H gene positive plants and wild type plants was detected by ultra performance liquid chromatography tandem triple quadrupole mass spectrometer(UPLC-QQQ MS).Quinoline alkaloid content.The original protopine,dihydrosanguinarine and sanguinarine,stems and leaves of the positive plants were increased compared with the wild-type bovine plants,which increased by about 2-3 times.5.By comparing the gene expression levels of materials transferred to the three different parts of the roots,stems and leaves of the positive plants and the wild type plants of the target gene P6 H.It was found that the expression level of P6 H gene in the roots,stems and leaves of M.cordata positive plants was significantly improved.The relative expression level in the roots of the positive plants was 6.27 times that of the wild plants;After transferring to the P6 H gene,the downstream DBOX gene expression was also significantly increased.The relative expression of the positive plants in the roots was 12.73 times that of the wild plants.