The Effect Of Overexpression Of Berberine Bridge Enzyme On Benzylisoquinoline Alkaloid Of Macleaya Cordata

Macleaya cordata(Willd.)R.Br.is an important medicinal plant that can produce isoquinoline alkaloids such as sanguinarine,berberine,chelerythrine,etc.Its market prospect is huge broad.By using of plant genetic engineering methods to overexpress key genes in the sanguinarium pathway can increase the production of active alkaloids and resistance to plants of M.Cordata.In this research,the berberine bridge enzyme was over-expressed by cloning technology and integrating the key gene berberine bridge enzyme which has previously been verified on the sanguinarium pathway into the genome of M.Cordata.By using methods of GUS staining and RT-PCR,we can select the positive transformed plants from the all over-expressed plants.Then,the positive plants will be identified by fluorescence quantitative PCR and the product will be accurately quantified by the ultra high performance liquid chromatography with triple quadrupole mass spectrometer.Finally,we compared the four group parallel of the gene expression amounts of Mc BBE,PSBBE,2301 empty vector and blank control,and also compared the key alkaloid content in the sanguinarine pathway,which will provided good preliminary research basis for transformation of sanguinarine synthesis pathway.The results of this paper were as follws:1.By using vacuum assisted methods in the Agrobacterium-mediated process can increase the conversion rate.In addition,the conversion rate of stem segments is higher than that of the leaves under the vacuum conditions.2.By comparing the gene expression levels of Mc BBE,PSBBE,2301 empty vector and blank control,it was found that the expression level of BBE gene was significantly improved in plants transfected into Mc BBE.They were respectively3.2,2.6 and 3 times the number of plants transferred to the Ps BBE、2301 empty vector and the blank control.The SMT gene expression was also obviously increased,they were respectively 4.4,5.1,and 5.7 times that of Ps BBE,2301 empty vector,and blank control plants.3.By comparing the content of benzylisoquinoline alkaloids in the four materials of Mc BBE,PSBBE,2301 empty vector and blank control,it was found that(S)-Reticuline,(S)-Scoulerine,(S)-Cheilanthifoline,(S)-tetrahydrocolumbamine in the plants were transferred into Ps BBE has much higher levels than plants that were transferred to Ps BBE,2301 empty vector,and blank control.