Molecular Regulation Study On Morphine Biosynthesis And Metabolism In Opium Poppy

As a medicinal plant, opium poppy(Papaversomniferum L.), it has a high value in medical field. Opium poppy contains a lot of alkaloid with highest content of morphine that is used widely; but morphine is highly addictive and dependent. Therefore, it is of great significance to search for the unique efficacy and no dependence on alkaloids in opium poppy. The demand for thebaine is increasing, because it is an important metabolic constituent in the anabolic pathway of poppy morphine and has also been widely concerned. However, the low yield of thebaine in opium poppy that leads to relatively low utilization efficiency and economic benefits. Therefore, how to cultivate high thebaine content of poppy variety that would be have an important role to promote the development of medicine and social economy.Based on the biosynthesis pathways of Morphin in opium poppy, the two genes were cloned successfully that they encode key enzymes of CODM and T6 ODM in metabolic pathways of the morphine, and the RNAi expression vector of these two genes was constructed and verified in tobacco successfully. This study would lay the foundation for inhibiting genes expression of key enzymes in metabolic pathway of opium poppy and cultivating high thebaine content of poppy variety.The main contents and results are as follows:1. The Primer 5.0, Olig6.0 software was used to design the primer according to the known sequence of CODM(GQ500141) and T6ODM(GQ500139) gene. The full-length sequence of CODM and T6 ODM was cloned respectively by RT-PCR. The results showed that the c DNA sequence of these two genes shared 98% homology respectively with the nucleotide sequenc in Gen Bank.2. Ten sensitive targets of CODM and T6 ODM gene were found respectively based on the analysis of the E-RNAi network online. These sensitive targets contain more effective small interfering RNA. The highest score of target was selected and constructed the interference vector.3. The overlapping PCR technology was used to link the selected target gene and added the suitable enzyme digestion sites in the 5 ’and 3’ end, and then shall be insert into the basic vector of p HANNIBAL. The methods of stepwise digestion and connection were used to insert the positive and reverse fragment on both sides of intron PDK, and get the intermediate vector of p HACTFR. At last, the "Ca MV 35 S P :: :: PDK forward fusion fragment :: intron reverse fusion fragment :: OCS T" was inserted the between of SacⅠand PstⅠgene in the vector of p CEPSPS that contains the glyphosate resistance gene, and the vector of ihp RNAi that named p CEHACTFR was received. The vector of PCEHACTFR was introduced into Agrobacterium LBA4404 strain by freezing-thawing method, and the engineering bacteria were obtained.4. The tobacco seedlings was transformed by vacuum infiltration and Agrobacterium mediated transformation. After the molecular detection and identification of herbicide resistance in transformed tobacco, the positive tobaccos were obtained that have resistance to glyphosate. This results indicated that the ihp RNAi unit and the expression unit located between the LB and RB in the constructed vector have been integrated into the tobacco genome.5. The tissue culture regeneration system of opium poppy was initially established by study on medium, culture conditions and transformation system, and the suitable medium and the Agrobacterium mediated transformation system have been screened.