Screening Of The ABC Family Transporter Genes Of Intermediate Metabolites Of Vinblastine Synthetic Pathway In Catharanthus Roseus

Catharanthus roseus(L.)G.Don is an important medicinal plant.It can be used as medicine and it is a little toxic.At present,more than 130 alkaloids have been found in Catharanthus roseus,of which vinblastine is a secondary metabolite present in the Catharanthus roseus,a kind of alkaloid belonging to the terpenoid indole alkaloids(TIAs).It has therapeutic effects on malignant lymphoma,lung cancer,single cell leukemia and other diseases.Vindoline and catharanthine are important precursors for the synthesis of vinblastine.However,too little vindoline is synthesized in the wild Catharanthus roseus,making the vinblastine accumulation too small in the Catharanthus roseus,which affects the commercialization of the drug.Due to the large number of alkaloids and the extremely complex structure,the cost of using chemical methods is too high.Therefore,how to effectively increase the synthesis and accumulation of vinblastine by biological means becomes extremely important.In the synthetic route of vinblastine,the content of intermediate metabolites influences the accumulation and production of vinblastine,and the transporter gene synthesizes the transporter protein to positively or negatively regulate the biosynthesis of metabolites.The ABC(ATP-binding cassette)transporter is an ancient and large-scale super-family trans-membrane protein widely existing in the biological world.ABC transporters play an important role in the transport and accumulation of secondary metabolites in plants.Through software analysis,we selected three genes of the MDR subfamily in the ABC family and conducted a series of experimental studies to screen out genes that affect the synthesis of intermediate metabolites in the vinblastine synthesis pathway.In this paper,virus-induced gene silencing(VIGS)is used as the main research technology.The known key enzyme genes in the intermediate metabolite synthesis pathway of vinblastine are used as reference and real-time.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to analyze the expression of the key enzyme gene and the expression level of the target gene.High-performance Liquid Chromatography(HPLC)was used to analyze the relative contents of the two precursors of catharanthine and vindoline,so that the above experimental data can be effectively used to screen out the transport that has animpact on the intermediate metabolites of vinblastine synthesis.gene.After silencing the CrMDR1 and CrMDR2 genes,both vindoline and catharanthine increased significantly.CrMDR1’s corresponding vindoline showed a twofold increase,and catharanthine increased about 7 times;CrMDR2 had the highest change,Wenduoling.The growth was 2.5 times,while the growth of catharanthine was up to 15 times;there was almost no change in relative CrMDR3.The presumed results of CrMDR1 are in agreement with the experimental data.The results of CrMDR2 are somewhat contradictory,and the results of CrMDR3 are not ideal.The transporters of CrMDR1 and CrMDR3 may be involved in the transport of signal molecules.Therefore,after the target gene is silenced,the key enzyme genes are overexpressed.In summary,it is determined that CrMDR1 participates in the related reactions of intermediate metabolites of the synthetic Changchun base,and is indirectly regulated;CrMDR2 may directly use intermediate metabolites as substrates,and the site of action may be in many places;the result of CrMDR3 is not ideal,and there is no direct regulatory relationship with intermediate metabolites.