Application Of Catharanthine Transport Gene CrTPT2 In Catharanthus Roseus Hairy Roots

Catharanthus roseus is an important model plant for Terpenoid Indole Alkaloids(TIAs)production,since most of TIAs have very good biological activity for healing human diseases.Among these TIAs,In C.roseus,catharanthine has antibacterial and hemostatic effects;in addition,ajmalicine can dilate blood vessels and lower blood pressure,but their content is minimal.C.roseus has a long growth cycle and is susceptible to environmental influences,resulting in resource shortage of this plant.The C.roseus is a perennial dicotyledonous plant that is susceptible to infection by Agrobacterium rhizogenes,thus producing the Hairy root.Hairy roots are suitable for optimization of fermentation culture and secondary metabolites production due to their stable metabolic genetic properties and rapid growth rate.Firstly,we established an optimal induction strategy for hairy roots of C.roseus:We used Agrobacterium rhizogenes C58C1 to invasive the leaves of sterile seedlings which had been induced on 1/2 MS medium for 2 days.In this process,the highest induction rate could be obtained by 5 seconds of ultrasound.Secondly,we transformed pBI121GD-CrTPT2 into C58C1 and successfully constructed transgenic C.roseus hairy roots with the best induction method.Thirdly,we used Real-Time PCR to detect the expression of CrTPT2 and used HPLC to detect the contents of catharanthine and ajmalicine in wild-type and transgenic hairy roots.The results showed that the expression of CrTPT2 in transgenic hairy roots is visibly higher than the wild types,and the content of catharanthine is increased about four times,and the content of ajmalicine is also increased about two times.In conclusions,this study established an optimal solution for the induction of hairy roots in C.roseus and successfully constructed transgenic hairy roots that overexpress CrTPT2.And we found that the accumulation of catharanthine and ajmalicine in this transgenic hairy roots has been successfully increased.This work will provide an efficient and feasible solution for subsequent studies related to metabolic regulation.