Study On The Induction And Regulation Mechanism Of Aspergillus Flavus Fungal Elicitor On Biosynthesis Of Terpenoid Indole Alkaloids In Catharanthus Roseus Cambial Meristematic Cells

Purpose:Study on the induction and regulation mechanism of Aspergillus flavus fungal elicitor on the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus cambial meristematic cellsMethods:(1)Preparation of A.flavus fungal elicitor:after A.flavus was cultured,the mycelium and the culture medium were separated and homogenized with distilled water.(2)Investigate the effect of A.flavus fungal elicitors on production of alkaloids in CMCs of C.roseus.A.flavus fungal elicitors were added into CMCs which was pre-cultured for 4 d or 6 d,making the final concentration of A.flavus fungal elicitors to be 5,15,25 mg/L,respectively.After induction for 12,24,36,48,60 h,CMCs were harvested.Concentration of vindoline,catharanthine and ajmalicine was determined by HPLC.The optimal induction condition was obtained.(3)Under the optimal condition,the effect of A.flavus fungal elicitor on the transcriptome of C.roseus CMCs was analyzed.(4)pCRISPR/Cas9-ORCA3,a knockout vector,was constructed and transformed into Agrobacterium tumefaciens strain EHA105 to infect CMCs.After screening and cultivation,ORCA3 knockout C.roseus CMCs was obtained.(5)Under the optimal condition,the expression of TIA biosynthesis-related genes,such as ORCA3,were detected by qRT-PCR.Then,the regulation mechanism of TIA biosynthesis in C.roseus CMCs induced by fungal elicitor was investigatedResult:(1)The optimum condition for the treatment of the A.flavus elicitor was as follows:after pre-cultivation of C.roseus suspension CMCs for 6 d,the A.flavus mycelium elicitor was added at 25 mg/L and the CMCs was induced for 48 h.Under this condition,the content of vindoline,catharanthine and ajmaline was up to 8.79,2.81 and 8.95 mg/L,respectively,which was 1.45,3.29,and 2.14 times that of the control group,respectively.(2)Transcriptome analysis showed that D4H,G10H,GES,IRS,LAMT,SGD,STR,TDC and ORCA3 were involved in the regulation of this induction process.(3)The qRT-PCR results showed that A.flavus elicitor could increase the expression of TIA biosynthesis-related genes by upregulating the transcription level of ORCA3,which in turn promoted the accumulation of vindoline,catharanthine and ajmaline in C.roseus CMCs.Conclusion:A.flavus fungal elicitor could stimulate the biosynthesis of TIA in C.roseus CMCs.Furthermore,the regulation mechanism of A.flavus fungal elicitor on the induction of alkaloid was related to the upregulation of ORCA3.