Effects Of Elicitors On Aconitine Biosynthesis And Its Induction Mechanism

Aconitine is a C19 diterpene diester alkaloid.Because of its good insecticidal and bactericidal effects,it can be developed as a new type of botanical biological pesticide.The traditional method for obtaining aconitine has disadvantages such as low yield,long culture period and harsh growth conditions,which seriously affect the application of aconitine in production practice.The purpose of this paper is to increase the yield of aconitine,take the screened endophytes producing aconitine as the research object,and use different kinds of elicitors to induce the fermentation broth to obtain the optimal elicitor and the best induction conditions.The induction mechanism of the inducer methyl jasmonate（MJ）on the biosynthesis of aconitine in SNGWT-002 strain was also explored,which provided a theoretical basis for the biosynthesis of aconitine.The main research contents of this paper are as follows:1.The seeds and tuberous roots of Aconitum are used as raw materials to separate and purify its endophytes,and then the isolated endophytes are subjected to liquid fermentation.After the fermentation broth is processed,different extraction methods are used to crudely extract aconitine.-MS method for qualitative and quantitative analysis of aconitine.The results showed that 20 strains of aconitum endophytic fungi and bacteria were isolated and purified from the seeds and tuberous roots of Aconitum,followed by LC-MS method,and 3strains of aconitine-producing strains were screened out,and the fermentation of each strain was determined by HPLC.The content of aconitine in the liquid,among them,the SNGWT-002 strain has a higher yield of aconitine.After extraction with ammonia water and ether,the content of aconitine is 1.874 mg/L.After 16S r DNA identification,the homology between this strain and Aspergillus flavus is 98%,and supplemented by morphological characteristics,it was determined to be Aspergillus flavus,and finally determined as the target strain.2.SNGWT-002 strain was took as the research object,appropriate amounts of biological elicitors（yeast extract YE,seed extract SE and symbiotic bacterial fermentation broth Bac）,metal elicitors(Co²⁺,Ag+and Cu²⁺)and Chemical elicitors（methyl jasmonate MJ,salicylic acid SA and sodium nitroprusside SND）were used to conduct induction experiments to explore the effects of different elicitors and different induction concentrations on the biosynthetic yield of aconitine.The results showed that an appropriate concentration of elicitor could increase the production of aconitine,while an excessively high concentration of elicitor would inhibit the biosynthesis of aconitine.Among them,the induction test of abiotic elicitors showed that the induction effect of Ag⁺and Cu²⁺was more significant when the concentration was 50μmol/L,and the yield was increased by about 10 times and 4.1 times respectively compared with the control group.MJ and SA also had significant induction effects at 100μmol/L,and the content of aconitine was about 11 times and 5 times that of the control group.Not obvious.Finally,100μmol/LMJ was selected as the best inducer for subsequent experiments.3.In order to explore the induction mechanism of MJ in the biosynthesis of aconitine,this thesis studied the effect of MJ on two initiation enzymes in the biosynthetic pathway of secondary metabolites—phenylalanine ammonia lyase（PAL）,tyrosine The effect of acid aminotransferase（TAT）.On this basis,the effects of MJ on the first rate-limiting enzyme in the MVA pathway of terpenoid biosynthesis,hydroxymethylglutaryl reductase（HMGR）,and the first rate-limiting enzyme in the MEP pathway,1-deoxyxylose,were further studied.-5-phosphate synthase（DXS）and the key enzyme in the synthesis of diterpenoids-geranylgeranyl diphosphate synthase（GGPPS）.The results of enzyme activity assay showed that the production of aconitine was positively correlated with the activity of PAL enzyme.The addition of MJ could enhance the activity of PAL enzyme,and attenuate the activity of TAT enzyme to increase the production of terpenoids（aconitine）.The total RNA of time SNGWT-002 strain was detected by q-RTPCR technique to detect the effect of MJ induction on the expression levels of key enzyme genes in aconitine biosynthetic pathway.The results showed that after MJ induction,the DXS gene expression of SNGWT-002 was up-regulated to the highest after 66 h of culture,which was about 8.02 times higher than that of the internal reference gene,and then the DXS gene expression level became stable.The expression level of GGPPS gene was up-regulated to the highest at 60h of culture time,which was about 4.72times higher than that of the internal reference gene.In the early stage of MJ induction（48h）,the expression of HMGR gene was up-regulated,and then the expression of HMGR gene began to show a downward trend.This result indicated that during the biosynthesis of aconitine,the addition of MJ allowed the precursors synthesized in the MVA pathway to participate in the MEP pathway,which together increased the production of aconitine.