The Preliminary Study On Synthesis Of Glucosinolates In Marchantia Polymorpha L

Glucosinolates are a kind of secondary metabolites that exist specifically in cruciformes.According to the difference of its side chain,glucosinolates can be divided into three types:indole glucosinolates,aliphatic glucosinolates and aromatic glucosinolates.Glucosinolates usually have no biological activity and can be degraded to produce a variety of bioactive degradation products under the action of myrosinase.Studies have shown that the metabolites of glucosinolates not only have insect-resistant and disease-resistant effects on plants,but also have a variety of benefits to human health.In addition,glucosinolates can also be used as biological pesticides in agricultural production.Some degradation products of glucosinolates,such as the famous sulforaphane,can effectively inhibit the occurrence of tumor and have anticancer effect.It has been found that indol-3-ylmethyl glucosinolate(I3M),also known as indole-3-carbinol(I3C),the degradation product of glucosinolates from brassica,can inhibit the expression of oncogenes.Although there is I3 M in common cruciferous vegetables,the treatment of tumor is far from enough because of its low content,so the artificial synthesis of glucosinolates is of great significance to exert its anticancer effect effectively.Marchantia polymorpha L.belongs to the bryophyte phylum of higher plants.Although it can not synthesize glucosinolates,it has many advantages,such as easy mass culture,short generation cycle,high transformation efficiency,easy to obtain homozygotes and so on.Therefore,this study chose Marchantia polymorpha L.as a bioreactor to synthesize indole glucosinolates I3 M.According to the length of the gene,His,HA and FLAG tags and stop codons were added to the core structure synthesis genes in the indole glucosinolate metabolic pathway by using multi-gene linking strategy.CYP97B2,UGT74B1 and GSTF9 add His tags;SOT16 and CYP83B1 add FLAG tags;SUR1 and GGP1 add HA tags.The entry vector was constructed by Gateway: BP reaction,and the single gene expression vector was constructed by LR reaction.The CYP97B2-GGP1-UGT74B1-GSTF9 gene was cloned into the plant expression vector p GWB402 by Golden gate cloning and the kanamycin resistance polygene expression vector was successfully constructed.The SOT16-SUR1-CYP83B1 gene was cloned into the plant expression vector p GWB501 by Gibson assembly and the hygromycin resistance multi-gene expression vector was successfully constructed.The constructed multi-gene vector was transformed by Agrobacterium,and the mustard glucosinolate metabolic pathway gene was transiently expressed in tobacco.Indole glucosinolates were detected in transformed tobacco.It was confirmed that the indole glucosinolates metabolic pathway gene was successfully transferred into tobacco and produced I3 M.On the basis of the success of transient expression in tobacco,the above expression vector was transferred into Marchantia polymorpha L.by Agrobacterium mediated induction method and stably expressed,in order to realize the artificial synthesis of I3 M in Marchantia polymorpha L.,and to explore the feasibility of artificial synthesis of indole glucosinolates,so as to provide useful data reference for the metabolic engineering of glucosinolates.The results of this thesis are as follows:1.The genes encoding CYP79B2,CYP83B1,GSTF9,GGP1,SUR1,UGT74B1 and SOT16 in the synthesis of indole glucosinolates core structure were cloned successfully.2.The entry vector and single gene expression vector containing CYP79B2,CYP83B1,GSTF9,GGP1,SUR1,UGT74B1 and SOT16 genes were constructed.3.CYP97B2-GGP1-UGT74B1-GSTF9 multi-gene expression vector and SOT16-SUR1-CYP83B1 multi-gene expression vector were constructed.4.Indole glucosinolate I3 M was successfully transiently expressed in tobacco.5.The process of genetic transformation was optimized,and the synthetic pathway of I3 M was transferred to the Marchantia polymorpha L.to realize the artificial synthesis of I3 M.