Functional Identification And Application Of CYP71Z18 Involved In Maize Terpenoid Phytoalexin Biosynthesis

Maize(Zea mays L.)is the most widely cultivated crop all over the world,which has important economic value.However,the decline of yield and quality of maize caused by stresses has brought great losses to agricultural production.When the corn is attacked by pathogens,it will produce phytoalexins to withstand stress.Terpenoid phytoalexins is a kind of phytoalexins,and it is a terpenoid derivatives formed by cytochrome P450 monooxygenase(P450).P450s in the biosynthetic pathway of rice diterpenoid phytoalexins have been extensively studied,however,P450s involved in the biosynthetic pathway of maize terpenoid phytoalexin have not been reported till now.Therefore,identification of P450s is an important step to clarify the whole biosynthetic pathways of maize terpenoid phytoalexins and will provide the basis for metabolic regulation and resistance breeding.In this study,by digging the transcriptome data of maize infected by Fusarium graminearum,and with the help of phylogenetic analysis of homologous genes from rice,we obtained CYP71Z18 related to the biosynthesis of maize terpenoid phytoalexins.By cloning and recombinant expression in metabolic engineering system in Escherichia coli,catalytic products were isolated and analyzed by GC-MS and NMR for structure identification.Data showed the function of CYP71Z18 was to catalyze sesquiterpene to produce sesquiterpenoid phytoalexin zealexin A1.By analyzing the expression pattern of CYP71Z18 and upstream genes,CYP71Z18 was demonstrated to work in zealexin A1 biosynthesis in maize.This P450 had a wide range of adaptability,and reacted with ent-(iso)kaurene to produce putative kauralexin A1 and B1.It also catalyzed oxidation of a number of rice diterpenes,among which syn-pimaradiene was oxidized sequentially by CYP71Z18 and with rice P450 CYP99A3 to form a new compound.Based on these results,we overexpressed CYP71Z18 in rice to explore the new strategy of constructing rice diterpenoid phytoalexin biosynthetic pathways by using its wide variety of catalytic function.The main results of this study are listed as followed:1.By analyzing the transcriptome data of F.graminearum infected maize,CYP71Z18 was found to be highly induced.Phylogenetic analysis showed that CYP71Z18 had close relationship to CYP71Z6 and CYP71Z7 which play a role in rice diterpenoid phytoalexin biosynthesis.So we inferred that CYP71Z18 was involved in the biosynthesis of maize terpenoid phytoalexins.2.In metabolic engineering system in E.coli,CYP71Z18 exhibited sequential oxidation on maize sesquiterpene(S)-?-macrocarpene.By comparing the mass spectra obtained from GC-MS analysis and NMR structure identfiation,we confirmed that CYP71Z18 catalyzed sequential oxidation of ?-macrocarpene to make three oxdative products,?-macrocarpen-15-ol,?-macrocarpen-15-al and ?-macrocarpen-15-oic acid,the maize sesquiterpenoid phytoalexin zealexin A1.3.CY71Z18 showed an inducible expression pattern.It was induced in maize leaves infected by F.graminearum.On the other hand,?-macrocarpene synthase genes ZmTPS6/11 also showed a strong induction in the early stage.It was speculated that this early induction offered a substrate.for the subsequent induction of CYP71Z18 and they were all involved in the biosynthesis of zealexin A1.4.CYP71Z18 catalyzed the formation of new products from various rice phytoalexin related diterpenes.Among them,CYP71Z18 catalyzed the formation of a putative new compound by C15-C16 epoxidation of syn-pimaradiene,which was thought to be further hydroxylated by rice P450 CYP99A3 at C19.CYP71Z18 also reacted with syn-stemarene,syn-labdatriene,syn-stemodene,ent-cassadiene and ent-sandaracopimaradiene,to produce new compounds which were presumed to be hydroxylated or carboxylated.5.In this research,we constructed the TO generation of CYP71Z18 overexpression lines in rice,which provide the basis for metabolic engineering rice diterpenoid phytoalexins.Conclusions:In this research,the first P450 in the biosynthetic pathway of maize sesquiterpenoid phytoalexin zealexin A1 was identified as CYP71Z18.CYP71Z18 exhibited sequential oxidation on sesquiterpene ?-macrocarpene to form ?-macrocarpen-15-ol,?-macrocarpen-15-al and zealexin A1,among which zealexin A1 was the main product.At the same time,CYP71Z18 showed an inducible gene expression pattern.It constitutively expressed in the whole seedling.It was able to be induced by F.graminearum infection within 48 h,which was consistent with zealexin A1 accumulation pattern.This suggested that CYP71Z18 was the P450 involved in the biosynthetic pathway of maize sesquiterpenoid phytoalexin zealexin A1.In view of the fact that CYP71Z18 also has the oxidative activities on various rice diterpenes,this study attempted to utilize it to reconstruct the biosynthetic pathway of rice diterpenoid phytoalexins.Thus overexpression lines of rice was constructed to provide a basis for further research in taking advantage of the gene function to improve crop resistance.