Exploration Of The Biosynthetic Mechanism Of Rice Diterpenoid Phytoalexin Ent-10-oxodepressin And Function Identification Of Maize Sesquiterpenoid Synthase ZmEDS

Terpenoids,as the most diverse natural products in nature,play an important role in plant growth and development,defense response,chemical production,pharmaceutical development and so on.Terpenoid phytoalexins,formed by multi-step modification of terpenes,have broad antibacterial properties and play an important role in the response to stress of crops,i.e.maize and rice.Rice blast is an important disease in rice production,which seriously affects the yield and quality of rice.The infection of Magnaporthe oryzae leads to the accumulation of ent-10-oxodepressin,a diterpene phytoalexin derived from ent-casbene,which exhibits obvious inhibitory effect on the spore germination and growth of M.oryzae,but its biosynthetic pathway is still unclear.By means of Escherichia coli metabolic engineering,NMR analysis and rice transgenic plant construction,we characterized the rice biosynthetic gene cluster c7BGC participating in the biosynthesis of rice diterpenoid phytoalexin ent-10-oxodepressin.that is,OsTPS28（OsECBS）catalyzed GGPP to ent-casbene,and Os CYP71Z21/22 continuously oxidized on C10 position of ent-casbene to generate ent-10-keto-casbene,and then Os CYP71Z2 reacted on C5 position to form ent-10-oxodepressin.This study provides target genes for transcriptional regulation and rice resistance breeding.The main results are described as follows:1.Based on ent-casbene is the skeleton of ent-10-oxodepressin,OsTPS28 was selected as a candidate rice casbene synthase gene based on homology retrieval in the rice genome database in which the sequence of identified Rc CBS（Ricinus communis casbene synthase）was used as a query.The main product of OsTPS28 reacting on GGPP was ent-casbene identified by metabolic engineering system,NMR analysis and specific rotation measurement.Hence,OsTPS28 was termed as OsECBS.2.Genes related to biosynthesis of many terpenoid phytoalexins are clustered on chromosomes.terpene.Analysis of the gene information near the location of OsECBS on the rice chromosome 7 showed that there were four CYP genes（Os CYP71Z2/21/22/30）in the 141k B region.The gene expression patterns of each gene in different developmental stages and tissues of rice,and under different treatments were analyzed based on the transcriptome data from Ricex Pro server and q RT-PCR analysis.The results indicated no expression of Os CYP71Z30 was detected whereas the other four genes were in similar pattern.Therefore,this region was named as c7BGC.3.OsECBS and each CYP were co-expressed respectively to identify the product profiles.The results showed that Os CYP71Z30 could not catalyze the product of OsECBS.Os CYP71Z2 could continuously oxidize at the carbon 5（C5）position of ent-casbene to form5S-hydroxy-ent-casbene and ent-5-keto-casbene.While Os CYP71Z21/22 could continuously oxidize at the C10 position of ent-casbene to produce 10S-hydroxy-ent-casbene and ent-10-keto-casbene.4.Ent-5-keto-casbene and ent-10-keto-casbene were fed into the fermentation cultures containing corresponding CYP respectively,to identify whether new products were generated.The results showed that Os CYP71Z2 can react on the C5 position of ent-10-keto-casbene to form ent-10-oxodepressin.And Os CYP71Z21/22 can continuously oxidize on the C10 position of ent-5-keto-casbene to generate ent-10-oxodepressin.5.OsECBS over-expression（OE）and CRISPR-Cas9 knock-out（ko）lines were constructed respectively in the background of rice cv.Kitaake.The accumulation of casbane-type compounds in the corresponding rice materials was analyzed respectively,and three related compounds,ent-casbene,ent-10-keto-casbene and ent-10-oxodepressin,were detected.None related products was detected in the ko lines.Compared with WT,ent-casbene accumulated significantly in the OsECBS-OE lines,and ent-10-keto-casbene and ent-10-oxodepressin were increased after Cu Cl₂ treatment.Moreover,the growth of M.oryzae in OE lines was inhibited,while the propagation of pathogens in OsECBS knockout lines was significantly increased,compared to WT.The various biological functions of terpenoids are determined by functional groups.So far most of identified terpenoid synthases only can catalyze their substrates to produce terpenoids containing at most one oxygen atom,and the addition of other groups can only be formed by further modification of subsequent oxidases,which greatly limits the production of the introduction of functional groups and terpenoids.In this study,we found the first sesquiterpenoid synthase Zm EDS that can produce two hydroxyl products in maize.Homologous modeling and site-directed mutagenesis were used to further analyze the catalytic mechanism,which provides a research basis for exploration of the functions of maize sesquiterpenoids and the directional modification of terpenoid synthases in the future.The main results are as follows:1.The main product of Zm TPS17 catalyzing FPP was sesquiterpene diol eudesmane-2,11-diol identified by means,like E.coli metabolic engineering system,the transient overexpression system of tobacco leaves.Therefore,Zm TPS17 was termed as Zm EDS.While,Zm EDS was highly expressed in maize seedling root and eudesmanediol was detected in the maize root exudates.2.The predicted Zm EDS protein structure was obtained by homology modeling using Nicotiana benthamiana 5-epi-aristolochene synthase protein structure as template.The molecular structure of substrate,product and intermediate were docked into the Zm EDS protein structure,respectively.Combined with sequence alignment results,multiple sites,i.e.F303,were selected for subsequent study.Site-directed mutagenesis and product analysis were performed with the selected sites respectively in which multiple single mutants significantly changed the product composition of Zm EDS.3.The result of in vitro catalytic experiments with deuterium oxide labeling showed that in the formation of eudesmanediol,a proton from the solvent was involved in the formation of its skeleton and added at C6 position.4.The compounds with high yield in Zm EDS or the mutants were purified and characterized the chemical structures,such as eudesmols,cryptomeridiols and hedycaryol.The effects of related sites in the catalytic reaction and the potential catalytic mechanism of Zm EDS were deduced in which hedycaryol was considered as an intermediate in the formation of major product.