The Key Genes Research Of Triterpenoid Saponin Biosynthesis And Regulation In Camellia Oleifera

Camellia oleifera,as the most important edible oil tree species,has been cultivated in a huge area in our country.Triterpenoid saponins are one of the main secondary metabolites in seed meals of C.oleifera,commonly known as tea saponin or theasaponin.They have a variety of biological activities such as foaming,detergent properties,anti-cancer and anti-inflammatory,and have broad application prospects in medicine,agriculture,industry and environmental protection.Current studies have shown that the triterpenoid saponins of C.oleifera are diverse in variety and complex in structure,and the separation of monomers is difficult.The biosynthesis and regulation mechanisms of triterpenoid saponins of C.oleifera are still unclear,which seriously hinders the research and development of biological activity mechanism and utilization of triterpenoid saponins.In this study,the biosynthesis and regulation mechanisms of triterpenoid saponins in C.oleifera were studied based on the whole genome sequencing data of ’Huashuo’(hexaploid),a common C.oleifera variety approved by the state.The primary results are as follows:1.Whole-Genome identification of all functional genes of triterpenoid saponins biosynthetic pathway in C.oleifera.Based on the whole genome data of common C.oleifera variety ’Huashuo’(hexaploid),and comparative analysis with genomes of Arabidopsis thaliana,Camellia sinensis,Actinidia chinensis,Panax ginseng,and Medicago truncatula,all functional genes involved in the triterpenoid saponin biosynthetic pathway were identified by gene family analysis.There were 18 triterpenoid skeleton biosynthetic gene family members,including mevalonic acid pathway(MVA),2-C-methyl-D-erythritol 4-phosphate pathway(MEP),farnesyl diphosphate synthase(FPS),squalene synthase(SS),squalene epoxide(SE)and 2,3-oxidosqualene cyclase(OSC).And two members of the triterpenoid skeleton modification gene family,including cytochrome P450 monooxygenase(CYP450)and UDP-glycosyltransferase(UGT).The results showed that there were 143 triterpenoid skeleton biosynthetic genes,1,169 CYP450s and 1,019 UGTs in C.oleifera.The results of cluster analysis of triterpenoid skeleton biosynthetic genes showed that the raw materials for triterpenoid skeleton biosynthesis of C.oleifera were mainly derived from MVA pathway.OSC,3-hydroxy-3-methylglutaryl-CoA reductase(HMGR)and SE play important roles in triterpenoid skeleton biosynthesis.A module highly related to triterpenoid saponins biosynthesis was screened out by weighted gene co-expression network analysis(WGCNA),in which 11 CYP450s and 14 UGTs were co-expressed with triterpenoid-related OSCs,and participated in triterpenoid skeleton modification in C.oleifera.Based on the above results,a biosynthetic pathway of triterpenoid saponins in C.oleifera was constructed in this study.2.Identification and functional study of the key enzyme OSC gene family of triterpenoid saponin biosynthesis in C.oleifera.2,3-oxidosqualene cyclase(OSC)determines the triterpenoid skeleton type and is the most critical enzyme in triterpenoid saponin biosynthetic pathway.Phylogenetic tree analysis showed that the triterpenoid-related OSCs in C.oleifera was mainly divided into β-amyrin synthase(bAS)and dammarenediol synthase(DDS).Five OSCs were cloned from the cDNA library of C.oleifera,named CobAS1,CobAS2,CobAS3,CoDDSl and CoDDS2,respectively.Subcellular localization results showed that all the five OSCs were located in the endoplasmic reticulum.Heterologous expression in yeast showed that CobAS1 and CobAS2 were single-function OSC,and the product was β-amyrin.CobAS3 is inactive;CoDDSl and CoDDS2 are multifunctional OSC,and the products are α-amyrin and β-amyrin,the content ratio of the two products is about 4.5:1.Sequence alignment revealed that Serine(S)at the 270th position of CobAS was one of the key amino acids affecting the activity of this protein.Further analysis of the expression patterns of C.oleifera showed that CobAS were mainly expressed in seeds and CoDDS in roots,while CobAS and CoDDS1 were expressed in stems and leaves,and the expression of CobAS was higher than that of CoDDS.Those results indicated that triterpenoid saponins are oleanane-types in C.oleifera seeds,while in roots,stems,and leaves they are oleanane-and ursulane-types.In addition,phylogenetic analysis with the OSC families of 216 angiosperms showed that OSC evolution was relatively independent between monocots and dicots.In dicots,functionally characterized DDS and multifunctional OSCs whose products are α-amyrin andβ-amyrin are located in the same branch,which is consistent with the results ofα-amyrin and β-amyrin products of CoDDS.3.Studies on transcription factors and regulation of triterpenoid saponins biosynthesis in C.oleifera.Based on the triterpenoid saponin biosynthetic pathway constructed in C.oleifera,promoters of 20 key genes for triterpenoid saponin biosynthesis and 12 key transcription factors(including 4 bHLHs,4 MYBs,2 ERFs,1 B3 and 1 GRF)were screened and cloned.The results of promoter cis-acting element analysis showed that the biosynthesis of triterpenoid saponins in C.oleifera was regulated by various hormones,such as jasmonate,ethylene,abscisic acid,salicylic acid,and auxin,and participated in light response and various stress response pathways.The results showed that 17 promoters could be directly used in yeast one hybrid test.The results of point-to-point yeast one hybrid showed that CoERF-1 could bind to and interact strongly with the promoters pbAS1 and pUGT73-1b.CobHLH-2 can bind to promoter pDDS1 and have strong interaction.To further verify the targeted interaction between transcription factors and promoters,a dual luciferase transient expression assays was performed.The results showed that the luciferase activity driven by pbAS1 and pUGT73-1b could be significantly increased by co-expression of CoERF-1 in tobacco,and the luciferase activity was 4.1 and 4.4 times that of the promoter expressed alone,respectively.Co-expression of CobHLH-2 significantly increased the luciferase activity driven by promoter pDDS1,which was 4.1 times that of promoter expressed alone.In conclusion,CoERF-1 regulates the biosynthesis of triterpenoid saponins in C.oleifera by binding and activating the promoters of CobAS1 and CoUGT73-1.CobHLH-2 is involved in regulating the biosynthesis of triterpenoid saponins in C.oleifera by recognizing and binding pDDS1 to activate the expression of CoDDS1.In conclusion,all functional genes of triterpenoid saponins biosynthetic pathways in C.oleifera were mined in this study,and the raw materials for triterpenoid skeleton biosynthesis of C.oleifera were mainly derived from MVA pathway.The function of OSC gene family,a key enzyme in triterpenoid saponins synthesis,were characterized,making the types of triterpenoid saponins in seeds,roots,stems and leaves of C.oleifera clear.The promoters and transcription factors involved in the biosynthesis of triterpenoid saponins in C.oleifera were explored and the regulatory mechanisms of promoters and transcription factors were preliminarily revealed.This study laid a theoretical foundation for fully revealing the biosynthesis process and regulatory molecular mechanism of triterpenoid saponins of C.oleifera,and provided scientific basis and technical approach for molecular breeding of C.oleifera with higher quality.