Cloning And Functional Characterization Of Key Genes Involving In Polyphenols And Terpenoids Biosynthesis From Liverworts

Bryophytes,which placed between algae and ferns,are the second largest taxa in plant kingdom and are widely distributed in nature.Liverworts,a class of bryophytes,are rich in a large number of secondary metabolites,mainly the terpenoids and polyphenol compounds,with variety of chemical structures and bioactivity,such as antitumor,antifungal,antioxidant and antifeedant.However,liverworts are usually small and grow in extreme environment,so it is difficult to collect a sufficient biomass for further research,which limits the development of the finding for new drugs.With the development of biotechnology,the target compounds can be biosynthesized using the method of metabolic engineering,which may effectively solve the problem.Dissecting the biosynthesis pathway of the active natural products is of great importance in producing or improving the output of the target compounds,by the method of combinatorial biosynthesis and genetic modification.By screening the transcriptome sequencing data and cDNA library,we got a number of genes involved in terpenoids and polyphenols biosynthesis pathway from liverworts.And several of them were studied in great detail.We obtained two diterpene synthase(diTPS)from the liverwort Conocephalum conicum,which was rich in terpenoids,and their functions were also studied.A PAD-encoding gene was isolated from the liverwort species Conocephalum japonicum,it exhibited high PAD activity and its sub-cellular localization was also conducted in Nicotiana benthamiana.We got seven 4-Coumarate:coenzyme A ligase(4CL)from the liverworts Plagiochasma appendiculatum and Marchantia paleacea.They were all heterologously expressed in Escherichia coli and site-directed mutations were also conducted with Pa4CL1.Their functions in vitro were analyzed using HPLC,showing different substrate activities.Additionally,the analysis of the expression level induced by abiotic stress methyl jasmonate(MeJA),abscisic acid(ABA)and salicylic acid(SA),was conducted.We selected two enzymess with higher activities and both transferred to the Agrobacterium tumefaciens strain EHA105,and then A.tumefaciens-mediated transient expression in Arabidopsis thaliana was performed.The flavonoids and lignin contents of transgenic A.thaliana were analyzed.The main research contents and results are as follows:1.Cloning and functional Characterization of two diterpene synthase(diTPS)from the liverwort Conocephalum conicumTwo diterpene synthases(diTPS)were identified and cloned from the liverwort Conocephalum conicum,namely CcCPS and CcSS.CcCPS showed high sequence homology with AtCPS and PpCPK/KS and harbored a conserved DXDD motif,which belonged to Class II terpene synthases,while CcSS harbored a conserved DDXXD motif,belonging to Class I terpene synthases.Both the full length cDNAs and the truncated versions lacking the transit peptide regions were amplified and heterologously expressed in E.coli and the purified protein were used for function analysis in vitro.Provided with substrate GGPP.the reaction products were analyzed and identified by GC-MS.The results showed that CcCPS could catalyzed GGPP to ent-copalyl diphosphate(ent-CPP),while CcSS subsequently converted ent-CPP to form ent-kaurene and ent-sandaracopimaradiene,with the latter being the main product.2.Cloning and functional Characterization of 4-coumarate CoA ligases from liverworts Plagiochasma appendiculatum and Marchantia paleacea4-Coumarate:coenzyme A ligase(4CL)was the third enzyme in the phenylpropanoid synthesis pathway,controlling the direction towards the flavonoids,lignin and coumarins.Three 4CLs were cloned from the liverwort Plagiochasma appendiculatum,namely Pa4CL1,Pa4CL2 and Pa4CL3,while four 4CLs were isolated from the liverwort species Marchantia paleacea,namely Mp4CL1,Mp4CL2,Mp4CL3 and Mp4CL4.They were all heterologously expressed in E.coli and site-directed mutations were also conducted with Pa4CL1.Pa4CL1,Pa4CL2,Mp4CL1 and Mp4CL2 exhibited a high level of 4CL activity and preferred substrate was p-coumaric acid;in particular,the enzyme could also convert dihydro-p-coumaric acid into dihydro-p-coumaroyl CoA,the precursor for bisbibenzyl synthesis,albeit less efficiently than the p-coumaric acid substrate.However,Pa4CL3,Mp4CL3 and Mp4CL4 displayed no activities with any of the substrates.Site-directed mutagenesis demonstrated that Met-247 and Ala-251 were involved in hydroxycinnamate binding and had nothing to do specificity of the enzyme.Pa4CL2 and Mp4CLl with higher enzymes activities were both transferred to the Agrobacterium tumefaciens strain EHA105,and then A.tumefaciens-mediated transient expression in Arabidopsis thaliana was performed.The lignin contents of Pa4CL2 and Mp4CL1 transgenic A.thaliana increased,while the flavonoids contents of Pa4CL2 and Mp4CL1 transgenic A.thaliana seedlings decreased.Both the transcription of Pa4CL1 and Mp4CL1 were induced when treated with either salicylic acid or methyl jasmonate.Pa4CLl,Pa4CL2,Mp4CLland Mp4CL2 were all sub-located in the cytoplasm and nucleus.3.Cloning and functional Characterization of a phenolic acid decarboxylase from the liverwort Conocephalum japonicumTranscriptome sequencing from the liverwort species Conocephalum japonicum has identified a PAD-encoding gene,namely CjPAD,which share a measure of sequence identity with microbial PADs.but are typically much longer proteins.CjPAD harbors the four conserved catalytic residues Tyr-60,Tyr-62,Arg-90 and Glu-114.When the gene was heterologously expressed in E.coli,its product exhibited a high level of PAD activity when provided with either p-coumaric or ferulic acid as substrate,along with the conversion of caffeic acid and sinapic acid to their corresponding decarboxylated products.Both N-and C-terminal truncation derivatives were non-functional.Both GFP-CjPAD and GFP-CjPAD-Tr were expressed in the cytoplasm and nucleus,showing that the N-terminal domain of CjPAD made no contribution to the gene product's subcellular localization(even though it did affect its catalytic activity).It is first time a PAD was characterized from plants and the present investigation provided a candidate gene for catalyzing the formation of volatile phenols.