Mining And Functional Analysis Of Cytochrome P450 Monooxygenase Genes Putatively Involved In The Biosynthesis Of Tritepenoid Saponins From Llex Asprella

ObjectiveTriterpenoid saponins are the most characteristic components of Ilex asprella(Hook.et Am.) Champ.ex Benth, a medicinal plant widely used in southern China. Low yield in herbal production and difficulties in separation have hampered further study on this sort of interesting compounds. Therefore, it is of great importance to study the biosynthesis of triterpenoid saponins in I. asprella. The aim of this study is to screen and characterize of cytochrome P450 monooxygenase (CYP)candidate genes involved in oxidation modifications in the biosynthesis of triterpenoid saponins from Ilex asprella.Methods1. Screening of CYP candidate genesWith 12 well characterized CYPsrelating to the biosynthesis of triterpenoid saponins from other plants, homology-based BLAST and phylogenetic analysis against the transcriptome of I. asprellawere performed. The results in combination with KEGG metabolic pathways analysis reveal some potentical CYP genes.2. Gene cloning and construction of expression plasmidsTotal RNA of I. asprella was extracted by commercial kit, and then transcripted into cDNA. Using the cDNA as template, The candidate genes was amplified by PCR and then the genes was inserted intothe Saccharomyces cerevisiae shuttle expression vector to give expression plasmids by utilizing Gateway technology, In-Fusion technology or traditional method with T4 ligase.3. Functional analysisThe Saccharomyces cerevisiae WAT11 strain carrying CPR genes from Arabidopsis thaliana. was transformed with the constructedexpression plasmids using a standard lithium acetate protocol. The empty vectors (pESC-TRP、pYES-DEST52) were used as negative controls. The recombinant yeasts were cultured and induced with 2%(w/v) galactose.Western Blot was used to analyze the expression of target proteins.Recombinant yeast strains expressing both amyrin synthase and CYP(WAT11/pEXP3079+ pESC-TRP-410) were induced with 2% (w/v) galactose and the metabolites both in cells and supernatants were analyzed with MS, using authentic olean-12-ene-3,24-diol as reference standard.Results1. CYPs candidate genesThe results of BLAST and Phylogenetic analysis in combination with that of KEGG pathways analysis showed4 candidate genes may take part in the biosynthetic pathway of triterpenoid saponins in Ilex asprella. Among them, CL410.contigl might encode an a-/β-amyrin-24-oxidase gene, CL3010.contigl and CL3010.contig2 might be a-/β-amyrin-28-oxidase genes and CL3008.contigl be responsible for the oxidation of catalyzing the conversion of β-amyrin to its12,13-epoxide with one additional hydroxyl group.2. Cloning of candidate genes CL410-1 and CL3010-1 and plasmid constructionTwo of the 4 candidate genes CL410.Contigl and CL3010.Contig (renamed as CL410-1 and CL3010-1) were cloned using RNA isolated from the root of I. asprella. And the corresponding yeast expression plasmids pEXP410、pESC-TRP-410, pESC-TRP-3010-1 were successfully constructed. Sequence analysis revealed that the coding sequence (CDS) of CL410-1 is 1545.bp long, encoding a protein of 514 aa., and the CDS of CL3010-1 is 1440 bp, encoding a protein of 476 aa.3. Functional analysis of CL410-1The results of Western blot showed a protein with the expected molecular weight of CL410-1 was detected in the cell lysate of recombinant yeast WAT11/pEXP410 and the protein expression reached its peak within 4h after induction. Similarly, proteins with the expected molecular weights of IaAS3079 and CL410-1, respectively, were detected in recombinant yeast WAT11/pEXP3079+pESC-TRP-410, although the production of the latter was much less.MS analysis showed that the cell extract solution and supernatant of recombinant yeasts (WAT11/pEXP3079+pESC-TRP-410)didnot containcharacteristic fragmentions (m/z422 and m/z409)corresponding to authentic olean-12-ene-3,24-diol.ConclusionThrough bioinformatics analysis, totally 4 candidate genes were screenedfrom the transcriptome of I. asprella,which might encode CYPs involved in terpene-relating pathways in I. asprella. Among them, CL410-1 was chosen for protein functional assayin vivo, but noa-/β-amyrin oxidation activity was observed, implying CL410-1 do not have predicted function and its real function should be further studied.