Biochemical Function Identification Of 10-Hydroxygeraniol Oxidoreductase In Gardenia Jasminoides

Objective:Gardenia jasminoides Ellis is a dried fruit of Gardenia jasminoides,which is a medicinal and food resource.It has the functions of protecting liver and biliary,lowering blood pressure,stopping bleeding,clearing heat and detoxifying,etc.Its chemical composition is very complicated,including the main compounds such as iridoids,flavonoids,organic acid esters,alcohols,polysaccharides,long-chain alkanes and aldehydes,and the most researched and used is iridoids and yellow pigments.Gardenoside are the active ingredients in the 2015 version of the Pharmacopoeia,which belongs to the group of iridoids as the geniposide.Pharmacological studies have shown that they have antitumor,hepatoprotective,antidepressant and hemostatic effects.Antitumor and antidepressant have always been the focus of research,however the active ingredients are obtained from plants in a relatively low amount.So it is very important to clarify the synthesis and regulation of secondary metabolites of medicinal plants.At present,the research on Gardenia is focused on the separation of chemical components,pharmacological activity and clinical application,but the research on the synthesis and regulation of its secondary metabolites is still rare.In order to elucidate the biosynthesis pathway of iridoid glycosides in Gardenia jasminoides Ellis,10-hydroxygeraniol oxidoreductase(10-HGO)which is the key enzyme gene of iridoid biosynthesis pathway was selected as the research object,and its gene cloning and biochemical function analysis were carried out.Methods:The 10-HGO gene was screened from the Gardenia jasminoides Ellis transcriptome.The sequence structure was analyzed by protein sequence alignment and phylogenetic tree construction,and specific primers were designed to analyze the transcriptome expression level by real-time fluorescent quantitative PCR and the genomic structure analysis was by general PCR.The 10-HGO gene was cloned with specific primers and constructed on pMAL-C5X prokaryotic expression vector.Then the protein was expressed and purified by the E.coli expression system and the MBP tag(maltose fusion protein)purification system.then the purified protein was applied to the substrates of 10-Hydroxygeraniol,10-Oxogeraniol,Geraiol,P-coumaraldehyde,Sinapylaldehyde and Coniferylaldehyde.Gas chromatography-mass spectrometry(GC-MS)and Ultra Performance Liquid Chromatography(UPLC)was used to detection and analysis the catalytic products,and the enzyme kinetics experiment was carried out with the microplate reader.Subsequently,mutants were obtained by site-directed mutagenesis,and the protein expression and purification of the mutants were carried out,and quantitative analysis was performed to study the changes in the biochemical functions of the mutants.Finally,molecular modeling was used to find the key amino acid residues of the 10-HGO gene.Results:Three candidate 10-HGO candidate genes were screened out in this paper,named Gj10-HGO1,Gj10-HG02,and Gj10-HG03.The protein sequence results showed that the three Gj10-HGO genes had high similarity with HGO family,geraniol dehydrogenase(GEDH)family and cinnamyl alcohol dehydrogenase(CAD)family,and have conserved domains such as NADP/NADPH and Zn2+ binding sites.The phylogenetic tree showed that the three Gj10-HGO genes were clustered into one class with the HGO family and had high homology with the GEDH family.Real-time fluorescent quantitative PCR results showed that the expression level of Gj10-HGO1 was higher in fruits and lower leaves,the expression level of Gj10-HGO2 was highest in flowers,and the expression level of Gj10-HG03 was highest in lower leaves.Analysis of the genomic structure showed that the full length of the Gj10-HGO1 gene was 2229 bp;the full length of the Gj10-HGO2 gene was 1786 bp;the full length of the Gj10-HGO3 gene was 2334 bp,and all of them contained 4 introns and 5 exons.Subsequently,three coding sequences of Gj10-HGO genes were cloned and constructed into the pMAL-C5X vector.The recombinant plasmid was transformed into E.coli to induce expression,and the protein was purified.Biochemical function analysis results showed that all three Gj10-HGO genes were active to 10-Hydroxygeraniol,10-Oxogeraniol,Geraiol,P-coumaraldehyde,Sinapylaldehyde,Coniferylaldehyde and other substrates.Enzyme kinetics results showed that Gj10-HGO1 had the highest activity on Geraniol,Gj10-HGO2 had the highest activity on 10-Hydroxygeraniol and Gj10-HGO3 had the highest activity on P-coumaraldehyde.Then seven mutants were obtained by site directed mutagenesis,and their proteins were obtained successfully.The results of biochemical function and quantitative analysis showed that except the G250Y mutant completely lose its biochemical function,the other six mutants had no significant changes in the biochemical activity of 10-Hydroxygeraniol,10-Oxogeraniol,Geraiol,P-coumaraldehyde,Sinapylaldehyde,Coniferylaldehyde and so on.Finally,molecular models of Gj10-HGO1,G250Y mutant,YA100VG mutant and V190L mutant were obtained.Through structural comparison,key amino acid residues such as 43-47,LYS,VAL,LEU,TYR,CYS;328-329,ILE,PRO;351-352,ILE,ASP were found.Conclusion:Three Gj10-HGO genes were cloned and identified in this paper.They were found to have the functions of three types of enzyme genes,namely HGO,GEDH and CAD.Combined with various analyses,it is believed that Gj10-HGO1 is most likely to participate in the biosynthetic pathway of gardenia iridoid glycosides.Through site-directed mutagenesis,a mutant with complete loss of function was obtained,and some key amino acid residues were found through molecular comparison of structural simulations.These have filled a blank for the synthetic pathways of gardenia iridoid glycosides,and also provided a basis for the analysis of the biochemical function of the 10-HGO gene.