| Olive(Olea europaea L.)was one of the four woody oil plants throughout the world,and was regarded highly values both in nutrition and economy for containing unsaturated fatty acids and many bioactive substances,such asterpenes,polyphenols,flavonoids.Terpenoids were the main constituents of the secondary metabolites of olive fruits,and about 90%of the total terpenoids in olive fruitwerebitter glycosides,which had antibacterial,anti-oxidation and anti-inflammatory effects.There were two metabolic pathways of terpenoids in higher plants:mevalonate(MVA)pathway and deoxyxylulose-5-phosphate(DXP)pathway.3-hydroxy-3-methylglutaryl coenzyme-A reductase(HMGR),the first rate-limiting enzyme involved in terpenes biosytnthesis,catalyzed the hydroxy-3-methyl-glutaryl coenzyme A(HMG-CoA)to mevalonate in the cytosolic mevalonate pathway(MVA),andthe enzyme gene could regulate the synthesis of plant terpenoidsTherefore,the study of the HMGR gene had important research value in the synthesis of metabolic pathway olive.In this study,three HMGR genes of olive were cloned whichregulate the synthesis terpenoids.In order to identify their function,the three genes were induced by prokaryotic expression in E.coli,and the expression of recombinant OeHMGR protein by in vitro enzyme activity verification.In addition,in order to reveal the relationship between the expression of the gene family and the biosynthesis and accumulation of the terpenoids in the HMGR gene,using quantitative PCR analysis the expression of HMGR gene in different tissues andfruits at different developmental stages of olive.The main findings were as follows:(1)Designated as OeHMGR1,OeHMGR2,OeHMGR3(theaccessionnumbers was KX783226?KX783227?KX783228)were firstly reported.Bioinformatic analysis showed that the length of mRNA open reading frame(ORF)of the OeHMGRs were 1713bp,1773bp,1737bp,which encoded 570,590,578 amino acids respectively.OeHMGR1 protein isoelectric point(PI)was 6.39,the molecular weight of 61.42KDa,a stable protein.OeHMGR2 protein isoelectric point(PI)was 6.14,the molecular weight of 62.31KDa,which belonged to the unstable protein;OeHMGR3 protein isoelectric point(PI)was 6.38,the molecular weight was 62.05KDa,the protein was unstable.OeHMGR-encoding proteins contained two transmembrane regions,and without signal peptide.Homologous sequence alignment and phylogenetic tree analysis indicated that the amino acid sequences of the three OeHMGR proteins shared more than 80%homology with the HMGR protein amino acid sequences of various plants,OeHMGR protein was clustered into the plant HMGR group,where OeHMGR2 and OeHMGR3 were in the same branch,and the phylogenetic relationship was close,while OeHMGRl was alone in a branch,so the OeHMGRs were belonged to HMGR multigene family.(2)pET30b(+)-OeHMGR1,pET30b(+)-OeHMGR2,pET30b(+)-OeHMGR3 were constructed and transformed successfully into BL21 of E.coli.Under the five IPTG concentration,the proteins were expressed,and the expression was highest under the condition of 0.2 mmol/L.The result of SDS-PAGE showed that the molecular weight of recombinant protein were all in 66.2?68.0 kDa.The separation and purification of recombinant protein,purified protein concentrations were 50 g/mL,98 g/mL and 59 g/mL The function of recombinant protein was confirmed,and the target peak of reaction product was detected by GC-MS,while the control group was not detected,indicating that the protein had 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGR)catalytic activity.(3)Quantitative real-time PCR analysis of the three OeHMGRsindifferent tissuesindicated thatthe expression level of the gene in the tissue was fruit>leaf>flower>root,the expression level in the fruit and leaf was higher,while the expression level in the flower and root was lower.Quantitative real-time PCR analysis of the three OeHMGRs in different growth stages of olive fruit showed that low expression levels with small changes were present at 45d,90d,120d after flowering,but relative higher expression level were observed at 165d after flowering. |
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