Cloning And Functional Verification Of Enzyme Genes DXS And DXR In MEP Pathway Of Olive

Olea europaea L.,an evergreen tree of Oleaceae,is one of the “four woody oil plants”in the world.Olive oil Olive oil obtained by cold pressing of olive fruits is not only rich in monounsaturated fatty acids,but also contains secondary metabolites such as terpenoids and squalene.Among them,terpenoids are important components that affect the quality and taste of olive oil,and also have medicinal value such as antibacterial,anti-inflammatory and cancer prevention.Olive leaf contains a large amount of olivopicrin,which is widely used in cosmetics industry and medicine,and has important market value.There are two pathways for terpenoids synthesis and metabolism in plants,namely methylovalerate(MVA)pathway and 2-C-methyl-D-erythritol-4-phosphate(MEP)pathway.1-deoxy-D-xylose-5-phosphate synthase(DXS)and 1-deoxy-xylose-5-phosphate reductase isomerase(DXR)are key enzyme and rate-limiting enzyme in MEP pathway,respectively.At present,there are few reports about DXS and DXR genes in olive.Therefore,in this study,two DXS and two DXR genes were cloned from olive and their function were verified by color complementary reaction.In order to reveal the differences of transcription levels of Oe DXS and Oe DXR in different tissues of olive,the expression levels of Oe DXS and Oe DXR family in different tissues of olive were analyzed by real-time fluorescence quantitative PCR.To provide theoretical basis for furture improving terpenoids content and varieties of olive.The main results are as follows:(1)Two DXS genes and two DXR genes were identified from olive,named Oe DXS1,Oe DXS2,Oe DXR1 and Oe DXR2,respectively.Bioinformatics results showed that the length of ORF of these four genes were 2172 bp,2139 bp,1425 bp and 1425 bp,encoding 723,712,474 and 474 amino acids respectively.The molecular weight of the four protein were178.00 KDa,76.811 KDa,51.34 KDa and 51.576 KDa,respectively.The theoretical isoelectric point(p I)of the four proteins were 4.94,7.14,5.87 and 5.70.The proteins are belonged to stable protein except Oe DXR1.Oe DXS1,Oe DXS2,Oe DXR1 and Oe DXR2 proteins all have no transmembrane proteins and no signal peptide.Sequence alignment results show that the amino acid sequences of Oe DXS1/Oe DXS2 and Oe DXR1/Oe DXR2 proteins are more than 80% consistent with the DXS and DXR amino acid sequences of many plants.Oe DXS1 and Oe DXS2 proteins have pyrimidine binding motifs,TPP binding domains and transketolase C terminal motifs.Both Oe DXR1 and Oe DXR2 proteins contain NADPH binding motifs,DOXP binding conserved sequences of substrates and highly conserved sequences rich in proline at N-terminal.Phylogenetic tree analysis showed that Oe DXS1 belonged to type I DXS and Oe DXS2 was clustered into type II DXS.Oe DXR1 and Osmanthus fragrans DXR belonged to the same phylogenetic branch,which indicated that Oe DXR1 was closely related and consistent with the traditional taxonomic system,while Oe DXR2 was a separate evolutionary branch.(2)The expression vectors p Trc-Oe DXS1,p Trc-Oe DXS2,p Trc-Oe DXR1 and p Trc-Oe DXR2 were successfully constructed and transformed into E.coli containing p AC-BETA plasmid.The TOP10,TOP10+p Trc-Oe DXS1/Oe DXS/Oe DXR1/Oe DXR2、TOP10 +p AC-BETA、TOP10+p Trc+p AC-BETA strains as control.The five different strains were streaked on the double-antibody plate and the function of the four genes verified by color complementary reaction.The results showed that the plaque color of the two plasmids containing p Trc-Oe DXS1/Oe DXS2/Oe DXR1/Oe DXR2 and p AC-BETA was lighter than that of the TOP10+p Trc+p AC-BETA.The other three control groups could not grow on the double-antibody plate,indicating that the expression of Oe DXS1/Oe DXS2 and Oe DXR1/Oe DXR2 genes in E.coli enhanced the production of β-carotene in E.coli.Therefore,Oe DXS1/Oe DXS2 and Oe DXR1/Oe DXR2 genes have catalytic functions.(3)The expressions of Oe DXS1/2 and Oe DXR1/2 in the roots,stems,leaves,fruits and flowers of Olive were detected by q RT-PCR using β-actin as internal reference gene.The results showed that the expressions of the four genes were different in all tissues,the expression level of Oe DXS1/Oe DXS2/Oe DXR1/Oe DXR2 gene in leaves was much higher than that in other tissues.The oleuropein in different tissues was extracted by microwaveassisted extraction and detected by HPLC.The results showed that the content of oleuropein in leaves was the highest,followed by fruits,and the content in other tissues was relatively short.The analysis of the correlation between the content of oleuropein and the relative expression of Oe DXS1/Oe DXS2/Oe DXR1/Oe DXR2 found that content of oleuropein and the expression of the four genes showed significant positive correlation,respectively.