Cloning And Characterization Of The Flavonoid And Lignin Synthesis Related Gene 4CL In Olive.

Olea europaea L.belongs to the Olea linae evergreen tree of Olea ceae.The olive oil extracted from the fruit is an edible oil with potential medicinal functions and is rich in biologically active ingredients such as carotene,phospholipids and flavonoids.Flavonoids have antioxidant,bacteriostatic,anti-inflammatory,anti-ultraviolet radiation and other functions.Lignin not only enhances the support strength of plants,but also improves the tolerance of plants to pathogen infection.4-coumarate:Co A ligase(4CL),as a key enzyme regulating the biosynthesis of flavonoids and lignin,has been studied in many plants,but the specific functions of the 4CL gene family have not been reported domestic and overseas in olive.Therefore,in this study,the 4CL gene family was cloned from olive tree and expressed in E.coli.Enzyme kinetics analysis of 4CL fusion proteins.At the same time,the expression of Oe4CL1 gene was correlated with lignin contents and flavonoid contents in olive roots,stems,leaves and fruits.Determine the metabolic pathway regulated by 4CL gene and lay the foundation for further study on the biosynthesis of lignin and flavonoid.The main results are as follows:(1)Four genes of Oe4CL1,Oe4CL2,Oe4CL3 and Oe4CL4 were cloned from olive by RT-PCR.The ORF were 1629 bp,1572 bp,1560 bp and 2400 bp,Separately.Four Oe4 CLs were displayed by bioinformatics analysis.The amino acid number of the proteins range from519 to 799,the relative molecular mass is between 56-90 k Da,Oe4CL1,Oe4CL3 and Oe4CL4 are acidic proteins,and Oe4CL2 is a weakly basic protein.Except that Oe4CL3 is an unstable protein,the other three proteins are stable.None of the four-entry proteins have a signal peptide and transmembrane domain.Multiple sequence alignment of 4CL homologous proteins revealed that only the Oe4CL1 gene has the family’s strictly conserved BOX I and BOX II.The other three genes have highly conserved sequences that are highly similar.BOX I is an AMP binding region,and BOX II is related to protein stability and catalytic activity.Therefore,the Oe4CL1 protein is predicted to have a catalytically active function.Phylogenetic tree analysis showed that the Oe4CL1 gene belongs to class I.It is presumed that Oe4 CL may regulate the accumulation of lignin in olive.The other three genes are not class I,class II and class III,but close to 4CL Phylogenetic relationship.(2)Two prokaryotic expression vectors pET30b-Oe4CL1 and pET30b-Oe4CL2 of the target gene were constructed and successfully expressed in the host strain BL21(DE3).The concentration of IPTG was determined to be 0.2 mmol/L by optimizing the expression conditions.Incubate at 16℃ for 16 h.After separating and purifying the protein.The target protein consistence were measured to be 0.5888 mg/m L and 1.4552 mg/m L,respectively.Using ferulic acid,p-coumaric acid,caffeic acid,cinnamic acid and sinapic acid as substrates,the enzyme kinetics of the two proteins were studied.The consequent manifest that ferulic acid was the optimal substrate for Oe4CL1 fusion protein;Oe4CL2 fusion protein had no catalytic activity for all five substrates.In olive,ferulic acid is a reactant of an important precursor of lignin monomer,indicating that Oe4CL1 is likely to control the biosynthetic pathway of lignin monomer.(3)Using qRT-PCR technology,the relative expression of Oe4CL1 gene in root,stem,leaf and fruit of olive was measured.The consequence showed that the expression level of Oe4CL1 gene had tissue specificity.The contents of flavonoid and lignin in the corresponding tissues was also determined.The correlation between Oe4CL1 gene expression level and flavonoid as well as lignin contents was analyzed.The study reviews that the expression level of Oe4CL1 gene was significantly correlated with lignin contents,and it was a key enzyme gene for controlling the biosynthesis pathway of lignin monomer.