Molecular Identification And Enzymatic Properties Of Laccase?in Diamondback Moth Plutella Xylostella

Laccase?EC1.10.3.2?is a kind of glycoprotein in the copper oxidase family.It uses molecular oxygen to oxidize a variety of aromatic and non-aromatic compounds.Laccase exists widely in fungi,bacteria,animal,according to the difference of the physiological effects of laccase can be divided into type I?Lac1?and type II?Lac2?.Lac1 plays an important physiological role in detoxification or ion metabolism in insects,and Lac2 plays an important role in the process of insect cuticle melanization.The type II gene cDNA from laccase of Plutella xylostella 3 instar larvae by PC R and RAC E?Px Lac2?cDNA sequence and molecular biology analysis;Determination of Px Lac2 in different stages and instars of the relative expression by using fluorescence quantitative PCR;from the diamondback moth pupa skin crude extract of laccase the basic solution,and enzymatic properties of laccase research.The results of this study laid the foundation for revealing the biological function of Lac2.The results are as follows:1.Full length Px Lac2 sequence of 1944bp contains an open reading frame?ORF?of1794bp,a 89bp 5'non encoding region?5'UTR?and a 61bp 3'non encoding region?3'UTR?,its deduced amino acid sequence with the amino acid methionine as the starting,the length of597 amino acid,molecular weight of 66.09kDa and isoelectric point of p I was 5.23.The half-life of Lac2 was 30h,and the coefficient of instability was 32.52.The chemical formula is C2940H4589N789O887S28.2.Through homology modeling,we established a model of the structure of the three level.After the analysis of amino acid sequence,we found that there were about three copper binding domains,which were located in 67-177,183-325,452-555 amino acids.This amino acid sequence contains the uniq ue C-X-R-X-C region of insect laccase.The presence of shear sites in 21 amino acids of 1²0 amino acids that signal peptide,has no transmembrane domain,alpha helix and random coil accounted for 14.41%,accounting for 59.63%,which is the maximum structural element for Px Lac2,extension chain accounted for 25.96%.3.The amino acid sequence of Plutella xylostella was compared with the sequence of the21 kinds of insect laccase.The results showed that the sequence of HWHG?X?9DG?X?5QCPI was found in the sequences of laccase sequences.Px Lac2 and Amyelois transitella Lac1?XP₀13194946?,Papilio Polytes Lac2?GenBank No.AB531135?,Papilio xuthus Lac2?GenBank No.AB499123?,Papilio machaon Lac2?GenBank No.AB531133?affinity closer,and amino acid homology were 61%,54%,56%,63%.4.Px Lac2 in the adult stage of relatively low expression,is set to 1.0,in the 3 instar larvae and pupae was higher,respectively 3.41 times and 4.84 times of adult;expression in 4instar larvae,pupa and adult stages of the re latively low level of expression of the 4 instar and pupa was 0.7 times higher than that of adult and 1.27 times.5.The characteristics of laccase enzyme analysis showed that the optimum temperature of laccase was Vm,and the optimum p H was 3.0.Under this condition,the Michaelis Menten constant K m was 0.97mM,and the maximum reaction rate was 56.82U/mL.When the final concentration of Cu²⁺was lower than 0.8mM,the activity of laccase was activated,which was higher than 0.8mM,and the inhibitory effect(IC₅₀)was 1.26mM.The activation energy?Ea?of laccase catalyzed ABTS oxidation was 17.36KJ/mol.