Identification And Characterization Of A Herbivore-induced Lipoxygenase Gene In The Leaves Of Camellia Sinensis Fed By Ectropis Obliqua

The jasmonic acid（JA）is important signal molecule fed by insects, lipoxygenase is the first key enzyme synthesised JA, so this enzyme is concerned for a long time, but the research of lipoxygenase is little in tea plant（Camellia Sinensis）. This paper got a EST of LOX（GenBank：GW342753）, so we plan to use RACE, prokaryotic expression, HPLC, qRT-PCR to research the functional characterization and the gene expression pattern of LOX in tea leaves.（1）We obtained full-length cDNA of CsLOX3by RACE, the Open reading frame（ORF）of lox3was2706bp in length, encoding901amino acids, we named it CsLOX3（GenBank：HM440161）, we calculated the molecular mass and pI were101.84kDa and6.39. the amino acid sequence homology of CsLOX2and CsLOX3was80%by comparison, CsLOX3was located in the chloroplast for having a chloroplast transit peptide, we also found the key amino acid residues of His₅₅₄,His₅₅₉, His₇₅₁,Asn755, Ile₉₀₁ which integrated with Fe3+in CsLOX3.（2）We known that the molecular mass of fusion protein was about120kD using prokaryotic expression and fusion protein purification by Co2+-TALON Resin and then we discovered the highest activity of CsLOX3at pH5.0(22.57mmol min^-1 mg^-1) and temperature of45°C(24.6mmol min^-1 mg^-1). At last we analyzed that the product type of CsLOX3activity using linolenic acid was13-HPOT by HPLC, so we clasiffied CsLOX3as13-LOX by its product type.（3）The results of qRT-PCR shown us that the CsLOX3was micro expression in the beginning period of tea plant flowering and tea fruit formation, but the gene expression of CsLOX3was increased to the maximum in mature stage of tea flowering and tea fruit formation, the mature stage was respectively15.3times and16times than the beginning period of tea plant flowering and tea fruit formation CsLOX3has been in a state of regulated expression during6-24h after feeding by Ectropis obliqua, but the expression pattern was different in different tea variety, upregulated expression of the start time and the time of maximum amount of shuchazao are later than longjing43, but the gene expression of them was similar, the attacked was higher10times and13.6times than the control the expression pattern was similar with the feeding of tea geometrid and wounding, but the expression pattern was different in different tea variety. the gene expression of CsLOX3in shuchazao was significantly greater than longjing43, the wounded was respectively10.6times and3.6times than the control the expression of CsLOX3rapidly increased to17.3times than the control after2days and then declined sharply after4days treated with MeJA, but the wounded had little effect on the Shuchazao, the maximum expression level was only2.1times than the control.