Clonging And Expression Analysis Of Lipoxygenase(LOX) Of Vitis Vinifera L.cv.Cabernet Sauvignon

The full length sequences of two LOX genes,were isolated from Cabernet Sauvignon using reverse transcription PCR approaches,and were named VvLOX1 and VvLOX2 respectively.They were expressed in prokaryotic cell.The expression levels of VvLOX1 and VvLOX2 were investigated during berry development,upon treatment with wounding and methyl jasmonate;the change of fatty acid,and LOX enzymatic activity of Cabernet Sauvignon skin were studied.The main results were as follows:1.The grape LOX open reading frame was predicted and the primers were designed based on the grape genome(PN40024)and other registered plant LOX genes.The obtained open reading frame of VvLOX1 was 2,577 bp,encoding 859 amino acids;the open reading frame of VvLOX2 was 2,748 bp,encoding 916 amino acids.Sequence analysis showed that the encoded amino acids were highly similar to LOX proteins of other species.Phylogenetic tree analysis indicated that grape LOX1 belonged to 9-LOX,and LOX2 belonged to 13-LOX.2.Quantitative RT-PCR analysis showed that the expression levels of both VvLOX1 and VvLOX2 genes changed due to the mechanical wounding and MeJA treatment.The higher expression levels of VvLOX1 and VvLOX2 were expressed in flower,leaves,skin and root.3.The VvLOXl and VvLOX2 were successfully subcloned into pGEX-6P-1,and constructed their prokaryotic expression vectors pGEX-VvLOXl and pGEX-VvLOX2.They were transformed into E.coli BL21(DE3),and then induced by 1 mmol·L-1 IPTG for 6 hours.SDS-PAGE showed that recombinant proteins were 123 kD,129 kD respectively.Because the vector itself coding a protein of 26 kD,the obtained proteins of 97 kD,103 kD encoded by VvLOX1 and VvLOX2 respectively were in accordance with the predicted molecular weight of LOX1 and LOX2.The recombinant LOXs were efficiently expressed in E.coli BL21(DE3).4.The LOX activity assay showed that LOX activity increased and peaked at the 12th weeks after anthesis,3 hours after wounding and 24 hours after MeJA treatment.5.The study of fatty acid composition showed that 12 fatty acids were identified mainly in grape peels.Hexadecanoic acid and stearic acid were the major saturated fatty acids,linoleic acid and linolenic acid were the major unsaturated fatty acids.The contents of fatty acids increased during berry development,and reached the peak at 15 weeks after anthesis.There were two peaks for the changes of fatty acid after wounding;the contents of hexadecanoic acid,linoleic acid,linolenic acid,and stearic acid were higher than control level.The results showed that the contents of fatty acids were affected during berry development,upon treatment with wounding and MeJA treatments.