Preliminary Functional Analysis Of Cucumber Lipoxygenase CsLOX2 Involved In The Synthesis Of C9 Volatiles

Lipoxygenase?LOX?is a group of non-heme,iron-containing enzymes which is ubiquitous in plants and animals and can catalyze o xygenation reaction.Previous studies have shown that C6 and C9.volatile compounds are the main aroma substance in cucumber fruit,among which C9 aldehydes are the most important contricutors to consumer liking of fresh cucumber.There are 12 LOXs fruit-specific expression genes in cucumber which have different function.While,it is not clear that which one is involved in the production of C9volatiles.We preliminarily studied the role of CsLOX2 in C9 production by determinating aroma composition and expression pattern of CsLOXs during fruit development.Conclusions we obtained are as follows:1.C6,C9 aldehydes and corresponding alcohols are the main aroma compounds during fruit development.The C6 compounds content was relatively high at the early stage but decreased during the ripening.The C9 compounds content which was significantly higher than that of C6 showed the trend of first rise and then decrease.The C9 compounds are the main aroma volatiles of cucumber characteristic flavor.2.There were different expression patterns of CsLOXs in the two varieties by semiquantitative and real-time PCR amplilification assay during fruit development.Analyzing the relationship of C9 volatiles and genes expression with heatmap and PC A,we found that CsLOX2 has the strongest correlation with the production of C9 compounds.CsLOX2 may be involved in the formation of C9 volatiles.3.Overexpressionvectorp B₂HA-CsLOX2andRNAinterferencevector pART27-CsLOX2RNAi were obtained via Gateway molecular cloning technology and“restriction enzymes-ligase”technology,respectively.And in cucumber,we got 13overexpression transgenetic plants and 17 interference transgenetic budsby PCR identified and selection of Basta preliminarily.4.We got the CsLOX2 recombinant protein which is about 84 k Da after constructing the prokaryotic expression vector 32a-CsLOX2.