Analysis Of CmLOX18 Promoter And The Roles Of CmLOX18 In High Temperature And Synthesis Of Aroma Volatiles In The Melon

Oriental melon（Cucumis melo var.makuwa Makino）,which has a sweet and crisp taste,juicy flesh,intense volatile aromas,is popular with consumers and is one of the most important economic crops.Both yield and quality are the main impact factors on the economic benefits of the melon,which can be improved by means of improving the stress resistance of the plant and increasing synthesis of aroma volatiles in the fruit.LOX plays an important role in the stress resistance and synthesis of aromas volatiles,therefore the study of LOX genes can provide theoretical basis for improving stress resistance and fruit quality of oriental melon by gene engineering.The LOX genes family of melon have eighteen members from Cm LOX01 to CmLOX18,and Cm LOX18 is type II 13-LOX.In order to identify function of Cm LOX18 gene,the oriental melon cultivar‘Yumeiren’was used as experimental material to study;Cm LOX18 promoter were cloned and analysis for study the promoter activity in the different stresses;CmLOX18-silenced plants were obtained by VIGS for study the role of CmLOX18 in defense against high temperature stress;meanwhile Cm LOX18 overexpression vector was constructed and expressed transiently in the fruit at different times after anthesis for exploring the role of Cm LOX18 in volatile aromas synthesis.The main results are described as followings:1.The 1500bp fragment of CmLOX18 promoter,obtained using TA-cloning method from the DNA genome of oriental melon’s tender leaves,was named p18T-LOX18pro.A number of putative cis-acting elements of CmLOX18 promoter sequences were discovered using PlantCARE and PLACE database,containing multiple cis-elements and inducibled expression elements by adverse environments:abiotic stresses（stress,high temperature,low temperature,salt,drought）,hormones（GA,IAA）and signal substance（ABA）.To identify the role and pivotal region of the promoter in response to the stress,the five 5’deletion expression vectors of CmLOX18pro:pBI121 GUS were constructing and named PC1,PC2,PC3,PC4,PC5respectively.2.The tobacco（Nicotiana benthamiana）were used as experimental material,the recombination expression vectors of CmLOX18pro were transiently over-expressed in tobacco leaves using the method of leaf injection by agrobacterium GV3101.Then the leaves inoculated were treated by abiotic stress,hormone and signal substances.The results of GUS histochemical staining and fluorimetric assay show that-1140^-724bp（PC1-PC3）and-463^-261bp（PC4-PC5）of promoter sequences were the vital regions in response to mechanical injury,high temperature stress and low temperature stress;-463^-261bp（PC4-PC5）was the main region containing the crucial sequences that were responsive osmotic stress（salt and drought）;-1400^-1178bp（PC1-PC2）and-463^-261bp（PC4-PC5）were the core regions in where the promoter response to GA₃ and IAA;-1400^-1178bp（PC1-PC2）,-1178^-261bp（PC2-PC5）,-1400^-463bp（PC1-PC4）were also the useful regions in response to the signal substances MeJA,H₂O₂,ABA,respectively.3.The 2724bp ORF nucleotide sequences of Cm LOX18 was isolated using TA-cloning method from full-length cDNAs of oriental melon fruit,then the over-expression vector pCAMBIA3301-LUC-CmLOX18 was constructed with the In-Fusion HD Cloning method.Meanwhile,300bp nucleotide sequences of CmLOX18 conserved region were amplified using PCR and pTRV2-LOX18 was also constructed for TRV-based gene silencing.4.The TRV-based Cm LOX18 gene silencing plants were obtained using seed absorption:the GFP fluorescence protein was high level expression and transferred from bottom to top in the plant;meanwhile,Cm LOX18 gene expression was suppressed,which the transcriptional level decreased one third to half as much as the control TRV2-0.At the period of four leaves,the plants infected were treated with high temperature（42℃）,TRV2-LOX18 compared to the control TRV2-0,the genes expression level of CmLOX18 and CmHPL,Cm AOS that were the genes of LOX downstream pathways related stress resistance declined sharply,the electrolyte leakage and the MDA content enhanced obviously,and the proline content significantly decreased.The results that the stress resistance of Cm LOX18 gene silenced plants was weakened indicated Cm LOX18 was involved in the stress-resistance pathway of plants responding to high temperature stress and played an important role.5.CmLOX18 were successfully transiently over-expression in melon fruits above the equator near the pedicel at 15DPF/25DPF/32DPF using EHA105-based injecting fruit flesh.The results suggested that the melon fruit flesh injected were presented water spots and detected the expression of fluorescent protein LUC,which were the evidences of the fruit infected by EHA105;comparing to the control LUC-0,the LUC-LOX18 had high transcriptional level of CmLOX18 gene and enhaned enzyme activity of LOX;besides,the gene expression level of Cm HPL,CmADHs and CmAATs increased sharply,which played a important role in synthesis of volatile aromas.Among the three different fruit periods,CmLOX18 gene had the highest transient expression level at 32DAP,for the reason the fruit released much ethylene in 32DAP and LOX is positively regulated by ethylene.Above all,Cm LOX18 promoter play positive role in defense response,and had sharply response on high temperature stress,ABA stress and H₂O₂ stress;CmLOX18 play the important role in response to high temperature stress and synthesis of fruit aroma volatiles in oriental melon.