The Function Of CmADHs In Aroma Volatiles Synthesis Of Oriental Melon(Cucumis Melo Var.makuwa Makino)and Functional Identification Of CmADH4 And CmADH12

Oriental melon is one of the most important econmic crops in China,which is widely grown in Chian and east aAsian countries.The aroma of melon is loved by consumers but the aroma of different oriental melon is varieties depend on the varieties or clutives of oriental melon,which is so rich.Previous study shows that LOX pathway is one of the key pathways for aroma compounds synthesis in plants.As one of the key enzymes in LOX pathway,ADH plays an important role in the convent of straight or branch-chain alcohols/aldehydes.Along with the publication of melon genome sequence in 2012,a large numbers of ADH-like DNA fragments have been found,but their functions have not been validated.In this study,we compared the quality characteristics of three aroma types of oriental melon called "Caihong 7 hao","Cuibao"and"Caigua" and studied two CmADH genes named CmADH4 and CmADH12.Main results are as follows:1.Aroma Characteristics of three aromatic types of oriental melon,high-aromatic,Cai Hong 7 Hao",aromatic "cuibao" and non-aromatic" Caigua" was compared at their mature stage.And the incubation experiment of "Cuibao" using ethanol,acetaldehyde and 4MP inhibitor was performed.we obtained the following conclusion:the key aroma compounds account for the differents of three aroma types of melon are ethyl acetate,hexyl acetate,(E,Z)-3,6-nonadienol and 2-methyl-2-hexene-l-ol.CmADH1/2/3/6/12 are the highest expressed in "Caihong" and "cuibao" fruit and they also changed along with fruit ripening processing and may be the main gene of fruit aroma synthesis;CmADH1/4/7/12 have similar characteristics in the expression of "Caigua","Caigua" is the only melon that CmADH4 highly expressed,thus CmADH4 may play a major role in the formation of"Caigua" aroma characteristics.In addition,ethanol and 0.1 mM 4MP up-regulated the expression of CmADH4 and CmADH12 in orientlal melon "Cuibao",acetaldehyde treatment increased the expression of CmADH1,CmADH4 and CmADH12.The expression of other CmADHs gene family members were inhibited by exogenous treatment.The effect of ethanol on the inhibition of ADH activity was more significant than that of 4MP.The changes of CmADH3 and CmADH12 were similar to those of ethyl acetate and hexyl acetate after incubation,suggesting the relationship between CmADH genes and the yield of aroma compounds.2.The CmADH4 and CmADH12 ORF without stop condon were cloned and the nucleotide length of CmADH4 was 1146 bp,and the nucleotide length of CmADH12 was 1908 bp.There is a substatution mutation between the sequence of CmADH4 and melon genome sequence on the site 1159 th at the base T replacement for C originally,but the amino acid sequence does not change after translation,which may be related to codon preference of different plants.Nucleotide sequence and amino acid sequence of CmADH12 is completely consist with and melon genomic sequence.The bioinformatics analysis of the reliability and translation of protein of the two genes were predicted to related their features and functions,the results show that CmADH4 and CmADH12 have medium-chain alcohol dehydrogenase(MDR)structure,CmADH12 also has a short-chain alcohol dehydrogenase(SDR)structure.Neither of them is secreted proteins or membrane proteins.3.The CmADH4 and CmADH12 ORF without stop condon were constructed to the prokaryotic expression vector pET30a,the Escherichia coli BL21(DE3)expression system was used to get the target protein.The target protein was purified by Ni affinity column chromatography and the purified recombinant protein was used to measure the enzyme activity of CmADH4 recombinant protein with different kinds of aldehydes as substrate,temperature and pH gradient.The results showed that CmADH4 recombinant protein was successfully induced and purified.The protein is about 41KD in size as forecast the optimum reaction temperature is 30 C and the optimum pH is 6 for recombinant CmADH4 protein.It has higher catalytic ability of aliphatic aldehyde aldehyde and hexanal,low affinity of branched aldehydes and aromatic aldehydes.4.The complete CmADH4 and CmADH12 were constructed to the expression vector pCAMBIA3301-LUC.EHA105 was ultimately used to inject oriental melon fruit flesh above the equator after screening different Agrobacterium and injection conditions.The results showed that CmADH4 and CmADH12 were transiently expressed in melon fruits,which significantly increased the abundance of CmADH4 and CmADH12 The over expression of CmADH4 can significantly improve the total ADH enzyme in melon flesh accompanied by the excess accumulation of ethyl acetate,hexyl acetate and(E,Z)-3,6-nonadien-1-ol content in melon injected.The content of acetaldehyde has the biggest change,to 5 times of control.