The Role Of CmCAD2 And CmCAD5 In Aroma Synthesis During The Late Period Of Melon Fruit Development (Cucumis Melo Var. Makuwa Makino)

Oriental melon (Cucumis melo var. makuwa Makino) is an important resource of melon varieties in China, and it has a good taste and unique fragrance. The aroma is one of the important indicators of the qualities of melon, and alcohol dehydrogenase plays a key role in aroma synthesis. As so far, eighteen alcohol dehydrogenase genes of melon have been identified from Melonomics database, including five cinnamyl alcohol dehydrogenase genes (cinnamyl alcohol dehydrogenase, CAD, EC 1.1.1.195) named CmCAD1-CmCAD5, respectively. The preliminary study has found that the CmCAD2 and CmCAD5 may play an essential role in fruit ripening and aroma synthesis, so this experiment uses "Rainbow 7" as the test material to study the expression features of CmCAD2 and CmCAD5 during melon fruit development and in melon fruit disks, to analyze some characters of CmCAD2 and CmCAD5 proteins by the bioinformatics tools, to study the characteristics of the recombinant protein by building prokaryotic expression vector. The main results in this research are as follows:1. With the change of endogenous ethylene content in the process of climacteric during the late period of melon fruit development, the varieties of fruit quality indexes are in consistency with endogenous ethylene content:firmness declining, color being yellow, soluble solids content rising first then fall, gradually. With the increase of endogenous ethylene content, the expression of CmCAD2 and CmCAD5 also rises gradually, and maintains a high level. At the same time, lignification occurring in melon fruit shows the consistency with the change of CAD enzyme activity, and aroma compounds such as cinnamaldehyde. All observations above indicate that CmCAD2 and CmCAD5 may be involved in the development of melon fruit, and CAD may participate in the melon fruit lignification process and biosynthesis of aroma substances.2. Fruit disks experiments demonstrate that cinnamaldehyde can upregulate the expression of CmCAD2 and CmCAD5, and promote aldehydes to convert into the corresponding alcohols. Benzaldehyde can suppress CmCAD2 and CmCAD5 expression and CAD enzymatic activity, but the aldehydes conversion ratio doesn’t change obviously. These results reveal that cinnamaldehyde may promote the transcription of CADs to produce more corresponding proteins, which promote cinnamaldehyde to turn into cinnamyl alcohol. These results indicate CAD may participate in the biosynthesis of fruit aroma substances.3. The bioinformatics analysis exhibits that CmCAD2 and CmCAD5 of which protein molecular weight is about 39.33 kDa and 38.83 kDa, respectively, encode the proteins of 356 and 368 amino acids respectively. The two proteins contain disulfide bond, and neither have hydrophobic domains, trans-membrane domains and N-terminal signal peptides. The subcellular localization prediction shows that both of proteins are located in the cytoplasm. Blastp analysis on NCBI reveals that CmCAD2 and CmCAD5 have high identity with MnCADl(76%) and PtSAD(76%), respectively. The homology between CmCAD2 and CmCAD5 is only 51%. But the results of multiple sequence alignment show that both CmCAD2 and CmCAD5 have three conservative domain structures which exist in most of plants, as follows Znl binding site [GHEX(2)GX(5)GX(2)V], Zn2 binding site [GDX(11)-CX(2)CX(7)C],and NADPH binding domain (GLGGXG). CADs from plants can be divided into five types, the results show that CmCAD2 and CmCAD5 belong to the Class I and Class Ⅱ, respectively. Secondaiy and tertiary structure prediction display that both CmCAD2 and CmCAD5 are mainly based on coiled coil and contain Rossmann fold, they are all belong to mixed type protein. CmCAD2 and CmCAD5 severally exist in monomer form and dimer form.4. The results of prokaryotic expression indicate that quantities of CmCAD2 recombinant proteins dissolve in the supernatant, and it is soluble protein. The reasons of less CmCAD5 may be improper expression vector, inadequate expression conditions or incorrect trial operation. This paper only analyzes the characteristic of CmCAD2 recombinant protein, which optimum pH and temperature are 6 and 30℃, respectively. CmCAD2 recombinant protein also has different substrate specificities, and it has the highest affinity for cinnamaldehyde followed by coniferaldehyde and least for sinapaldehyde. It has the highest catalytic efficiency when uses coniferaldehyde as the substrate.