Genome-Wide Identification And Analysis Of Valaciclovir Hydrolase And Stay Green Genes Family In Melon

Melon (Cucumis melo L.) is one of the top ten fruits in the world with great economic value, also a model plant of fruit development and mature research of the cucurbitaceae family. The whole genome of melon sequencing has been completed and published in June 2012, providing mass information for studying novel genes and their functions via bioinformatics methods. In this study, we identified the valacyclovir hydrolase (VACVase), stay green protein (staygreen protein, SGR) and NYC (non-yellow coloring) of the melon genome via bioinformatics methods, and performed bioinformatics, expression, subcellular localization and function analysis.1) We identified two VAC Vases in melon, and named CmVACVasel and CmVACVase2. Bioinformatics analysis showed that the two proteins were highly conserved and no signal peptide structures were found, instructing them to be non-secretory proteins. CmVACVase1 or CmVACVase2 has 4 and 3 trans-membrane structure, containing 24 and 15 phosphorylation sites respectively. Multiple hormone response elements, tissue-specific expression elements and spatio-temporal expression regulatory elements were found besides the core elements (CAAT-box?TATA-box?5'UTR Py-rich and so on) through promoter analysis. Two highly homologous proteins interacting with 10 and 8 proteins were found mapping to CmVCAVase1 and CmVCAVase2 respectively via analysis of protein-protein interaction network. Two miRNAs were found for each gene, and one miRNAdegraded the target mRNA via complementary forms, and the othermiRNA couldinhibit the expression of the target genesthroughrepressingthe translation of the target mRNAs.We identified one NYC and four SGR proteins from the melon genome via bioinformatics methods. SGR is the homologous gene of NYC, and the genes were named CmNYC and CmSGR1?CmSGR4 respectively. A conservative motif (G-X-S-X-G/A) of esterase/lipase family was found in CmNYC, dictating it to be a weak acidic and instable protein. It has no chloroplast transit peptides structure in N-terminal, and may be located in Chloroplast liposome. It has 2 trans-membrane structures which belong to transmembrane proteins and 27 phosphorylation sites, instructing the catalytic reaction or signal transduction functions in chloroplast membrane. As a homologous gene family of NYC, SGR owns the same functions. The SGR gene family contains a highly conserved motif (C-X3-C-X-C2-F-P-X5-P) in C-terminal except for CmSGR3 and CmSGR4. SGRs have no structure of chloroplast transit peptides or transmembrane structures2) In this article, we cloned the cDNA of CmVACVase1, CmVACVase2, CmNYC, CmSGR1 and CmSGR2 from the melon genome, encoding 458,398,499,216 and 257 amino acids respectively, with molecular weight between 24.8 and 55.4 kDa.3) RT-qPCR results showed that CmVACVase1 and CmVACVase2 gave the highest expression levels in fruit 45 DAP, which were 355 and 2.6-fold respectively compared to those of leaf.Their expression levels were very low in early stages of fruit development, indicating them to be inducible expressed genes during fruit developing process. CmNYC showed the highest expression in leaf and cotyledon. The expression of it in fruit was much lower, but slowly increased along with the development of the fruit. The expression level of CmSGR1 and CmSGR2 were the highest in case of 45 DAP, and became even higher during the developing process. The expression levels of CmSGR3 and CmSGR4 in leaf, stem and cotyledon were high, but started to drop after fruit pollination. However they increased a little at 30 DAP.4) CmVACVase1 and CmVACVase2 subcellular localization vectors were constructed and named as pUC18-CmVACVase1-GFP and pUC18-CmVACVase2-GFP. The results of onion epidermal cell transformation showed that CmVACVase1 and CmVACVase2 were located at the cell membrane, dictating them to be membrane proteins, consistent with the formal predictions.5) The result showed that the fruits all presented staygreen phenotype after injections of overexpression vector RNAi vector. The expression levels of p35S-VACVasel and p35S-VACVase2 were 2.21 and 2.25-fold higher than those of control group.Xyloglucan endotransglucosylase/hydrolase (XTH) gene expression level was 8.1-fold higher than that of control group in case of CmVACVase1 overexpression. Expansion (EXP) and phytoene synthase (PSY) gene expression level were 1.7 and 1.5-fold higher than that of control group in case of CmVACVase2 overexpression.ACC oxidase (ACO) and ACC synthetase (ACS) gene expression level was 3.2 and.2.2-fold higher than that of control group in case of CmVACVasel RNAi. Expressions of other associated gene were significantly inhibited. The expression levels of beta-galactosidase (?-gal) and EXP were 51 and 23-fold higher than those of control group in p35-VACVase2 injection.The fruits presented yellow phenotype after the injection of overexpression vector p35S-NYC, p35S-SGR1 and p35S-SGR2, chlorophyll degradating obviously, while the fruits injected with RNAi vectors pART27-NYC, pART27-SGRl and pART27-SGR2 showed the stay green phenotype.In this study, CmVACVasel, CmVACVase2, CmNYC, CmSGR1 and CmSGR2 genes were first identified via bioinformatics methods and their cDNA were cloned in melon. Their structure, evolution relationships, motifs and distribution among family members were analyzed via bioinformatics. Their functions have been studied via transient expression. The results proved useful material for further research of their function mechanism, shedding light on the insights into the catalysis functions of CmVACVase1 and CmVACVase2 in plants, providing basal data for further study of the functions of this gene family. The results laid the foundation for further understanding the molecular mechanism of CmNYC/CmSGRs in chlorophyll degradation, aging and ripening.