2.1. Transcriptome analyses
We obtained 28 days fruits of ‘M15’ and ‘Baogua’ after pollination (Fig. 1), and compared their gene expression levels using strand-specifific mRNA sequencing. The results showed that there were a total of 6817 differently expressed genes. Compared to ‘M15’, there were 4370 genes up-regulated and 2377 genes down-regulated in ‘Baogua’ (Fig. 2). We found that the expression levels of 12 MYB transcription factors were significantly different between the two varieties, including MELO3C013071.2, MELO3C012039.2, MELO3C012926.2, MELO3C009678.2, MELO3C010833.2, MELO3C015228.2, MELO3C035029.2, MELO3C017134.2, MELO3C021555.2, MELO3C028590.2, MELO3C020701.2, MELO3C006728.2.
The transcriptome sequencing results of ‘M15’ and ‘Baogua’ were analyzed for significant enrichment in the GO (gene ontology) database, including biological process, molecular function, and cellular component.The results show (Fig. 3) that the differently expressed genes of the two involve multiple metabolic pathways. The proportion of differently expressed genes involved in cell components is highest in the cell periphery, followed by the membrane, component of membrane, extracellular region, external encapsulating stracture, and cell wall also accounts for a higher proportion. Oxidoreductase activity accounts for the highest proportion in molecular functional classification, followed by catalytic activity and protein kinase activity. Differently expressed genes involved in biological processes are mainly concentrated in oxidation-reduction process, followed by response to stimulus, and also involved in aminoglycan catabolic process, and chitin metabolism Chitin metabolic process, chitin catabolic process, cell wall macromolecule catabolic process, amino sugar catabolic process, compound containing glucosamine Glucosamine-containing compound metabolic process, glucosamine-containing compound catabolic process, protein phosphorylation, and so on.
KEGG pathway enrichment analysis showed that differentially expressed genes are mainly involved in starch and sucrose metabolism, MAPK signaling pathway-Plant, amino sugar and nucleotide sugar metabolism (Amino sugar and nucleotide sugar metabolism) and phenylpropanoid biosynthesis(Fig. 4).
2.2. CmMYB1 protein subcellular localization analysis
Throughlaser confocal scanning microscopy, it was observed that CmMYB1 protein is distributed in the cytoplasm and nucleus . However, the green fluorescent signal of CmMYB1-GFP was mainly concentrated on the plasma membrane(Fig. 5). These results indicate that the CmMYB1 protein may be a transcription factor that plays a role in the nucleus and cytoplasm.
2.3. Phylogenetic analysis of CmMYB1
Phylogenetic relationships between CmMYB1 and MYB genes in other plants were explored by constructing an evolutionary tree of amino acid sequence using MEGA 5.1 software. The relationship between CmMYB1 and a gene from the Cucumis sativus (XP004147900.1) was the closest, with 98.19% homology. However, homology with Morella rubra was only 57.68%(Fig.6). This suggests that the MYB gene may have evolutionary differences.
2.4. Phenotypic observation of transgenic A. thaliana lines
The phenotypes of wild-type and transgenic A. thaliana showed no significant difference between the vegetative and reproductive growth phases (Fig. 7).
2.5. RT-PCR to detect the expression of CmMYB1 under salt stress
RT-PCR results showed that, compared with the control, 200 mM NaCl solution treated melon seedlings at 1 h and 3 h showed a significant increase in CmMYB1 expression and began to decline after 6 h. After 24 h, CmMYB1 expression was increased (Fig. 8), indicating that it functions in the early response to salt stress.