| Summary: | Watermelon (<i>Citrullus lanatus</i>) is an economically important cucurbit crop. Its pulp is rich in antioxidant carotenoids, which confer a variety of flesh colors. <i>ClPsy1</i> (Phytoene Synthase) is the rate-limiting enzyme for carotenoid synthesis; however, the promoter activity of <i>ClPsy1</i> is still unknown. In the present study, promoter sequences were isolated from four watermelon accessions: Cream of Saskatchewan pale yellow (COS), canary yellow flesh (PI 635597), golden flesh (PI 192938), and red flesh (LSW-177), all of which express <i>ClPsy1</i> at extremely high levels. Sequence alignment and cis-element analysis disclosed six SNPs between the four lines all in COS, two of which (at the 598th and 1257th positions) caused MYC and MYB cis-element binding sequence variations, respectively. To confirm <i>ClPsy1</i> gene promoter activity, full-length and deletion fragments of the promoter were constructed and connected to a β-D-glucosidase (GUS) vector and transferred into tomato fruits. GUS staining was performed to analyze the key segment of the promoter. The activity of the PI 192938 <i>ClPsy1</i> full-length promoter exceeded that of COS. The deletion fragment from −1521 bp to −1043 bp exhibited strong promoter activity, and contained a MYB transcription factor-binding site mutation. We combined RNA-seq with qRT-PCR to analyze the gene expression pattern between the MYB transcription factor <i>Cla97C10G196920</i> and <i>ClPsy1</i> gene and found that <i>Cla97C10G196920</i> (ClMYB21) showed the same expression trend with <i>ClPsy1</i>, which positively regulates carotenoid synthesis and metabolism.
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