Isolation and Activity Analysis of Phytoene Synthase (ClPsy1) Gene Promoter of Canary-Yellow and Golden Flesh-Color Watermelon

Watermelon (Citrullus lanatus) is an economically important cucurbit crop. Its pulp is rich in antioxidant carotenoids, which confer a variety of flesh colors. ClPsy1 (Phytoene Synthase) is the rate-limiting enzyme for carotenoid synthesis; however, the promoter activity of ClPsy1 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 ClPsy1 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 ClPsy1 gene promoter activity, full-length and deletion fragments of the promoter were constructed and connected to a beta-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 ClPsy1 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 Cla97C10G196920 and ClPsy1 gene and found that Cla97C10G196920 (ClMYB21) showed the same expression trend with ClPsy1, which positively regulates carotenoid synthesis and metabolism.