Figure 4.

Functional validation of two genotypes of GhRPRS1 in cotton. (a) The WMV067‐AADA vector was employed for overexpressing the GhRPRS1 in ZM24. (b) Two genotypes of GhRPRS1 cloned from white petal and red petal were overexpressed in ZM24. Lines RPRS1‐5 and RPRS1‐6 were generated by overexpression GhRPRS1 from white petal, while RPRS1‐9 and RPRS1‐10 were generated by the overexpression of GhRPRS1 from red petal. ZM24 served as controls for white petals. (c) The measurement of anthocyanin content in GhRPRS1 overexpression lines. (d–f) The expression levels of GhRPRS1 in overexpression lines were detected by qPCR and semi‐quantitative PCR. GhActin served as an internal control and western blotting of GhRPRS1 in transgene lines. (g) WMVC016 was utilized for gene editing of GhRPRS1 in ZYH. Three mutants with base deletion were observed at the target position. rprs1‐10, rprs1‐4, and rprs‐2 were the knockout lines in ZYH. (h) The phenotype of three GhRPRS1 knockout lines in ZYH, ZYH served as controls for red petals. (i) The measurement of anthocyanin content in GhRPRS1 knockout lines. (j) Comparison of petal colours between ZM24 and ZYH. A total of 13 stages were photographed from little bud to flowering. (k) Expression level of the GhRPRS1 in 13 stages as shown in panel j. (l) Comparison of petal colours before and after flowering between ZM24 and ZYH. (m, n) Comparison of expression level of GhRPRS1 between ZM24 and ZYH in four stages as showed in panel (k). Data were analysed using GraphPad Prism (v8.0.2, GraphPad Software, United States) software. Statistical testing was applied using a Student's t‐test, statistical significance is defined as P < 0.05.