Objective The flesh of Prunus humilis has bright color, unique flavor and significant economic value. However, the molecular regulation mechanism of flesh color remains unclear. In this study, the yeast cDNA libraries of P. humilis flesh were constructed, and Y1H method was used to screen upstream regulators of the anthocyanin biosynthesis gene PhLDOX, aiming to lay a preliminary theoretical foundation for revealing the color regulation mechanism of P. humilis flesh, and provide important gene resources for future molecular breeding of P. humilis.

Method Flesh samples from red- and yellow-fleshed P. humilis at different developmental stages were used as materials. Yeast cDNA libraries were constructed via Gateway BP and LR recombination reactions. The expression pattern of PhLDOX and cis-acting elements in its promoter were analyzed using RT-qPCR and PlantCARE. The promoter sequence of PhLDOX was cloned and used to construct a bait vector. The regulatory factors were screened from the libraries by YIH and further identified in combination with transcriptome data.

Result The quality identification of the primary library revealed that the library capacity and library titer were 1.6 × 10⁷ CFU and 8.0 × 10⁶ CFU/mL, respectively. The secondary library had a capacity of 1.6 × 10⁷ CFU and a titer of 7.8 × 10⁶ CFU/mL, the recombination rate was 100%, and the average length of insert fragment as more than 1 000 bp. The expression pattern analysis showed that the expression level of PhLDOX gene in red flesh was higher than that in yellow flesh. Promoter sequence prediction showed that the PhLDOX gene promoter contained various light-responsive elements, hormone-responsive elements, and transcription factor binding elements. Y1H combined with expression pattern analysis demonstrated that three transcription factors, including AP2/ERF (Ph0214288) and WD40 (Ph0209298, Ph0209443), could bind to the PhLDOX gene promoter.

Conclusion In this study, high-quality yeast cDNA libraries of different colors flesh from P. humilis with were successfully constructed. Three regulatory factors, which may be involved in anthocyanin synthesis by regulating the expression of PhLDOX, were identified. The results provide an important theoretical basis and key candidate gene resources for further analysis of the molecular regulation mechanism of flesh coloration in different colors of P. humilis, and have important application value for subsequent molecular breeding and quality improvement of flesh color traits in P. humilis.