Cloning of PRE6-like gene and analysis of its function in anthocyanin synthesis in apple

Abstract：【Objective】The PREs gene, also known as the polyoxazole resistance gene, belongs to the basic/helix-loop-helix (bHLH) transcription factor family. It lacks a DNA binding domain and typically forms homodimers or heterodimers to regulate the expression of target genes, thereby influencing plant morphology, cell size, pigment metabolism and response to abiotic stress. This study aimed to verify the function of the atypical member of the polyoxazole resistance protein gene (PRE6-like) in apple and explore its regulatory role in anthocyanin biosynthesis.【Methods】The Oregon Spur Ⅱ, which is highly prone to bud mutation, exhibits significantly improved fruit color compared to the original variety. A new mutant strain of the Oregon Spur Ⅱ apple was discovered, which exhibits early and intense coloring, with a reddish fruit surface at maturity, and stable variation traits. Based on transcriptome sequencing of the skin of fruits on the bud mutation branch from the Oregon Spur Ⅱ apple, a flower pigment synthesis-related regulatory gene, MdPRE6-like was identified. The coding sequence (CDS) of the MdPRE6-like gene (MD14G1197600) was obtained from the apple genome website, and primers were designed using Primer 5.0 to amplify the cDNA from Oregon Spur Ⅱ the skin tissue of fruits on the bud mutation branch. Bioinformatics analysis software was used for biological information analysis, and tis-sue- specific expression was analyzed using the fluorescence quantitative PCR. The CDS region was cloned and ligated into the linearized expression vector pCAMBIA1301- GFP. Escherichia coli DH5α was heat-shock transformed, and Agrobacterium tumefaciens GV3101 was transformed to construct the overexpression vector. Transient expression was performed in Golden Delicious apple fruit, and the color changes of the skin under intensive light conditions (12 000 lx) were observed to preliminarily identify the gene’s role in anthocyanin synthesis. Further functional validation was conducted through Agrobacterium-mediated transformation of apple callus and by the floral dip method in Arabidopsis.【Results】The MdPRE6-like is located on chromosome 14, with an open reading frame of 279 bp, encoding 92 amino acids. The molecular formula is C444H745N137O149S2, with a relative molecular weight of 10 450.75 Da and a theoretical isoelectric point (pI) of 6.41. The total hydrophobicity is -0.654, which is a hydrophilic protein with a lipid solubility index of 97.50 and an instability index of 85.86. It belongs to an unstable protein, and its secondary structure shows that the protein α spiral structure accounts for 66.30%, irregular curls account for 32.61%, and extended chains account for 1.09%, so there are three α-The atypical bHLH proteins composed of a spiral structure, which is consistent with the protein’s tertiary structure. The subcellular localization prediction results of MdPRE6-like protein indicated that it may be localized in the nucleus. Multiple sequence alignment of its conserved domains with similar species revealed that the MdPRE6-like protein was an atypical bHLH protein containing an HLH domain. Organizational specific expression analysis showed that the expression level of MdPRE6-like gene in flowers and fruit peel was significantly higher than that in roots and leaves, indicating that this gene had a certain impact on the growth and development of apple fruit. Instantaneous expression of the Golden Delicious apple showed that MdPRE6-like significantly promoted the accumulation of anthocyanins at the injection site of the Golden Delicious apple peel, and increased the expression level of structural genes related to the anthocyanin synthesis pathway. The UFGT gene, also known as the 3-O-flavonoid glucosyltransferase gene, was significantly different from the control (p≤0.001); The overexpression of MdPRE6-like gene in apple callus and Arabidopsis thaliana revealed a significant increase in anthocyanin content in transgenic apple callus compared with the control. The expression level of MdPRE6-like gene was 5.22 times higher than that of the control, and the relative expression level of structural genes in the anthocyanin synthesis pathway increased. The differences in CHI, DFR and F3H compared to the control were extremely significant (p＜0.001), being 5.22, 18.40 and 8.96 times higher, respectively. The accumulation effect of total anthocyanin content in the leaf veins of transgenic Arabidopsis was significant, and the expression level of MdPRE6-like gene in OE1 and OE3 strains was 9.22 and 7.14 times higher than the control, with extremely significant differences compared to the control.【Conclusion】 MdPRE6-like gene can positively regulate the synthesis and accumulation of anthocyanins and promote the expression levels of structural genes in the anthocyanin synthesis pathway. Therefore, the results can provide theoretical reference for the MdPRE6-like gene to participate in improving the quality of apple fruit in the future.