PcHY5甲基化调控‘红巴梨’和‘巴梨’的花青苷生物合成和转运

doi:10.1016/j.jia.2023.07.017

摘要/Abstract

摘要：

梨果皮的红色主要是由花青苷合成积累导致，以‘巴梨’（‘Bartlett’, BL）和‘红巴梨’（‘Max Red Bartlett’, MRB）为代表的芽变品种是研究梨果皮花青苷合成积累分子机制的理想材料。虽然早前的研究已通过遗传图谱定位了‘红巴梨’果皮色泽的数量性状基因座（QTL），但是决定色泽突变的关键基因及调控机制尚不明确。因此，本研究以‘巴梨’和‘红巴梨’为研究试材，通过对其果皮组织的转录组和DNA甲基化差异比较分析，发现‘红巴梨’的PcHY5 DNA甲基化水平低于‘巴梨’，且PcHY5基因的表达量高于‘巴梨’，由此推测PcHY5 DNA甲基化水平可能与‘巴梨’和‘红巴梨’果皮颜色差异有关，并利用双荧光素酶试验证实了PcHY5不仅能激活花青苷合成相关转录因子PcMYB10和PcMYB114，也能激活花青苷合成基因PcUFGT和转运基因PcGST，说明PcHY5不仅能调控花青苷的合成，还调控了花青苷的转运。进一步，对‘巴梨’和‘红巴梨’PcHY5的关键差异甲基化位点进行了分析，发现‘红巴梨’PcHY5内含子区域的低甲基化水平与果皮红色形成显著相关，而‘巴梨’同一位点的高甲基化水平与果皮绿色显著相关。因此，基于‘巴梨’和‘红巴梨’PcHY5基因差异表达和差异甲基化，结合基因的调控功能验证，推测‘红巴梨’PcHY5 通过DNA低甲基化水平促进其自身基因表达，并调控花青苷合成和转运相关基因的表达，从而促进果皮着色。

Abstract:

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’ (BL) and the red-skinned derivative ‘Max Red Bartlett’ (MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus (QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars; MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

Key words: pear , PcHY5 , DNA methylation , anthocyanin , biosynthesis and transport

WEI Wei-lin, JIANG Fu-dong, LIU Hai-nan, SUN Man-yi, LI Qing-yu, CHANG Wen-jing, LI Yuan-jun, LI Jia-ming, WU Jun. PcHY5甲基化调控‘红巴梨’和‘巴梨’的花青苷生物合成和转运[J]. Journal of Integrative Agriculture, 2023, 22(11): 3256-3268.

WEI Wei-lin, JIANG Fu-dong, LIU Hai-nan, SUN Man-yi, LI Qing-yu, CHANG Wen-jing, LI Yuan-jun, LI Jia-ming, WU Jun. The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett’ and ‘Bartlett’ pears[J]. Journal of Integrative Agriculture, 2023, 22(11): 3256-3268.

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