山楂 CpPG 基因家族鉴定及表达模式分析

Identification and expression analysis of hawthorn CpPG gene family

摘要

【目的】不同山楂品种间质地差异显著，但其调控果实质地变化的分子基础尚不清晰。本研究旨在以质地差异显著的山楂品种为材料，全基因组鉴定果实软化的关键因子多聚半乳糖醛酸酶（PG）基因家族成员，探究其在山楂果实软化和品种间质地分化过程中的表达特异性，寻找与山楂果实硬度相关的的候选 PG 基因，以期为解析山楂果实质地的调控机制奠定前期理论基础，也为后期通过分子设计育种改良山楂果实品质提供候选基因资源。【方法】基于山楂基因组数据鉴定 PG 基因家族成员，分析其理化性质、亚细胞定位、系统发育、基因结构、保守基序、启动子顺式作用元件及转录因子结合位点，解析染色体分布、基因复制类型、选择压力及跨物种共线性；测定‘金如意’和‘豫北红’果实发育期的硬度和果胶含量；通过 qRT-PCR 分析 CpPG 表达模式，并进行基因表达量与表型的相关性分析。【结果】（1）在山楂基因组中共鉴定到 68 个 CpPG 成员，理化性质差异较大；且约 44% 定位于细胞核中。（2）系统发育分析将其划分为 6 个亚族，各亚族成员间基因结构较为保守，所有 CpPG 均包含 Glyco_hydro_28 催化结构域和 5 个保守 Motif。（3）启动子顺式作用元件分析显示， CpPG 启动子区域富含激素响应与低温响应相关元件。（4）基因复制分析表明，全基因组复制与分散复制是 CpPG 家族扩张的主要方式，纯化选择是其进化保守的主要驱动力。（5）两个果实硬度差异的品种‘金如意’和‘豫北红’在果实软化过程中果实硬度均逐渐降低，总果胶含量均呈先升后降趋势，而水溶性果胶含量随成熟度持续上升。（6）qRT-PCR 结果表明， CpPG4 在果实发育后期特异性高表达的 G1 组，且在‘金如意’中的表达量极显著高于‘豫北红’。（7）仅 CpPG4 表达量与果实硬度呈极显著负相关，与水溶性果胶含量呈极显著正相关，推测该基因在调控山楂果实软化和品种间质地分化过程中发挥作用。【结论】山楂基因组中鉴定出 68 个 CpPG 基因，启动子普遍富含低温及激素响应元件，家族扩张主要依赖分散复制和全基因组复制，纯化选择维持进化保守性。 CpPG4 通过促进果胶溶解驱动果实软化，其品种间差异表达是果实质地分化的关键分子基础。本研究为解析山楂果实质地形成的分子机制提供了理论依据，也为通过分子育种改良果实硬度提供了关键基因资源。

Abstract

[Objective] Significant differences in fruit texture exist among different hawthorn varieties, yet the molecular basis underlying this variation remains unclear. This study aims to identify members of the polygalacturonase (PG) gene family, a key factor in fruit softening, through a genome-wide analysis using hawthorn varieties with contrasting fruit texture, to investigate their expression specificity during fruit softening and varietal texture differentiation, and to screen for candidate PG genes related to hawthorn fruit firmness. The findings will provide a theoretical foundation for deciphering the regulatory mechanisms of hawthorn fruit texture and offer candidate gene resources for future molecular design breeding to improve hawthorn fruit quality. [Method] Based on hawthorn genomic data, PG gene family members were identified, and their physicochemical properties, subcellular localization, phylogeny, gene structure, conserved Motifs, cis-acting elements in promoters, and transcription factor binding sites were analyzed. The chromosomal distribution, gene duplication types, selective pressure, and interspecies collinearity were also resolved. The fruit firmness and pectin content of ‘Jinruyi’ and ‘Yubeihong’ during fruit development were measured. CpPG expression patterns were analyzed by qRT-PCR, and correlation analysis between gene expression levels and phenotypic data was performed. [Result] (1) In the hawthorn genome, a total of 68 CpPG genes were identified. Their physicochemical properties varied considerably, and approximately 44% of the CpPG proteins were predicted to be localized in the nucleus. (2) Phylogenetic analysis divided them into six subfamilies. The gene structure of members within each subfamily was relatively conserved, and all CpPG contained the Glyco_hydro_28 catalytic domain and five conserved Motifs. (3) Analysis of cis-acting elements in the promoters revealed that the promoter regions of CpPG are enriched in hormone-responsive and low-temperature-responsive elements. (4) Gene duplication analysis indicated that whole-genome duplication and dispersed duplication are the main modes of CpPG family expansion, and purifying selection is the major driving force for its evolutionary conservation. (5) In the two varieties with contrasting fruit firmness, ‘Jinruyi’ and ‘Yubeihong’, fruit firmness gradually decreased during fruit softening. Total pectin content showed a trend of first increasing and then decreasing, while water-soluble pectin content continuously increased with ripening. (6) qRT-PCR results showed that CpPG4 was specifically highly expressed in the G1 group during the late stages of fruit development, and its expression level in ‘Jinruyi’ was extremely significantly higher than that in ‘Yubeihong’. (7) Only the expression level of CpPG4 was extremely significantly negatively correlated with fruit firmness and extremely significantly positively correlated with water-soluble pectin content, indicating that this gene functions in the regulation of fruit softening and texture divergence among hawthorn varieties. [Conclusion] A total of 68 CpPG genes were identified in the hawthorn genome. Their promoters are generally enriched in low-temperature-and hormone-responsive elements. The family expansion mainly relies on dispersed duplication and whole-genome duplication, and purifying selection maintains its evolutionary conservation. CpPG4 drives fruit softening by promoting pectin solubilization, and its differential expression between varieties is a key molecular basis for fruit texture differentiation. This study provides a theoretical basis for deciphering the molecular mechanism of fruit texture formation in hawthorn and offers key gene resources for improving fruit firmness through molecular breeding.

关键词

山楂 / 多聚半乳糖醛酸酶（PG） / 基因家族 / 果实质地 / 软化机制 / 表达模式 / 分子育种

Key words

Crataegus pinnatifida / polygalacturonase (PG) / gene family / fruit texture / softening mechanism / expression pattern / molecular breeding

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基金资助

中国林科院中央公益性基本科研业务费专项(CAFYBB2024MA005)

科技部科技基础资源调查专项(2024FY100502-9)