水稻（Oryza sativa L.）是全球重要的粮食作物，其品质改良对确保粮食供应稳定及适应消费需求升级具有关键作用。稻米外观品质是决定市场价值与消费者选择的首要指标，而垩白（胚乳中白色不透明区域）作为核心表型特征，直接影响稻米碾磨品质、蒸煮特性及商品价值。垩白形成受遗传调控与环境因子互作的共同影响，其分子机制的解析是突破水稻优质育种的瓶颈问题。目前研究表明，籽粒灌浆缺陷和胚乳贮藏物质代谢异常是垩白形成的主要原因，但关键调控网络仍有待完善。

目前研究报道籽粒灌浆和胚乳贮藏物质缺陷是造成垩白形成的主要原因。本研究从 9311 和 高垩白材料 PA64S 构建的染色体片段置换系（CSSL）群体中筛选出的高垩白家系，将其命名为 C51 染色体片段置换系（Chromosome Segment Substitution Line, CSSL），旨在解析其高垩白表型的遗传基础，明确 7 号染色体主效数量性状位点（Quantitative trait locus, QTL）qPGWC-7 的候选基因，为垩白形成的分子机制研究提供新见解。研究结果如下：

连续多年多点试验表明，C51 的垩白粒率和腹白粒率均显著高于 9311，且受低温和低氮条件显著诱导。对 9311 与 C51 连续发育时期胚乳横切面观察发现 C51 在开花后（Day After Flowering, DAF）18 天出现腹白，且扫描电镜观察发现，在 15 DAF 时 C51 胚乳腹白区域淀粉颗粒开始出现呈不规则椭圆形，排布疏松且表面黏附物增多，与 9311 致密六面体结构形成鲜明对比。

2. 发育中胚乳细胞学观察发现 C51 腹部淀粉颗粒在胚乳发育早期（6-12 DAF）合成速率减缓，直径减小，淀粉沉积面积占比下降。生理指标测定发现 C51 成熟籽粒总淀粉含量显著低于 9311，直链淀粉与支链淀粉比例失衡，快速黏度分析（Rapid Visco Analyser, RVA）谱特征值（峰值粘度、崩解值等）显著降低。表达水平分析进一步揭示 C51 中淀粉合成关键基因在胚乳发育过程中显著下调。

3. 在 qPGWC-7 定位的 7 号染色体 44 kb 区间内，通过一代测序、基因注释以及表达模式分析筛选出 6 个候选基因并构建基因组互补转基因材料。多世代表型分析表明，ORF4（Open Reading Frame 4） 转基因家系（T1-T3代）垩白粒率和腹白粒率与 C51 无显著差异，且低温低氮环境下表型稳定性增强。扫描电镜显示 ORF4 转基因家系成熟籽粒胚乳腹白区域淀粉颗粒呈不规则椭圆形，排布疏松且表面黏附物增多，与 C51 表型高度一致。ORF4 启动子区测序发现，9311 与 C51 启动子区域具有多个单核苷酸多样性（Single Nucleotide Polymorphism, SNP）与 插入缺失（Insertion-Deletion, InDel）位点，导致多个转录结合元件的差异。ORF4 表达差异分析显示 C51中 ORF4 表达量在 18 DAF 胚乳中显著高于 9311。

4. 亚细胞定位表明 ORF4 编码线粒体定位蛋白，系统发育分析表示其在双子叶与单子叶植物中具有高度保守性。转录组分析显示，C51 胚乳中淀粉与蔗糖代谢通路相关基因在 15 DAF 显著下调，而支链氨基酸降解通路相关基因上调。为深入解析 ORF4 的功能，构建了一系列遗传材料，包括启动子精准编辑、敲除突变体及标签融合转基因家系。这些材料为揭示 ORF4 在垩白形成中的分子调控网络及靶向改良水稻品质提供了重要工具。

综上，本研究鉴定出 C51 为稳定遗传且受低温低氮诱导的高垩白材料，确定 ORF4 为控制 qPGWC-7 位点的关键基因，表明其启动子区域的自然变异导致其在 9311 与 C51 18 DAF 胚乳中表达水平的差异，从而影响 C51 籽粒淀粉的合成，最终导致腹白的形成。此外，ORF4 编码的线粒体定位蛋白如何通过能量代谢影响胚乳淀粉合成，这为解析调控籽粒品质的分子机制提出了新的科学问题。

Rice (Oryza sativa L.), a globally essential food crop, plays a pivotal role in safeguarding stable grain supply and adapting to evolving consumer demands through quality improvement. Rice appearance quality serves as the primary determinant of market value and consumer preference, with chalkiness (the opaque white regions in endosperm) representing a core phenotypic trait that directly influences milling quality, cooking characteristics, and commercial value. Chalkiness formation is regulated by the interaction between genetic factors and environmental conditions, and deciphering its molecular mechanisms remains a critical challenge for premium-quality rice breeding. Current studies indicate that defective grain filling and abnormal metabolism of endosperm storage substances constitute the primary causes of chalkiness formation, though the key regulatory networks require further elucidation.

Current studies have reported that defects in grain filling and endosperm storage substances are the primary causes of chalkiness formation. In this study, a high-chalkiness line was screened from a Chromosome Segment Substitution Line (CSSL) population constructed using 9311 and the high-chalkiness material PA64S, which was designated as C51 (Chromosome Segment Substitution Line, CSSL). This research aims to elucidate the genetic basis of its high-chalkiness phenotype, identify the candidate gene of the major quantitative trait locus (QTL) qPGWC-7 located on chromosome 7, and provide new insights into the molecular mechanisms underlying chalkiness formation. The research results are as follows:

Multi-year and multi-location trials demonstrated that both the chalky grain rate and belly chalkiness rate of C51 were significantly higher than those of the recurrent parent 9311, with these phenotypes being markedly induced under low-temperature and low-nitrogen conditions. Comparative analysis of transverse endosperm sections at consecutive developmental stages revealed the emergence of belly chalkiness in C51 at 18 days after flowering (DAF). Scanning electron microscopy (SEM) observations showed that irregularly elliptical starch granules with loose arrangement and increased surface adhesives appeared in the belly region of C51 endosperm at 15 DAF, contrasting sharply with the densely packed hexagonal prisms observed in 9311.

Cytological examination of developing endosperm indicated reduced synthesis rate, smaller diameters, and decreased deposition area of starch granules in the belly region of C51 during early endosperm development (6-12 DAF). Physiological assays revealed significantly lower total starch content, imbalanced amylose-to-amylopectin ratio, and reduced Rapid Visco Analyser (RVA) profile parameters (including peak viscosity and breakdown value) in mature C51 grains. Expression profiling further demonstrated significant down regulation of starch biosynthesis-related genes during C51 endosperm development.

Within the 44-kb interval of qPGWC-7 on chromosome 7, six candidate genes were prioritized through Sanger sequencing, gene annotation, and expression pattern analysis. Genomic complementation transgenic lines were subsequently constructed. Multi-generational phenotyping revealed no significant differences in chalky grain rate or belly chalkiness between ORF4 (Open Reading Frame 4) transgenic lines (T1-T3 generations) and C51, with enhanced phenotypic stability under low-temperature and low-nitrogen stress. SEM analysis confirmed that ORF4 transgenic lines exhibited irregularly elliptical starch granules with loose arrangement and surface adhesives in mature endosperm, phenocopying C51. Promoter sequencing identified multiple SNPs (Single Nucleotide Polymorphisms) and InDel (Insertion-Deletion) between 9311 and C51, resulting in differential transcription factor binding motifs. Expression analysis demonstrated significantly higher ORF4 transcript levels in C51 endosperm at 18 DAF compared to 9311.

Subcellular localization confirmed that ORF4 encodes a mitochondrial-localized protein, while phylogenetic analysis revealed high conservation across dicotyledonous and monocotyledonous plants. Transcriptome profiling showed significant downregulation of starch and sucrose metabolism-related genes alongside upregulation of branched-chain amino acid degradation pathways in C51 endosperm at 15 DAF. To further elucidate ORF4 function, a series of genetic materials were developed, including precisely edited promoters, knockout mutants, and tag-fusion transgenic lines. These resources provide essential tools for dissecting ORF4's regulatory network in chalkiness formation and enabling targeted quality improvement.

In conclusion, this study identifies C51 as a stably inherited, environmentally responsive high-chalkiness material and establishes ORF4 as the key gene underlying qPGWC-7. Natural variations in the ORF4 promoter region drive its differential expression between 9311 and C51 endosperm at 18 DAF, thereby impairing starch biosynthesis and ultimately inducing belly chalkiness formation. Furthermore, the mitochondrial localization of ORF4 raises new scientific questions regarding how mediated by energy metabolism regulate grain quality, advancing our understanding of the molecular mechanisms governing chalkiness formation in rice.

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