大豆[Glycine max (L.) Merr.]籽粒中约含有30-46%蛋白质，并且氨基酸组成与动物蛋白质近似，是世界范围内重要的植物蛋白来源。但籽粒中含硫氨基酸（甲硫氨酸和半胱氨酸）含量偏低，低于人类日常膳食推荐水平，使其成为限制大豆籽粒蛋白品质的主要因素。随着人类日益提高的消费需求，通过传统育种或基因工程的方法提高大豆籽粒含硫氨基酸含量、改良大豆籽粒蛋白品质具有重要意义。

本研究通过籽粒含硫氨基酸相关性状的全基因组关联分析结合转录组数据确定候选基因GmCBL1和GmCBL2（Cystathionine β-lyase，CBL）。对其进行功能验证并研究其正调控大豆籽粒蛋白品质的分子调控网络。同时发现大豆籽粒蛋白品质性状与耐盐性状之间具有相关性，GmCBL1和GmCBL2也正调控大豆耐盐性，为设计高营养价值和高耐盐性的大豆品种提供策略。具体研究结果如下：

1） 本研究使用实验室已收集的三年不同环境下211份大豆自然群体籽粒含硫氨基酸相关表型数据，利用大豆高密度芯片数据NJAU 355K SoySNP，进行全基因组关联分析，结合实验室已有的含硫氨基酸极端材料转录组数据在19号染色体上确定影响大豆籽粒含硫氨基酸合成的候选基因，其功能注释为编码胱硫醚 β-裂解酶（Cystathionine β-lyase，CBL）。系统发育分析发现，其在栽培大豆基因组中3号染色体上存在同源基因，分别为GmCBL1和GmCBL2，后续对这一对同源基因展开功能研究。以栽培大豆品种Jack为遗传转化受体材料创制多个大豆转基因材料，OE-GmCBL1，OE-GmCBL2过表达转基因材料，gmcbl1单敲突变体，gmcbl2单敲突变体，gmcbl1/gmcbl2双敲突变体。籽粒品质表型鉴定结果显示，OE-GmCBL1和OE-GmCBL2转基因材料中包括甲硫氨酸和半胱氨酸在内的大多数氨基酸含量显著高于对照品种，总氨基酸含量也显著提高。与之相对的，在敲除突变体籽粒中甲硫氨酸含量整体呈现下降趋势。其中，gmcbl1单敲突变体下降11.86%-12.17%，gmcbl1-3-1株系降幅显著；gmcbl2单敲突变体变化不显著；双敲突变体显著下降。敲除突变体籽粒中半胱氨酸含量与对照相比没有明显变化；敲除突变体籽粒中总氨基酸含量与对照相比显著降低，且大多数其他氨基酸含量均降低。籽粒蛋白含量检测结果显示，OE-GmCBL1和OE-GmCBL2-3转基因材料籽粒蛋白含量显著提高；gmcbl1单敲突变体和gmcbl2单敲突变体蛋白含量显著下降，gmcbl1/gmcbl2双敲突变体籽粒蛋白含量下降0.80%-1.75%，其中gmcbl1/gmcbl2-4-2籽粒蛋白含量降幅明显。表明GmCBL1和GmCBL2正调控大豆籽粒含硫氨基酸的合成，提高籽粒氨基酸和蛋白含量，改善大豆籽粒蛋白品质。

2） 酵母双杂筛选出GmCBL1、GmCBL2的互作蛋白GmRBCS13，RBCS（RuBisCO small subunit）是光合作用关键酶RuBisCO小亚基多基因家族，GmCBL1和GmCBL2与GmRBCS13互作影响叶片中的净光合速率，OE-GmCBL1和OE-GmCBL2转基因材料叶片中蔗糖含量提高；同时叶片中含硫氨基酸合成通路上第一个关键酶基因GmSERAT表达量显著提高，在其催化下叶片中含硫氨基酸含量提高，“源”器官中营养同化物质合成积累增加。同时验证GmCBL1和GmCBL2与Gm7Sα（β-伴大豆球蛋白α亚基）的互作关系，OE-GmCBL1、OE-GmCBL2转基因材料和gm7Sα/gm7Sα’突变体材料籽粒球蛋白组分表型变化趋势一致，说明GmCBL1和GmCBL2与Gm7Sα互作影响籽粒球蛋白组分。酵母单杂交筛选GmCBL2启动子上游转录调控因子，结合实验室前期对GmCBL1启动子酵母单杂筛库结果共同筛选到GmZFHD11（Zinc Finger Homeodomain）转录因子家族成员。通过酵母单杂定向回转验证、荧光素酶报告系统并结合组织表达量在线预测发现位于7号染色体的GmZFHD11转录因子（GmZFHD11-7）可以直接与两个同源基因的启动子结合，并在种子中表达量较高。对下游GmCBL1起转录激活作用，对GmCBL2起转录抑制作用。在创制的OE-GmZFHD11-7转基因材料中GmCBL1表达量提高，GmCBL2表达量降低。籽粒品质鉴定结果显示OE-GmZFHD11-7转基因材料种子中含硫氨基酸、蛋白含量均显著提高，7S球蛋白（β-伴大豆球蛋白）含量降低，11S球蛋白（大豆球蛋白）含量提高，11S/7S比值提高。表明GmZFHD11-7通过调控GmCBL1和GmCBL2的表达，影响大豆籽粒蛋白品质。

3） 据报道氨基酸除了是蛋白质基本组成成分之外，同时在逆境耐受性中发挥多重生物学功能。本研究利用大豆自然群体籽粒品质性状（球蛋白组分相关性状11S，7S，11S/7S，11S+7S；含硫氨基酸相关性状Cys，半胱氨酸；Met，甲硫氨酸；Cys/SAA，半胱氨酸/含硫氨基酸；Met/SAA，甲硫氨酸/含硫氨基酸；Cys/Met，半胱氨酸/甲硫氨酸）和萌发期耐盐性状（GR，Germination Rate，发芽率；TGR，Relative Germination Rate，相对发芽率；GP，Germination Potential，发芽势；TGP，Relative Germination Potential，相对发芽势；GI，Germination Index，发芽指数；TGI，Relative Germination Index，相对发芽指数）进行相关性分析，结果显示大豆籽粒贮藏蛋白组成和含硫氨基酸含量与大豆植株耐盐性之间具有显著相关性。基于该结果进一步对GmCBL1和GmCBL2过表达和敲除转基因材料进行耐盐性状鉴定，过表达GmCBL1和GmCBL2后，大豆萌发期和苗期的耐盐性提高，敲除后则表现出相反的表型。酵母双杂筛出GmCBL1与GmCBL2的互作蛋白蝶呤-4-α-甲醇胺脱水酶（Pterin-4-alpha-carbinolamine dehydratase，PCD），在OE-GmCBL1和OE-GmCBL2转基因材料盐处理前后，GmPCD表达量较对照材料均提高，表明GmPCD可能是影响转基因植株耐盐性变化的关键蛋白。本研究还发现种子蛋白品质改良的gm7Sα/gm7Sα’突变体材料在萌发期和苗期也具有耐盐性，进一步支持了蛋白品质与逆境适应性之间可能存在的功能关联。

综上所述，本研究鉴定到含硫氨基酸合成途径上的关键酶基因GmCBL1和GmCBL2，功能研究发现GmCBL1和GmCBL2正调控大豆籽粒蛋白品质。分子调控机制研究表明，通过与GmRBCS13互作影响叶片中营养同化物质合成积累，最终可能影响籽粒品质；与Gm7Sα互作影响种子中球蛋白组分。同时GmCBL1和GmCBL2在上游GmZFHD11-7转录因子的调控下影响大豆籽粒蛋白品质。另外发现大豆籽粒蛋白品质与耐盐性状之间具有相关性，GmCBL1和GmCBL2过表达材料以及gm7Sα/gm7Sα’突变体材料在籽粒品质改良同时也表现出耐盐性。本文解析GmCBL1和GmCBL2影响大豆籽粒蛋白品质和耐盐性的分子机制，增加对大豆籽粒蛋白品质合成途径的了解，为育成高品质、高耐盐性大豆新品种提供策略。

Soybean [Glycine max (L.) Merr.] seeds contain approximately 30-46% protein, with an amino acid composition similar to that of animal proteins, making it an important plant-based protein source worldwide. However, the content of sulfur-containing amino acids (methionine and cysteine) in soybean seeds is relatively low, falling below the recommended dietary intake for humans, thus becoming a major limiting factor in soybean seed protein quality. With increasing consumer demand, improving the protein quality of soybean seeds by enhancing sulfur-containing amino acid content and optimizing globulin composition through conventional breeding or genetic engineering is of great significance.

In this study, the candidate genes GmCBL1 and GmCBL2 (Cystathionine β-lyase, CBL) were identified through the genetic dissection of seed sulfur-containing amino acid-related traits in combination with transcriptomic data. Functional validation of these genes was conducted, and their involvement in the positive regulation of seed protein quality in soybean was investigated through analysis of the underlying molecular regulatory network. Moreover, a correlation between seed protein quality and salt tolerance traits was observed, GmCBL1 and GmCBL2 were found to enhance salt tolerance in soybean. These findings provide a theoretical basis and genetic strategy for the development of soybean varieties with both high nutritional value and improved salt tolerance. The specific research results are as follows:

1)  In this study, phenotypic data on seed sulfur-containing amino acid-related traits were obtained from a natural population consisting of 211 soybean accessions, evaluated across three years under diverse environmental conditions. A genome-wide association study (GWAS) was conducted using high-density NJAU 355K SoySNP, in combination with transcriptome data from extreme materials, to identify a candidate gene on chromosome 19 that influences sulfur-containing amino acid synthesis in soybean seeds. Functional annotation revealed that this gene encodes cystathionine β-lyase (CBL). Phylogenetic analysis identified a homologous gene on chromosome 3 in the cultivated soybean genome, designated as GmCBL1 and GmCBL2. Subsequent functional studies were conducted on these two homologs. Transgenic soybean lines were generated using the cultivated variety 'Jack' as the transformation recipient, including overexpression lines (OE-GmCBL1, OE-GmCBL2), single knockdown mutants (gmcbl1, gmcbl2), and double knockdown mutants (gmcbl1/gmcbl2). Phenotypic analysis revealed that overexpression of GmCBL1 and GmCBL2 significantly increased most amino acids, including methionine and cysteine, as well as total amino acid content compared to wild-type. Conversely, methionine content decreased in gmcbl1 mutants (by 11.86%-12.17%), with a significant reduction in the gmcbl1-3-1 line. No significant changes were observed in gmcbl2 mutants, while double knockdown mutants (gmcbl1/gmcbl2) showed significant reductions. Cysteine content in mutants was unchanged, but total amino acid levels and most other amino acids were significantly reduced. Additionally, seed protein content increased significantly in OE-GmCBL1 and OE-GmCBL2-3 lines, but decreased in gmcbl1, gmcbl2, and gmcbl1/gmcbl2 mutants. These results indicate that GmCBL1 and GmCBL2 positively regulate the synthesis of sulfur-containing amino acids, enhancing total amino acid and protein content and improving soybean seed protein quality.

2)  Yeast two-hybrid screening identified GmRBCS13 as an interacting protein of GmCBL1 and GmCBL2. RBCS (RuBisCO small subunit) is a key enzyme in photosynthesis, and its interaction with GmCBL1 and GmCBL2 influenced net photosynthetic rate in leaves. Overexpression of GmCBL1 and GmCBL2 led to increased sucrose content in leaves, and the expression of GmSERAT, the first key enzyme in the sulfur-containing amino acid synthesis pathway, was significantly upregulated. This resulted in increased sulfur-containing amino acid content in leaves, promoting the accumulation of assimilates in source organs. Additionally, GmCBL1 and GmCBL2 was found to interact with Gm7Sα, influencing seed globulin composition. Yeast one-hybrid screening identified GmZFHD11, a transcription factor family member upstream of the GmCBL2 promoter. Further verification using yeast one-hybrid assays, luciferase reporter system, and expression analysis revealed that GmZFHD11-7, located on chromosome 7, directly binds to the promoters of GmCBL1 and GmCBL2, with a high expression level in seeds. GmZFHD11-7 acts as a transcriptional activator of GmCBL1 and a repressor of GmCBL2. In OE-GmZFHD11-7 transgenic lines, GmCBL1 expression was upregulated, while GmCBL2 expression was downregulated. Phenotypic analysis showed that sulfur-containing amino acid and protein content were significantly increased in OE-GmZFHD11-7 seeds, along with a reduction in 7S globulin and an increase in 11S globulin, leading to a higher 11S/7S ratio, consistent with the phenotype of OE-GmCBL1 seeds. These findings suggest that GmZFHD11-7 regulates GmCBL1 and GmCBL2 expression, thereby influencing soybean seed protein quality.

3)  Since amino acids function not only as protein components but also in stress tolerance, this study further examined the relationship between seed quality and salt tolerance. Correlation analyses were performed between seed storage protein traits (11S, 7S, 11S/7S, 11S+7S), sulfur-containing amino acid traits (Cys, Met, Cys/SAA, Met/SAA, Cys/Met), and germination-stage salt tolerance traits (GR, TGR, GP, TGP, GI, TGI) in the natural population. Results indicated a significant correlation between sulfur amino acid content, globulin composition, and salt tolerance. Salt tolerance evaluation of GmCBL1 and GmCBL2 transgenic lines revealed that overexpression of these genes not only improved soybean seed protein quality but also enhanced salt tolerance during germination and seedling stages, while knockdown mutants exhibited the opposite phenotype. Yeast two-hybrid screening identified pterin-4-α-carbinolamine dehydratase (PCD) as an interacting protein of GmCBL1 and GmCBL2. In OE-GmCBL1 and OE-GmCBL2 transgenic lines, GmPCD expression was significantly upregulated before and after salt treatment, indicating that GmPCD may be a key protein affecting salt tolerance in transgenic plants. Additionally, the gm7Sα/gm7Sα’ mutant with improved seed protein quality also displayed enhanced salt tolerance, further supporting a potential functional link between protein quality and stress adaptation.

In summary, this study identified key enzyme genes, GmCBL1 and GmCBL2, in the sulfur-containing amino acid synthesis pathway and demonstrated that they positively regulate soybean seed protein quality. Molecular regulatory analysis showed that their interaction with GmRBCS13 influences the accumulation of assimilates in leaves, ultimately affecting seed storage substance accumulation. Additionally, their interaction with Gm7Sα to modulate seed globulin composition. GmZFHD11-7, an upstream transcription factor, differentially regulates GmCBL1 and GmCBL2 expression, thereby influencing seed protein quality. Moreover, a positive correlation between seed protein quality and salt tolerance was established, with overexpression of GmCBL1, GmCBL2, and the gm7Sα/gm7Sα’ mutant exhibiting both improved quality and salt tolerance. This study elucidates the molecular mechanisms by which GmCBL1 and GmCBL2 influence soybean seed protein quality and salt tolerance, enhancing our understanding of soybean seed protein quality synthesis pathways and providing strategies for breeding high-quality, salt-tolerant soybean varieties.

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