大豆种子富含油脂和蛋白质，是人类和动物重要的蛋白质和油脂来源。随着我国对大豆需求量的增加，如何在耕地面积有限的情况下提高大豆品质和产量已成为亟待解决的问题。挖掘与品质性状相关的新基因并阐明其作用机制，对培育优质大豆品种具有重要意义。本课题组前期经Meta分析和转录组测序获得了与子粒蛋白油分合成通路相关的hub基因GmM28，并利用遗传转化获得该基因的转基因大豆植株。在此基础上，本研究完成了大豆稳转植株的品质和产量性状的表型鉴定，利用扫描电子显微镜对GmM28转基因成熟子粒进行细胞形态比较；利用多重组学联合分析，探究了GmM28的大豆蛋白和油分含量调控途径；利用2898份大豆种质资源群体分析GmM28的驯化特征并挖掘GmM28的优异单倍型。主要研究结果如下：

品质和产量性状表型分析：对T₃、T₄代GmM28转基因大豆子粒进行蛋白和油分含量测定，发现gmm28蛋白含量平均降低1.21%，总脂肪酸含量平均升高1.24%，GmM28^OE蛋白含量平均升高1.96%，总脂肪酸含量平均降低1.92%。透射电镜的结果显示，与对照组和GmM28^OE相比，gmm28成熟子粒中的蛋白体更小，油体排列更紧密。以上结果表明GmM28同时控制油分含量和蛋白含量。另外，对T₃、T₄代GmM28转基因大豆植株进行产量相关表型统计，结果表明GmM28^OE植株的粒长、粒宽、百粒重和株高相比DN50都显著增加。

GmM28的细胞形态学分析：通过扫描电镜对GmM28转基因成熟子粒进行细胞学形态观察和统计。与对照DN50相比，gmm28的细胞数量和细胞面积分别降低了20.93%、2.99%，而GmM28^OE的细胞数目和面积分别增加了4.27%、10.43%，这些结果表明，GmM28可能通过影响细胞面积和细胞数量共同调控粒重和子粒大小。

GmM28的多组学联合分析：基于GmM28转基因大豆植株的IP-MS/MS、转录组学和代谢组学（脂质）联合分析，发现GmM28可能是通过参与调控蛋白质、脂肪酸生物合成途径控制油分含量和蛋白含量的，同时根据测序数据的表达模式分析筛选了77个基因和10个代谢离子作为GmM28的调控靶基因。此外，通过对RNA-seq得到的差异表达基因进行表达模式分析，发现GmM28可以通过调节17个大豆品质和产量基因的表达控制蛋白含量、油分含量和粒重。并利用IP-MS/MS鉴定到的1522个蛋白，结合蛋白功能描述和有关文献分析，筛选与蛋白、油分生物合成或运输有关的GmM28候选互作蛋白。

GmM28的选择驯化和单倍型分析：利用大豆种质资源对GmM28基因ATG上游3000 bp序列进行单倍型分析，鉴定到两类GmM28单倍型GmM28^Hap1和GmM28^Hap2，GmM28^Hap2在野生品种、地方品种和改良品种中的频率分布分别为3.00%、52.06%和73.62%，呈逐渐扩张的趋势。两个单倍型分别与脂肪酸和蛋白含量进行关联分析，发现GmM28^Hap1蛋白含量高于GmM28^Hap2，GmM28^Hap2的油分含量高于GmM28^Hap1。此外，对两个单倍型进行转录活性分析，发现GmM28^Hap2的转录活性显著高于GmM28^Hap1。上述结果说明在大豆选择驯化和品种改良过程中，GmM28^Hap2控制的高油表型比例逐渐增加。

综上所述，本研究阐明了大豆驯化过程中GmM28基因控制大豆品质和产量性状的遗传变异机制，初步探究了GmM28的分子机制，为调控大豆子粒蛋白、油分积累的遗传机理提供了新见解，并为大豆高品质高产分子辅助育种提供理论基础。

Soybean seeds are rich in oil and protein, and are an important source of protein and oil for humans and animals. With the increase of soybean demand in our country, how to improve soybean quality and yield under the condition of limited arable land has become an urgent problem to be solved. It is of great significance to explore new genes related to quality traits and elucidate their mechanisms for cultivating high-quality soybean varieties. In our research group, the hub gene GmM28, which is related to the oil synthesis pathway of grain protein, was obtained by Meta analysis and transcriptome sequencing, and transgenic soybean plants with this gene were obtained by genetic transformation. On this basis, the phenotypic characteristics of soybean stable plants were identified, and the cell morphology of GmM28 transgenic mature seeds was compared by scanning electron microscopy. The regulation of soybean protein and oil content in GmM28 was investigated by multi-recombinant analysis. Domestication characteristics of GmM28 were analyzed from 2898 soybean germplasm populations and excellent haplotypes of GmM28 were discovered. The main findings are as follows:

 Phenotypic analysis of quality and yield traits: Protein and oil content measurements of T₃ and T₄ generation GmM28 transgenic soybean kernels revealed that gmm28 protein content was reduced by an average of 1.21% and total fatty acid content was elevated by an average of 1.24%, while GmM28^OE protein content was elevated by an average of 1.96% and total fatty acid content was reduced by an average of 1.92%. The results of transmission electron microscopy showed that the proteasomes were smaller and the oil bodies were more tightly arranged in gmm28 mature zygotes compared to controls and GmM28^OE. The above results indicate that GmM28 controls both oil content and protein content. In addition, yield-related phenotyping of T₃ and T₄ generations of GmM28 transgenic soybean plants showed that grain length, grain width, 100-grain weight, and plant height were significantly increased in GmM28^OE plants compared with DN50.

 Cytomorphologic analysis of GmM28: Cytological morphology of GmM28 transgenic mature zygotes was observed and counted by scanning electron microscopy. Compared with the control DN50, the cell number and cell area of gmm28 decreased by 20.93% and 2.99%, respectively, while the cell number and area of GmM28^OE increased by 4.27% and 10.43%, respectively, and these results suggest that GmM28 may co-regulate the grain weight and zygote size by affecting the cell area and cell number.

 Multi-omics coanalysis of GmM28: Based on the combined IP-MS/MS, transcriptomics and metabolomics (lipid) analyses of GmM28 transgenic soybean plants, it was found that GmM28 may be controlling the oil content and protein content by participating in the regulation of protein and fatty acid biosynthesis pathways, and 77 genes and 10 metabolite ions were also screened as regulatory target genes of GmM28 based on the expression pattern analysis of the sequencing data. In addition, by analyzing the expression patterns of differentially expressed genes obtained by RNA-seq, it was found that GmM28 could control protein content, oil content and grain weight by regulating the expression of 17 soybean quality and yield genes. The 1522 proteins identified by IP-MS/MS were also used to screen for GmM28 candidate interacting proteins related to protein, oil biosynthesis or transport, in conjunction with protein functional descriptions and relevant literature analysis.

 Selective domestication and haplotype analysis of GmM28: Haplotype analysis of the 3000 bp sequence upstream of the ATG of the GmM28 gene using soybean germplasm resources identified two types of GmM28 haplotypes , GmM28Hap1 and GmM28Hap2, and the frequency distributions of GmM28Hap2 in the wild, local and improved varieties were 3.00%, 52.06% and 73.62%, respectively, which showed a gradually expanding trend. The two haplotypes were correlated with fatty acid and protein content respectively, and it was found that the protein content of GmM28^Hap1 was higher than that of GmM28^Hap2, and the oil content of GmM28^Hap2 was higher than that of GmM28^Hap1. In addition, transcriptional activity analysis of the two haplotypes showed that the transcriptional activity of GmM28^Hap2 was significantly higher than GmM28^Hap1. These results indicated that the proportion of high oil phenotypes controlled by GmM28^Hap2 increased gradually during the process of soybean selection and domestication and variety improvement.

In summary, this study clarified the genetic variation mechanism of GmM28 gene controlling soybean quality and yield traits during soybean domestication, preliminarily explored the molecular mechanism of GmM28, provided new insights into the genetic mechanism of regulating soybean grain protein and oil accumulation, and provided a theoretical basis for molecular assisted breeding of soybean for high quality and high yield.

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