蛋氨酸代谢调控及富含蛋白表达对大豆蛋白蛋氨酸积累的影响

Soybean seeds provide an excellent source of vegetal protein for human and livestock nutrition. However, their nutritional quality is hampered by a low concentration of the essential sulfur amino acid, methionine (Met). Methionine is the main limiting factors affect their nutritional quality. As the content of Met limits the nutritional value of soybean, attempts to increase Met content have been made in soybean seeds by traditional breeding and selection of mutants. However, these efforts have yielded only limited success, and the plants with enhanced Met content are usually associated with a significant reduction in yield. Therefore, in recent years, researchers have genetically engineered soybeans to express heterologous seed proteins rich in sulfur-containing amino acids or through manipulation of Met biosynthesis pathways. Although in some transgenic seeds a net increase in soluble or total Met content was reported, the increase was not sufficient to meet the nutritional requirements of human, livestock, and poultry. In order to study the genes expression regularity and interaction of committed enzymes in Met metabolism pathway and seed proteins rich in Met, the transgenic soybean lines overexpressing AtCGS exhibit a higher Met content and some typical soybean varieties will be used. Meanwhile the transgenic soybean plant expressing heterologous Met-rich seed proteins will be bred. The second step, some novel transgenic lines will be obtained by crossing between the transgenic soybean lines overexpressing AtCGS and the lines expressing heterologous Met-rich seed proteins. This will create Met source-sink interaction. And their impact of these manipulations on various seed traits such as soluble and total Met contents, protein profiles, the levels of total proteins, as well as the germination rate and yield will be analyzed. This will allow us to gain novel insight into the ability of the seeds to tolerate changes in protein composition and/or heightened Met content. This research proposal can lead to better understanding of Met metabolism in soybean seeds and help addressing the need for soybeans with enhanced Met levels and improved nutritional quality, meeting the requirements for human consumption and animal feed. On the other hand, some molecular markers can be gotten for selection of soybean variety resources.

大豆是人类重要的植物蛋白来源，在畜牧业生产中也发挥着重要作用。但大豆蛋白存在蛋氨酸等含硫氨基酸含量偏低的缺点，限制了其氨基酸组成的均衡性，是影响其营养品质的重要限制因子。多年的育种实践表明，通过常规育种手段难以获得高蛋氨酸材料。近年来，通过调控代谢途径关键酶基因或表达富含含硫氨基酸的外源蛋白，尽管已创制出了一些游离蛋氨酸或种子蛋白蛋氨酸含量提高的大豆材料，但仍难以满足人类和动物的相应营养需要。本项目以过量表达外源CGS基因的高蛋氨酸转基因株系和常规品种为材料，探讨大豆蛋氨酸代谢途径关键酶基因和富含蛋氨酸蛋白编码基因的表达规律和相互关系，同时创制表达富含蛋氨酸外源蛋白编码基因的转基因材料，并通过两类材料的杂交，创制同时实现蛋氨酸代谢途径调控和外源富含蛋氨酸蛋白表达的新型材料，探讨其对大豆蛋氨酸含量提高效果的影响。在创制优异育种材料的同时，也为高蛋氨酸种质资源、优异亲本的筛选提供分子手段。

大豆是人类重要的植物蛋白来源，在畜牧业生产中也发挥着重要作用。但大豆蛋白存在蛋氨酸等含硫氨基酸含量偏低的缺点，限制了其氨基酸组成的均衡性，是影响其营养品质的重要限制因子。多年的育种时间表明，通过常规育种手段难以获得高蛋氨酸材料。近年来，通过调控代谢途径关键酶基因或表达富含含硫氨基酸的外源蛋白，尽管已创制出了一些游离蛋氨酸或种子蛋白蛋氨酸含量提高的大豆材料，但仍难以满足人类和动物的相应营养需求。本项目以过量表达外源CGS基因的高蛋氨酸转基因株系和常规品种为材料，探讨了大豆蛋氨酸代谢途径关键酶基因和富含蛋氨酸蛋白编码基因的表达规律和相互关系，明确了通过改变大豆生长环境或采取增施硫肥等栽培措施对GmGCS表达的影响较小，不足以改变其原有的表达特性，难以实现蛋氨酸积累的有效提升，创制高蛋氨酸材料是提升大豆含硫氨基酸水平的关键。针对组成型过量表达拟南芥AtDCGS基因的高蛋氨酸转基因大豆材料，系统分析了其蛋氨酸含量的变化，筛选到了游离蛋氨酸含量提高3.7-7.3倍、总蛋氨酸含量提高1.5-4.8倍的优选株系。新创制出以富含蛋氨酸的15kD玉米清蛋白编码基因（β-Zein）为目标基因的转基因大豆材料，筛选获得了蛋氨酸含量得到提高的株系，为大豆优质育种提供了新材料。通过杂交和双价载体转化手段，获得了同时含有外源蛋氨酸代谢调控关键酶基因（AtDCGS）和富含蛋氨酸的15kD玉米清蛋白编码基因（β-Zein）的转基因大豆特异材料，这类同时调控蛋氨酸代谢关键酶基因和富含蛋氨酸储藏蛋白基因材料，种子蛋白蛋氨酸含量得到了进一步提高，为大豆优质育种提供了新材料、新手段。进一步优化了已有的大豆遗传转化系统并在大豆中成功建立了CRISPR/Cas9基因组编辑技术体系，为定点编辑、调控大豆蛋氨酸代谢关键基因，从而提升大豆含硫氨基酸含量提供了新手段、新方法。