水稻籽粒赖氨酸累积的分子调控及其代谢关联研究

Plants collectively synthesize lots of metabolites, which exhibit diverse functions during plant growth, development as well as quality formation. Lysine is the first limiting essential amino acid in cereal crops, especially in rice. Efforts, including genetic engineering, thus far have not achieved a desirable level of lysine in rice. The synthesis and degradation of lysine is regulated by a complex metabolic network. In higher plants, the aspartate-family pathway, synthesizes, through several different metabolic branches, four essential amino acids including lysine. It has a strong metabolic connection with the other pathway such as the cellular energy metabolism, revealing the multifaceted role of aspartate-family pathway in plant metabolism. In previous studies, we engineered rice for increased lysine through expressing bacterial lysine feedback-insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance lysine biosynthesis; through RNA interference or knockdown of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down-regulate its catabolism; and by combined expressing AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. Results showed that free lysine content was greatly increased up to ~60-fold in seeds comparing with that in wild type. Unfortunately, the transgenic events with dramatic increase of lysine content did lead to observable changes in endosperm development, color appearance of kernels as well as seed germination. In present study, with the aim to better understand the metabolic regulation of lysine accumulation in rice, the different above transgenic lines will be used, and their transcriptome and metabolome will be compared among them by using system biological methods. Besides, the phenotype of kernel development and germination will also carefully be analyzed. The expected data and results will being made to understand biological processes associated with the lysine metabolism, as well as its regulation mechanisms, and its importance for optimal improvement of the nutritional quality of crops.

植物产生的众多代谢物与其生长发育及产量和品质形成密切相关。赖氨酸是禾谷类作物的第一限制必需氨基酸，其代谢调控较为复杂；负责赖氨酸合成的天冬氨酸族代谢途径与细胞能量代谢等多条通路关联，在植物代谢中具有多重角色。在前期研究中，申请者通过调控水稻赖氨酸代谢途径上几个关键酶的表达，选育了各类突变体和转基因系；籽粒中游离赖氨酸含量最高可增加60倍以上，但是对籽粒发育、外观色泽和种子萌发等产生显著影响。拟在已有基础上，利用所选育的与赖氨酸代谢调控相关的各类水稻突变体和转基因材料，借助转录组学、代谢组学和生物信息学等系统生物学手段，结合表型、生理和生化等性状鉴定，研究并揭示在水稻籽粒中调控赖氨酸代谢后的关联效应及其形成的分子和代谢机制。预期结果可为深入阐明水稻等单子叶植物中天冬氨酸族氨基酸的代谢调控及关联机制提供重要的理论依据，也可为培育高赖氨酸含量、表型正常的优质水稻新种质提供技术支撑。

稻米富含淀粉和优质蛋白，是人类主要的能量与蛋白来源。但稻米中缺乏赖氨酸，被称为第一限制性必需氨基酸，影响稻米的营养价值。植物中赖氨酸的代谢调控机制较为复杂，其它众多代谢通路相关联。本项目以粳稻品种武香粳9号及其来源的赖氨酸代谢调控相关转基因水稻新品系为材料，从分子鉴定、品质分析、田间表现、营养评价、代谢组与转录组比较、以及代谢关联等多个方面开展了研究。结果表明，HFL1和HFL2两个高赖氨酸聚合系种子中游离赖氨酸含量与亲本相比提高25倍，总赖氨酸、总游离氨基酸和蛋白质含量也相应增加；同时其稻米理化品质有所提高，而淀粉特性并未受到影响；除成熟种子呈现棕褐色表型外，其他主要农艺性状均表现正常。营养评价结果显示，食用含高赖氨酸转基因稻米的HFL1组和HFL2组SD大鼠在体重增长、食物利用率、表观消化率、蛋白质功效比和赖氨酸利用率等生物性能上均显著优于食用含非转基因对照大米的WT组，与添加相似赖氨酸剂量的WT20组相当。由此表明，转基因稻米中提高赖氨酸含量可显著提升其营养价值，且与外添相同剂量的野生型WT20组具有相同的效应。. 为了深入探析高赖氨酸成熟种子中棕褐色物质成因及其相关的代谢关联，通过代谢组学和转录组学结合功能验证结果显示，在HFL水稻中5-羟色胺的含量显著提高，并与棕褐色种子表型密切相关；进一步通过相关基因表达和代谢物分析以及过表达TDC3得以验证；此外，茉莉酸信号途径和TDC表达在HFL水稻胚乳发育后期均受到强烈诱导，与5-羟色胺积累和棕褐色种子表型一致；综上分析，提出赖氨酸积累与棕褐色表型间的代谢关联模型，即调控赖氨酸代谢通路使籽粒中赖氨酸有效累积，从而提高了植物胁迫响应相关途径的活性，进而诱导5-羟色胺积累，最终产生棕褐色表型。相关结果将有助于深化人们对水稻代谢组遗传基础的理解，同时可为利用代谢工程进行作物营养品质等的改良提供新的信息和方向。

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