大豆种子特异表达糖转运蛋白GmST1和GmST5的功能及作用机制研究

The transport of assimilate from the source (leaves) to the sink (roots, seeds and so on) tissues, especially to the developing seeds, is essential for the plant growth and seed yield. Previously, we carried out the transcriptome analyses in three early seed developmental stages using two genotypes with contrasting seed size. The study identified two novel sugar transporters, GmST1 and GmST5 that were highly and specifically expressed in early seed developmental stage，implying that they may play important roles in seed development and yield. This project aims to study the biological function and regulatory mechanism of GmST1 and GmST5. Scientific goals include, 1) to evaluate the expression profile of GmST1 and GmST5 by analyzing the expression of the reporters (GUS and YFP) in the transgenic soybean plants generated from the constructs of the endogenous promoters or whole length genomic sequence fusing to the reporter gene(s); 2) to identify the substrates of these sugar transporters using yeast complementation assay and the high-sensitivity fluorescence resonance energy transfer (FRET) sugar sensor assay; 3) to analyze the diversity and signatures of selection on GmST1 and GmST5 genes among wild soybean (Glycine Soja) cultivated and landrace soybean varieties; 4) to illustrate the association between the hapotypes of GmST1 and GmST5 genes, their expression levels and sugar contents in seeds, seed development, and the seed size using transgenic soybean plants that either overexpressed or knockout the GmST1 and GmST5 genes. Based on the results, the function of regulatory mechanism of the GmST1 and GmST5 genes will be explored. The project will define novel sugar transporters for soybean seed development and filling, which will provide a new venue for the engineering of soybean plants with enhanced transport of assimilates to developing seeds, therefore increase yield.

同化产物由源向库, 尤其是向发育种子的转运, 是植物生长和种子产量的关键。前期我们用不同籽粒大小的大豆品种, 对发育种子进行了转录组分析，发现糖转运蛋白家族中GmST1和GmST5特异地在种子发育早期高丰度表达，暗示其在种子发育和产量形成中起关键作用。本研究拟对其功能及作用机制进行研究。将: 1)利用基因启动子融合GUS等载体，在转基因大豆中分析GmST1和GmST5基因的时空表达特征；2)通过酵母互补、荧光共振能量转移传感等方法确定其转运底物；3)解析GmST1和GmST5基因在大豆品种群体中的差异及其选择驯化效应。4)培育GmST1和GmST5基因超(原位)表达和敲除转基因大豆，分析GmST1和GmST5基因的不同单倍型、表达丰度，与种子糖分积累、种子发育和种子大小的关联，阐明其功能及作用机制。研究结果将鉴定参与大豆种子发育的重要糖转运体成员，为培育高产大豆品种提供新候选基因和新种质。

同化产物由源向库转运是植物生长的关键，其中同化产物向发育种子的转运效率与种子产量密切相关。前期我们用不同粒重大豆品种对发育种子进行了转录组分析，发现糖转运蛋白家族中GmST1（GmSWEET10)和GmST5（GmSWEET15)特异地在种子发育早期高丰度表达，暗示其在种子发育和产量形成中起关键作用。本项目计划GmST1和GmST5进行功能及机制研究。在本研究，我们利用遗传学、分子生物学、生物化学、生物信息学和分子育种等方法，系统地解析了上述两个大豆种子糖转运蛋白的组织表达模式和功能。. 研究发现，大豆从野生大豆小籽粒、低含油量、高蛋白质的特点到栽培大豆大籽粒、高含油量和低蛋白的驯化和改良过程中，GmSWEET10a/b起到了关键的作用。研究发现，GmSWEET10a/b特异表达在大豆种皮的薄壁组织，介导蔗糖、葡萄糖及果糖从种皮向胚的运输。敲除GmSWEET10a或GmSWEET10b都会导致百粒重和油份显著下降，蛋白质含量显著提高。两个基因同时敲除，百粒重降低40.2%，油分含量降低40.7%，蛋白含量提高32.1%。群体遗传学发现，GmSWEET10a和GmSWEET10b发生了渐进式的变异和人工选择。其中，GmSWEET10a在大豆驯化过程中受到强烈选择，使栽培品种的籽粒变大、含油量提高、蛋白质含量降低；GmSWEET10b的驯化和完成选择的程度稍滞后于GmSWEET10a。在栽培大豆中过表达这两个基因，大豆种子百粒重及油分含量都显著提高，且单株产量提高达11%-20%。该研究对于通过分子育种提高大豆产量及品质有重要意义。该研究成果于2020年5月在《National Science Review》上发表，并受到国内外媒体的广泛关注。. 项目研究中我们还鉴定了两个旁系同源糖转运蛋白基因 GmSWEET15a 和 GmSWEET15b，它们在种子发育过程中子叶期的胚乳中高度表达，并且在非洲爪蟾卵母细胞中显示出蔗糖的流出和流入活性。 结果表明，GmSWEET15 通过在种子发育早期介导从胚乳到胚胎的蔗糖输出，对大豆胚胎发育至关重要。该研究成果于2019年发表于Plant Physiology。. 综上，本项目已完成了立项过程中提出的科学问题，主要研究结果发表高水平SCI论文6篇，获得专利一项。项目已取得了预期的成果，申请结题。