大豆种子硬度关联基因GmPG1a的克隆与功能验证

Hard seededness of soybean relates closely to storage and processing of soybean seeds.Cloning and studying of hardness-associated gene in soybean has important academic value to reveal the molecular mechanism of seed hardness, and it is more important to select and breed cultivars for different purposes. GmPG1a as the hardness-associated gene is preliminary confirmed by QTL positioning interval, resequencing data and relevant papers. We have cloned GmPG1a gene and identified that GmPG1a took part into absorption process of germination which directly related to seed hardness.We determined the GmPG1a genotypes, and then 1000 soybean materials were genotyped and carried out seed absorption- germination test. Correlation analysis showed polymorphism site of GmPG1a gene is very significant correlation to seed-absorption content/rate.In order to clarify the mechanism of how GmPG1a effects seed hardness, we need to further clarify its action type and regulation mode; In addition, we analyze whether GmPG1a influence on seed hardness though over-expression or RNAi transgenic lines. Last we research suppression effect of GmPGIPs and GmPG1a in order to reveal the regulation mode of seed hardness.We expect to further explore the molecular mechanism of controling soybean seed hardness through the study of GmPG1a gene.

大豆种子的硬实性与大豆储存和加工紧密相关，克隆和研究大豆种子硬度关联基因，不仅对揭示种子硬度分子机制具有重要的学术价值，更重要的是可据此选择和定向改良栽培品种以满足不同的用途需求。结合本课题组前期精确的QTL定位区间、重测序数据及相关文献报道，初步证实 GmPG1a是种子硬度关联基因。克隆GmPG1a基因，并鉴定到其参与种子硬度直接相关的种子吸水萌发进程；鉴定GmPG1a基因型，并对1000份大豆材料进行基因分型及种子吸水萌发试验，关联分析结果显示GmPG1a基因中的多态性位点与单粒种子吸水量/率非常显著相关。为了阐明GmPG1a影响种子硬度的机制，我们需要进一步明确其作用机理；通过超表达/RNAi转基因株系解析其对种子硬度的影响；鉴定其与GmPGIPs的抑制关系及参与调控种子硬度的方式。我们期望通过对GmPG1a基因的研究以进一步探讨控制大豆种子硬度的分子机制。

大豆广泛种植于全世界，也是重要的蛋白质和油料提供者，在国民经济中有着不可替代的位置。大豆的储存和加工是大豆利用中非常重要的环节，而种子硬度决定了大豆储存的质量、豆制品的品质及种子萌发能力，因此解析大豆种子硬度分子机制是十分必要的。.本研究精确定位了种子硬实性关联基因区间，结合亲本材料重测序数据，利用候选基因法初步推测多聚半乳糖醛酸酶基因GmPG1a是种子硬度QTL关联基因。接着我们分别从亲本材料中克隆了GmPG1a，发现其基因编码区内存在一SNP，导致其第289位氨基酸由组氨酸(H)突变为酪氨酸(Y)；鉴定GmPG1a基因型，并对1000份大豆材料进行基因分型及种子吸水萌发试验，关联分析结果显示GmPG1a基因中的多态性位点与单粒种子吸水量/率非常显著相关。该结果从遗传学角度充分证明GmPG1a基因与种子硬度相关。同时，在转录水平上验证了GmPG1a基因参与到种子硬度相关的种子种皮发育和种子吸胀进程。进一步，通过亚细胞定位发现野生型GmPG1a(289H)和突变型GmPG1aTK(289Y)蛋白均定位于细胞壁，亚显微解剖观察显示种子吸水前后的野生型GmPG1a和突变型GmPG1aTK种皮中的细胞壁结构发生变化，该结果充分说明GmPG1a编码的多聚半乳糖醛酸酶在细胞壁中发挥作用，分解细胞壁。最后我们把野生型GmPG1a转化到软粒GmPG1aTK大豆中，结果显示GmPG1a在植株中发挥了作用，它的导入使得转基因种子细胞壁结构比较坚固，种子不易吸水。其中原因有可能是GmPG1a的导入使得转基因种子内聚半乳糖醛酸分解成短半乳糖醛酸链增多，进而与周围的其他糖链、蛋白结合，导致细胞壁结构比较坚固，种子不易吸水。这些结果很好的映证了GmPG1a通过野生型到突变型的转化，引起了种子变硬及种子不易于吸水。我们期望通过对GmPG1a基因的研究以进一步探讨控制大豆种子硬度的分子机制。