GmINS1参与磷调控大豆根瘤发育的分子机制

It could bring great significance for agriculture in reducing fertilization while increasing efficiency to give full play to the biological nitrogen fixation (BNF) in soybean. Phosphorus (P) is not only one of essential nutrient elements for nodule growth and development, but also affects the energy metabolism in the process of BNF. Therefore, P nutrient status is closely associated with soybean nitrogen fixation efficiency. Previously, we had identified a low-P enhanced and nodule-preferred gene, GmINS1, from a suppression subtractive hybridization library. We found that altering the expression of this gene significantly affected soybean nodule development and BNF efficiency. The project is proposed on the basis of previous works to further study the molecular mechanisms of the GmINS1 involvement in P regulated nodule development, so as to provide the theoretical base and important candidate genes for genetic improvements of crops with coordinative high efficiency of nitrogen and phosphorus. Firstly, by using the systems of soybean transformation and tobacco transient expression, we will verify the tissue specificity of GmINS1 and the subcellular localization of its coding protein. Then, the promoter/transcriptional sequence analysis of GmINS1 will be carried out to investigate the key regulation elements/mutations involved in P regulated nodule development. Furthermore, using the yeast one-hybrid/ yeast two-hybrid system to screen the important transcription factors/interacted proteins of GmINS1 to comprehensively explore the molecular mechanisms and biological functions of the GmINS1 involvement in P regulated nodule development.

充分发挥大豆生物固氮作用，对农业减肥增效意义重大。磷不仅是根瘤生长发育必需的营养元素之一，还影响生物固氮过程中的能量代谢。因此，磷营养状况与大豆固氮效率密切相关。本实验室前期利用抑制缩减杂交文库，筛选到一个低磷增强、根瘤高表达的基因，GmINS1；并发现调控该基因的表达，显著影响大豆根瘤的发育及生物固氮效率。本项目拟在前期工作的基础上，深入研究GmINS1参与磷调控大豆根瘤发育的分子机制，以期为作物氮/磷协同高效的遗传改良提供理论依据和重要的候选基因。首先，利用大豆转基因体系结合烟草瞬时表达，验证GmINS1表达的组织特异性及其编码蛋白的亚细胞定位；然后进行GmINS1的启动子区段/转录序列分析，挖掘其参与磷调控根瘤发育的关键调控元件/突变；进一步利用酵母单杂/酵母双杂系统，筛选重要的转录因子/互作蛋白。以期综合解析GmINS1在参与磷调控大豆根瘤发育过程中的分子机制和生物学功能。

大豆是典型的生物固氮作物，固氮根瘤的形成和发育依赖于充足的磷供应。因此，探讨磷调控大豆根瘤发育的分子机理，揭示相关基因的功能及其调控机制，对大豆固氮能力的提高具有重要意义。本实验室前期利用抑制缩减杂交文库，筛选到一个低磷增强、根瘤高表达的基因，GmINS1；并发现调控该基因的表达，显著影响大豆根瘤的发育及生物固氮效率。本研究在前期工作的基础上，深入研究GmINS1参与磷调控大豆根瘤发育的遗传与分子机制。结果表明，GmINS1在细胞壁定位，并在根瘤的皮层，维管束及薄壁细胞中表达。利用175份F9：11重组自交系材料进行生物固氮相关性状的QTLs定位，发现GmINS1是控制大根瘤数目qBNF-11位点的重要候选基因。GmINS1序列在亲本中的差异主要在启动子调控区域。其中，4个SNP位点的差异导致多个潜在的与激素响应相关的调控位点的变异。通过对GmINS1启动子进行区段化，发现pro3含有SNP-C/G，能够驱动GUS基因的高表达及增强GUS酶活活性。暗示了，GmINS1 启动子含有SNP-C/G，是导致GmINS1高表达的关键。进一步利用dCAPs的标记技术分型，发现含有SNP C/G的大豆RIL材料及核心种质的生物固氮能力与GmINS1的表达密切相关。RILs群体及大豆核心种质材料中，GmINS1表达较高的遗传材料，结瘤能力较强，而低表达的则具有较少的根瘤数目。此外，利用构建的GmINS1整株转基因大豆材料进行结瘤试验，发现过表达GmINS1显著增加了高、低磷条件下的大根瘤比例，促进了根瘤发育，进而提高了大豆生物固氮效率。本研究明确了GmINS1在参与磷调控大豆根瘤发育过程中生物学功能及遗传机制，研究结果可为大豆氮/磷协同高效的遗传改良提供重要理论依据和候选基因资源。