硫氧还蛋白h及其靶蛋白在小麦种子萌发中的功能研究

Wheat pre-harvest sprouting is a worldwide climate disaster. Thioredoxin h (TRX h), a kind of thioredoxin generally present in organisms, can take part in many important biological processes such as regulating enzymes activities and signal transduction, by reducing the disulfide bond of target proteins.we had transferred the anti-trxs gene into wheat, and it had been proved that the transgenic wheats have the pre-harvest sprouting resistance. Since serine proteinase inhibitor (Serpin), protein disulfide isomerase (PDI) and xylanase inhibitor protein-I (XIP-I) were considered as the potential target proteins of TRX h，it is not enough to clarify the molecular mechanism of TRX h participating in wheat seeds germination. The major research contents are as follows. ① In order to clarify the molecular mechanism of TRX h participating in wheat seeds germination, the target proteins of TRX h in different germination stages will be screened with the combination of mBBr labelling/2D Electrophoresis method and Co-immunoprecipitation/liquid chromatography-tandem mass spectrometry （LC-MS/MS）. ② To confirm that Serpin, PDI and XIP-I are the trarget proteins of TRX h, the interaction between TRX h and the three proteins will be detected with bimolecular fluorescence complementation (BiFC) method. ③ The role of TRX h and the three target proteins in wheat seeds germination will be studied with the specific reaction system. The goal of the project is to establish the target proteins database of TRX h in wheat seeds germination, and to make clear the interaction between TRX h and the three proteins (Serpin, PDI and XIP-I), perfecting the molecular mechanism of TRX h participating in wheat seeds germination. These study will provide theoretical basis for the application of TRX h in crop improvement and breeding.

小麦穗发芽是一种世界性的自然灾害,硫氧还蛋白h（TRX h）是生物体内调控氧化还原平衡的一种多功能蛋白。本课题组将反义硫氧还蛋白基因转入小麦获得了抗穗发芽小麦，并对抗穗发芽机制进行初步研究，确定丝氨酸蛋白酶抑制蛋白（Serpin）、蛋白质二硫键异构酶（PDI）、木聚糖酶抑制蛋白（XIP-I）为TRX h的“潜在”靶蛋白，但其参与小麦萌发的机制尚不十分清晰。主要研究内容有：①mBBr标记/双向电泳法和免疫共沉淀/液相色谱-串联质谱法相结合分析小麦萌发中TRX h的靶蛋白；②双分子荧光互补技术分析TRX h与Serpin、PDI、XIP-I的相互作用；③构建反应体系分析TRX h和靶蛋白参与种子萌发的作用。本项目将建立TRX h在小麦萌发中的靶蛋白数据库，明确TRX h与Serpin、PDI、XIP-I的互作关系，完善其参与萌发的分子机制, 为TRX h在作物改良育种中的开发利用提供理论基础。

小麦穗发芽是一种世界性的自然灾害，本课题组前期将反义硫氧还蛋白基因转入小麦获得了抗穗发芽小麦，并对抗穗发芽机制进行初步研究。硫氧还蛋白h（TRX h）是生物体内调控氧化还原平衡的一种多功能蛋白，为了阐述TRX h在小麦种子萌发中的作用，本项目以转反义硫氧还蛋白基因小麦为材料，采用mBBr标记/双向电泳法、酵母双杂交法、双分子荧光互补等技术进行研究，得出以下主要结果：（1）分析筛选了小麦萌发中TRX h的靶蛋白，发现靶蛋白主要与糖代谢、蛋白质合成与降解、胁迫响应代谢等相关；（2）证明TRX h与Serpin、PDI、XIP-I三种蛋白质在体内存在相互作用；（3）通过酶活性及转基因小麦种子蛋白质组成的检测，进一步分析了TRX h和Serpin、PDI、XIP-I相互作用参与种子萌发的作用。TRX h通过对Serpin的还原作用使其失去蛋白酶抑制活性，进而提高蛋白酶的活性。TRX h能够还原XIP-I的二硫键使其失去抑制淀粉酶活性的能力。Trxh通过PDI的作用影响籽粒中谷蛋白大聚合体的形成，从而影响籽粒蛋白质的可降解性能，进而调控小麦萌发进程。本项目研究结果完善了TRX h参与小麦种子萌发的分子机制, 为TRX h在作物改良育种中的开发利用提供了理论基础。