水稻抗褐飞虱基因编码SCR蛋白在跨膜通道中迁移的研究

ɑ-hemolysin is the first proposed and widely used biological nanopore. It has shown attractive prospects as a single-molecule sensing technique based on the fabrication of a transmembrane protein channel in vitro, which was originally applied to nucleic acids detection with the long term goal of DNA sequencing. Although nanopore analysis for protein and/or polypeptides sequencing is still challenging at this moment, it is crystal clear that this kind of pore takes advantage of linear protein translocation and different conformations discrimination (ɑ-helical, β-sheet, disordered peptides). In this proposal, we aim at probing SCR protein, encoded by BPH resistance gene (for rice defence against brown planthopper insect) in rice, translocation through transmembrane protein channels. Firstly, we will investigate unfoldase or Guanidium-HCl mediated translocation behavior of SCR protein and peptides between different conformations within the nano-channel by current blockage observation. That will help people to simulate and reveal the mechanisms of biomacromolecules` traversing through lipid membrane. Furthermore, identify and characterize the secondary structures of SCR protein through conformational comparison by nanopore analysis, well-known protein sequence and 3D structure modeled by bioinformatics methods. According to the results, this study will provide us more useful theoretical and experimental basis to understand the molecular mechanisms of conferring resistance against BPH in rice, in particularly, disclose the interaction between protein structures and functions. This project would also expand the nanopore routine use in the fields of molecular genetics, structural biology and bioinformatics in crops.

ɑ-溶血素是应用最早，最为成熟的纳米孔。其在DNA测序为研究导向的核酸检测领域的成功应用，展现了这种基于跨膜蛋白通道体外构建的单分子检测技术的诱人前景。目前，纳米孔分析虽然在蛋白质/多肽测序领域仍面临严峻挑战，但在线性蛋白质穿孔和不同多肽结构鉴定（ɑ-螺旋，β片层，无规则结构）方面具有优势。为此，本项目提出对水稻抗褐飞虱基因BPH32编码SCR蛋白进行纳米孔研究。首先，在解折叠酶或盐酸胍调节下，通过阻遏电流变化探讨不同构象SCR蛋白和多肽片段在纳米通道中穿孔行为，模拟生物大分子跨膜运动的分子机理。 其次, 通过对不同构象的纳米孔分析，结合已知的蛋白序列,进一步对同源建模所获得蛋白二级结构进行认证和表征。 研究结果将为我们深入了解水稻抗褐飞虱基因的分子机理，特别是蛋白质结构与功能的相互关系，提供更多的理论依据和实验基础。拓展纳米孔在作物分子遗传学，结构生物学和生物信息学领域的实际应用。

纳米孔被用作蛋白质、多肽和氨基酸的单分子传感器，在生物医学鉴定中具有重要意义。 本项目我们第一次将其应用于水稻抗虫基因编码的多肽组学检测与分析。 以褐飞虱(Bph)为例，我们鉴定并克隆了Bph32基因，该基因编码SCR(“Bph32”)蛋白，与水稻抗褐飞虱(Bph)存在着直接作用关系。 然而，我们发现在易感品系中表达的同源蛋白却不具有这种抗虫性。 因此，分析Bph32蛋白的结构模块(肽域)可以为了解水稻对昆虫的反应提供必要的科学基础。 在此，我们结合生物信息学、生物化学和纳米孔分析的方法来分析具有不同性质的水稻多肽。 Bph32蛋白通过使用Swiss-Model workspace理论建模分为24个功能肽域。 然后，通过双缩二脲化学增强纳米孔电流调制信号强度，以此鉴定出22个水溶性抗虫多肽。 其中，通过读取电流调制光谱，可以在一个氨基酸分辨率下识别并区分出16个肽分子，从而提供抗虫肽分子的指纹图谱。 此外，研究发现纳米孔的调制电流与多肽分子质量呈现二次函数关系。 这些结果表明，纳米孔可以作为新一代的质朴检测传感器，用于更多的组学分析，特别是在需求强烈的农业领域。