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    GST-pull down技术筛选毛白杨天冬氨酸蛋白酶PtoAED3互作蛋白

    Identification of aspartic acid protease PtoAED3-interacting proteins through GST pull-down assays in Populus tomentosa

    • 摘要:
        目的   天冬氨酸蛋白酶属于蛋白水解酶家族,为了解析毛白杨天冬氨酸蛋白酶PtoAED3在植物生长发育中的分子调节机制,利用GST-pull down联合质谱技术,对PtoAED3的互作蛋白进行鉴定和分析。
        方法   通过同源克隆获得了毛白杨PtoAED3的CDS序列,构建含GST标签的原核表达载体pGEX-4T-PtoAED3。使用IPTG诱导GST-PtoAED3融合蛋白表达后,利用GST标签对PtoAED3蛋白进行纯化,纯化后的PtoAED3蛋白与毛白杨植株总蛋白共孵育,应用GST-pull down技术获得毛白杨总蛋白中与PtoAED3蛋白相互作用的候选蛋白,随后通过质谱技术对筛选到的候选蛋白进行鉴定与分析。
        结果   通过质谱技术鉴定这些蛋白的氨基酸序列,共筛选出128 个与PtoAED3特异性结合的候选互作蛋白,这些互作蛋白涉及到细胞进程、代谢过程、应激反应、生物调节、发育过程等多个生物学过程。
        结论   通过GST-pull down实验联合质谱技术,筛选出毛白杨中与PtoAED3互作的候选蛋白,为研究毛白杨PtoAED3与底物或复合物的互作及其影响毛白杨生长发育的分子调节机制提供了初步方向。

       

      Abstract:
        Objective   Aspartic acid protease belongs to proteolytic enzyme family. In order to further analyze the molecular regulation mechanism of PtoAED3 in plant growth and development, GST-pull down combined mass spectrometry technology was used to identify and analyze the interacting protein of protein PtoAED3 in Populus tomentosa.
        Method   The CDS sequence of PtoAED3 was cloned by homologous sequence from P. trichocarpa, and a prokaryotic expression vector pGEX-4T-PtoAED3 containing GST tag was constructed. The GST-PtoAED3 fusion protein was induced by IPTG and purified by GST beads. The purified protein PtoAED3 was co-incubated with the total protein extracted from P. tomentosa, then the candidate interacting proteins of PtoAED3 proteins were obtained by GST-pull down technique and analyzed by mass spectrometry technology.
        Result   Through the identification of amino acid sequences of these proteins with mass spectrometry, a total of 128 candidate proteins interacting with PtoAED3 were screened, which involve multiple biological processes such as cell process, metabolic process, stress response, biological regulation and development process.
        Conclusion   The GST-pull down combined mass spectrometry technology was used to screen out candidate proteins that interact with PtoAED3 in P. tomentosa, providing a preliminary direction for studying the interaction of PtoAED3 with substrates or complexes and the molecular regulatory mechanism affecting the growth and development of poplar.

       

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