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

  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.