Expression and Purification of Tobacco PR-1a Protein for Function Analysis

摘要

Pathogenesis-related proteins are assigned an important role in plant defense and in general adaptation to stressful environment.Although tobacco PR-1a is the first pathogenesis-related protein to be purified and characterized,its function is the least known of all PR-families.In the present study,coding sequence for tobacco mature PR-1a protein was amplified and subcloned in pGEX-5X-3 expression vector to overexpress soluble GST-PR la fusion protein(42 kDa)in Escherichia coli.The cleaved PR-1a protein(15.5 kDa)was isolated after removal of GST-tag by Factor Xa.Purified recombinant GST-PR 1a protein was used to prepare antiserum which can be used to detect the native tobacco PR-1 in the acidic extract of the TMV-infected leaves.Antifungal assay in vitro showed that both GST-PR la and cleaved PR-1a proteins had antifungal activity against Phytophlhora infestans,suggesting that the free N-terminus is not necessary for PR-1a in its antifungal activity.These results provide some clues for investigating the action mechanism of PR-1a.