Abstract:

【Objective】 The Pcpme3 gene of Phytophthora capsici was expressed in Eukaryotic expression system to obtain purified protein and specific antiserum, and to detect the function of Pcpme3 in phytophthora-infected pepper through Western blot. 【Method】 Mature peptide of Pcpme3 obtained by RT-PCR was cloned into pPIC9K, and the eucaryotic expression plasmid was constructed. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. After induced by methanol, the culture supernatant was analyzed by SDS-PAGE, and the antiserum was prepared. 【Result】 The high copy transformants with Mut+/His+ phenotype were selected by screening on MD, MM and G418 medium and PCR. The culture supernatant was analyzed by SDS-PAGE after induced by 1% methanol, and the results showed that a specific protein of about 43 kD was expressed. The specific antiserum was produced after the rabbit was immunized with recombinant protein, and Western blot indicated that Pcpme3 could be highly expressed in phytophthora-infected peppers, and the expressed level became stronger with the symptom became heavier. 【Conclusion】 Antiserum prepared from expressed protein can be used to detect the function of Pcpme3 in the pathogenesis of Phytophthora capsici.

Key words: Phytophthora capsici, pectin methylesterase, eukaryotic expression, antiserum