摘要
【目的】对辣椒疫霉果胶甲基酯酶Pcpme3基因进行真核表达,制备其特异性抗血清,为用Westernblot检测该基因在辣椒疫霉致病过程中的功能奠定基础。【方法】利用TR-PCR分离鉴定Pcpme3的成熟肽片段,克隆于pPIC9K载体。将获得的表达载体转化毕赤酵母GS115,甲醇诱导表达,对表达产物进行SDS-PAGE分析和免疫家兔制备特异性抗体。【结果】酵母转化子经MD、MM平板筛选和G418抗生素筛选与PCR鉴定,获得Mut+/His+表型高拷贝重组转化子,经1%甲醇诱导后,SDS-PAGE检测发酵上清液,检测出43kD的特异蛋白。重组蛋白免疫家兔制备特异抗血清,Westernblot检测该基因在游动孢子侵染辣椒叶片中的表达,随着病情加重,该基因的表达逐渐增强,从而证明该基因参与了病菌的侵染致病过程。【结论】利用真核表达获得的蛋白制成特异性抗体可以有效地检测Pcpme3在辣椒疫霉侵染寄主过程中的功能特性。
[Objective] The Pcpme3 gene ofPhytophthora 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 GS 115 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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2009年第6期1988-1993,共6页
Scientia Agricultura Sinica
基金
国家高技术研发(“863”)计划(2006AA02Z198)
关键词
辣椒疫霉
果胶甲基酯酶
真核表达
抗血清
Phytophthora capsici
pectin methylesterase
eukaryotic expression
antiserum
