根癌农杆菌介导的Pr1基因在贵阳腐霉的组成性表达

Improvement of Pythium guiyangense by constructive expression of Pr1 gene

摘要:

【目的】构建组成性表达贵阳腐霉Pr1基因的载体pCambia-hph-Pr1, 转化贵阳腐霉, 以获得高毒力的基因工程转化菌株。【方法】以双元载体pCambia-Pnos-PNN-hph为基本骨架，将Pr1基因置于组成型启动子PgpdA和终止子TtrpC之间, 获得含Pr1基因的表达元件。以贵阳腐霉菌丝体为受体材料，利用根癌农杆菌介导的遗传转化法转化贵阳腐霉，观察转化株的生物学特征和灭蚊毒力。【结果】转化株的菌丝生长速度和游动孢子产量与野生株无显著性差异。生物测定表明转化株对蚊幼虫平均致死率达到32.9%, 比野生株提高了约一倍,并且使受试蚊幼虫生长速度明显减缓。【结论】利用根癌农杆菌介导的遗传转化获得了遗传稳定的高毒力菌株。

Abstract:

[Objective] To construct a constructive expression vector pCambia-hph-Pr1 containing a Pr1 ORF of Pythium guiyangense for transformation of the fungus to get a genetic recombinant strain with enhanced virulence against mosquito larvae. [Methods] The Pr1 gene expression vector was constructed using a binary vector as basic frame, and Pr1 gene was placed between promoter PgpdA and terminator TtrpC. The recombinant strains were obtained by Agrobacterium-mediated transformation system, with P. guiyangense mycelia as acceptor, using vector pCambia-hph-Pr1 containing hygromycin B resistance gene as marker. The biological characteristics and virulence to Culex larvae of the transformant were observed. [Results] No distinct difference was found between wild-type stain and transformant in terms of growth rate and zoospore yield. A transformed strain of the fungus with significantly improved virulence was achieved. It caused an average larval death rate of 32.9% which was approximately double that of wild-type stain. Besides, the larvae exposed to transformant grew obviously slower. [Conclusion] A genetically stable strain with higher virulence was obtained by constitutive expression of Pr1 protease through Agrobacterium-mediated transformation.