增强型绿色荧光蛋白基因转化大豆疫霉菌及其表达

为了探讨大豆疫霉菌Phytophthora sojae的侵染过程及其在土壤中的生态学,采用CaCl2-PEG介导的原生质体转化方法,将外源增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因转化到大豆疫霉菌中,观察EGFP基因在大豆疫霉菌不同生育时期的表达,对转化子中的EG-FP基因进行PCR检测,并对转化子的生长、发育及致病性进行测定。结果显示,EGFP基因能够在大豆疫霉菌菌丝、游动孢子囊和卵孢子中稳定表达,并在475 nm蓝光激发下发出绿色荧光;转化子的菌丝生长速率与野生型无显著差异,个别转化子在无性繁殖和有性生殖方面与野生型有显著或极显著差异,其中一个转化子的致病型发生了改变。研究表明获得的EGFP基因转化子可以作为研究大豆疫霉菌侵染及定殖的材料。

Transformation and expression of enhanced green fluorescent protein gene in Phytophthora sojae

In order to study the infection process in soybean and ecology in soil of Phytophthora sojae, transgenic P.sojae trains that produced enhanced green fluorescent protein (EGFP) were obtained after stable DNA integration using the CaCl₂-PEG-mediated transformation method. The expression of EGFP gene in hypha, zoosporangia and oospores of P.sojae was observed using fluorescent microscopy. The EGFP gene in the transformants was detected by PCR and the growth development and pathogenicity of transformants were measured in the media. The results showed that EGFP gene can be stably expressed in hyphae, zoosporangia and oospores of P.sojae. There was no significant difference in the growth rate of hyphae between the transformants and the wild type, while remarkable or extremely remarkable difference was existed in the asexual and sexual reproduction between some transformants and the wild type, and one transformant has changed in the pathotype. So the transformants with EGFP gene we obtained can be used as a material for study of infection and colonization of P.sojae in host and in soil.