根癌农杆菌介导gfp基因转化水稻纹枯病菌及其对病原菌稳定性和致病力的影响

Agrobacterium Tumefaciens-Mediated Transformation of gfp Gene and Its Effects on Stability and Pathogenicity for Rhizoctonia solani

本文对根癌农杆菌介导gfp基因转化水稻纹枯病菌及其对病原菌稳定性和致病力的影响进行了系统研究。结果表明,30μg/m L潮霉素B可作为转化子的筛选浓度,水稻纹枯病菌遗传转化的最佳条件为：共培养AS浓度为250μmol/m L,预诱导时间为9 h,共培养温度为26℃,共培养时p H为5.8~6.0,共培养时间为22 h。分别对随机选取的5个转化子进行荧光显微镜观察、PCR扩增及测序。结果表明,绿色荧光蛋白基因gfp已成功转化到水稻纹枯病菌中。转化子稳定性及致病力鉴定结果表明,转化子连续转接5代后仍对潮霉素B具有抗性,表明gpf成功转化到水稻纹枯病菌中且稳定遗传。转化子与野生型水稻病菌导致的水稻发病程度基本一致,表明gfp遗传转化后对水稻纹枯病菌的致病力无显著影响。本研究结果为开展水稻纹枯病菌与水稻品种互作机制奠定了基础。

In this paper, a gfp gene were introduced into the rice sheath blight fungus by agrobacterium mediated transformation and its effects on the stability and pathogenicity of the fungus investigated. The results showed that 30μg/mL of selection agent hygromycin B could significantly inhibit the growth of Rhizoctonia solani on PDA medium. The optimized parameters for A. tumefaciens mediated transformation of Rhizoctonia solani were as follows： 250μmol / mL of acetosyringone （AS） during the co-culture, pre-inducement time for 9 h, culture temperature at 26 ℃, pH 5.8 - 6.0 during co-cuhure and the total culture time for 22 h. About 5 randomly selected transformants were an- alyzed by fluorescence microscope, sequencing and PCR. The results indicated that the gfp gene was transformed in- to Rhizoctonia solani. The stability and pathogenicity analysis of the transformed fungus showed that 5 transformants after continuous culture for five generations still showed resistance to the selection agent hygromycin B, confirming the stable inheritance of the gfp gene in the transformed rice sheath blight fungus. The transformed fungus exhibited similar disease severity as that of the wide type strain after inoculating rice, indicating that the pathogenicity of the Rhizoctonia solani was not significantly changed by introduction of the exogenous gfp gene. Our results would lay the foundation for further investigation on the interaction mechanism between Rhizoctonia solani and rice varieties.