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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, Agrobacterium tumefaciens mediated gfp gene transformation of Rhizoctonia solani and its effects on pathogenic bacteria stability and pathogenicity were systematically studied. The results showed that 30μg / ml hygromycin B could be used as the screening concentration of the transformants. The optimum conditions for the transformation of Rhizoctonia solani were as follows: co-culture AS concentration 250μmol / m L, pre-induction time 9h The culture temperature was 26 ℃, the co-culture p H was 5.8 ~ 6.0, co-culture time was 22 h. Five randomly selected transformants were observed by fluorescence microscopy, PCR amplification and sequencing. The results showed that the green fluorescent protein gene gfp has been successfully transformed into R. solani. The results of the transformant stability and virulence identification showed that transformants were still resistant to hygromycin B after five generations of continuous transformation, indicating that gpf was successfully transformed into R. solani and was stable and inherited. Transformants and wild-type rice pathogens caused by rice pathogenesis are basically consistent, indicating that gfp genetic transformation of Rhizoctonia solani pathogenicity had no significant impact. The results of this study laid the foundation for the interaction mechanism between R. solani and rice varieties.