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【目的】将农杆菌介导的转化应用于重要的工厂化栽培食用菌斑玉蕈中,建立稳定的农杆菌介导的斑玉蕈遗传转化技术。【方法】将构建的双元载体pYN6982转入农杆菌LBA4404菌株中,以斑玉蕈SIEF3133菌株打碎的双核菌丝为受体材料,利用根癌农杆菌介导的转化方法进行斑玉蕈转化试验。【结果】经潮霉素抗性筛选、PCR鉴定以及有丝分裂稳定性试验验证,表明潮霉素磷酸转移酶基因(hph)已经整合到斑玉蕈的基因组中;转基因斑玉蕈菌丝在荧光显微镜下可以观测到绿色荧光,表明增强型绿色荧光蛋白基因(egfp)已经在转基因斑玉蕈菌株中获得了表达;通过PCR检测,随机挑选的8个转基因斑玉蕈菌株中有2个可以扩增出载体转移DNA(T-DNA)边界重复序列外的卡那霉素基因(kan)序列。【结论】获得了稳定遗传和表达的斑玉蕈转基因菌株,建立了农杆菌介导的斑玉蕈遗传转化方法。农杆菌介导的斑玉蕈遗传转化中,存在载体T-DNA边界重复序列之外的DNA序列转移到转基因斑玉蕈中的现象,有待进一步研究。
【OBJECTIVE】 Agrobacterium-mediated transformation of Agrobacterium tumefaciens into Agrobacterium tumefaciens was carried out in an important industrialized culture. 【Method】 The binary vector pYN6982 was transformed into Agrobacterium tumefaciens strain LBA4404. The double nucleus mycelium broken by SIEF3133 strain was used as the receptor material, Agrobacterium tumefaciens transformation was carried out by Agrobacterium tumefaciens transformation test. 【Result】 The result of hygromycin resistance screening, PCR identification and mitosis stability test showed that the hygromycin phosphotransferase gene (hph) had been integrated into the genome of M. anisopliae. , Green fluorescence was observed, indicating that egfp has been expressed in the transgenic Agrobacterium tumefaciens strain; two of the eight randomly selected eight Agrobacterium mutans strains can be amplified by PCR The kan sequence out of the vector-transfer DNA (T-DNA) border repeats was generated. 【Conclusion】 The stable and inherited mutants of P. morifa were obtained, and Agrobacterium-mediated genetic transformation of P. hyodysentris was established. In Agrobacterium-mediated transformation of P. hyodysenteriae, there is a phenomenon that the DNA sequence other than the T-DNA border repeat of the vector is transferred into the P. morifolium, which needs further study.