农杆菌介导的斑玉蕈遗传转化

Agrobacterium-mediated transformation in Hypsizygus marmoreus

摘要:

【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。

关键词:斑玉蕈，农杆菌，EGFP，遗传转化

Abstract:

[Objective] Using Agrobacterium-mediated transformation as a tool, we built a stable genetic transformation of Hypsizygus marmoreus. [Methods] After binary expression vector pYN6982 was transformed into Agrobacterium tumefaciens LBA4404, we transformed H. marmoreus SIEF3133 blended vegetative dikaryotic mycelia via Agrobacterium-mediated transformation. [Results] Dozens of transgenic strains of H. marmoreus with genetic stability were obtained after hygromycin resistance selection, PCR identification and mitotic stability test. By fluorescence microscopy analysis of randomly selected transformants, green fluorescence can be observed. This enhanced green fluorescent protein gene (egfp) has expressed in transgenic H. marmoreus. Moreover, two of eight randomly selected H. marmoreus transformants contain kanamycin gene (kan) sequences from outside the transferred DNA (T-DNA) border repeats using PCR analysis. [Conclusion] Transgenic strains of H. marmoreus with genetic stability and egfp expression were obtained. Also, DNA sequences from beyond the classically defined T-DNA border direct repeats can be transferred into the H. marmoreus mycelia. This phenomenon requires attention in H. marmoreus functional gene study.

Key words:Hypsizygus marmoreus, Agrobacterium tumefaciens, EGFP, Genetic transformation