高效银耳芽孢遗传转化体系的建立

Development of Highly Efficient Genetic Transformation System of Yeast-Like Conidia of Tremella fuciformis

【目的】建立PEG介导的高效银耳(Tremella fuciformis)芽孢遗传转化体系。【方法】采用PEG介导的原生质体法把表达质粒pAN7-1(含有构巢曲霉gpd-An启动子和潮霉素抗性基因hph)和pLg-hph(含香菇gpd-Le启动子和hph基因)转化进银耳芽孢的细胞中;采用夹层法在含50μg·ml-1潮霉素的筛选培养基中初筛银耳芽孢假定转化子,之后转接于潮霉素含量为100μg·ml-1的PDA平板上进行第2次筛选。【结果】PEG4000浓度为25%时介导的银耳芽孢原生质体初转化率最高;pLg-hph假定转化子经过PCR鉴定及Southern杂交验证,结果表明hph基因能有效整合进银耳芽孢的基因组中,其转化率为110个/μgDNA。而pAN7-1转化子经过PCR鉴定及Southern杂交验证,结果表明只有部分转化子基因组中整合了hph基因,其转化率只有9个/μgDNA。银耳芽孢转化子在PDSA培养基中继代5次后仍检测到hph基因的存在,表明外源基因hph能在银耳芽孢继代中稳定遗传。【结论】25%的PEG4000能高效介导银耳原生质体的转化;香菇gpd-Le启动子是银耳芽孢表达外源hph基因的强启动子;外源hph基因能够在银耳芽孢继代培养中稳定遗传。

[Objective] The highly efficient genetic transformation of yeast-like conidia of T.fuciform was developed by PEG- mediated protoplast transformation. [ Method] The expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg·ml^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transfered on PDA plates containing 100μg·ml^-1 hygromycin for second round selection. [Result] Experimental results showed that the most optimum concentration of PEG4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 per μg DNA. However the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. Its transformation efficiency was only 9 per μg DNA. The presence of hph gene in the genome of transformants after 5 rounds of subculture on PDSA medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture. [ Conclusion ] The concentration of PEG4000 for highly efficient transformation was 25%. The gpd-Le promoter from L. edodes could drive effectively exogenous hph gene expression in the yeast-like conidia of T.fuciformis. The exogenous hph gene could be stably hereditary during sub-cultures of yeast-like conidia of T.fuciformis.