农杆菌介导的碱性成纤维细胞生长因子（bFGF）转化金针菇的研究

构建了金针菇表达载体p139035S-bFGF,并将重组质粒转入到根癌农杆菌EHA105中。以白金针菇Flammulina velutipes菌丝球为受体材料,用根癌农杆菌介导转入碱性成纤维细胞生长因子(bFGF)。金针菇菌株对潮霉素(Hyg)抗性测验结果表明,在PDA固体培养基上的潮霉素筛选浓度为9μg/mL,在液体培养基中为6μg/mL。探索头孢霉素(Cefotaxime)对菌丝的毒性实验、农杆菌的菌液浓度、侵染时间、乙酰丁香酮(AS)的添加及其浓度的控制、共培养的时间等因素对转化效率的影响。结果表明,Cef对金针菇菌丝几乎无抑制作用,最佳抑菌浓度为400μg/mL;农杆菌的菌液浓度OD600为0.5,侵染时间在30min左右,在AS为200μmol/L的共培养基上共培养72h,转化率最高。PCR与Southern杂交结果证明bFGF基因已整合到金针菇的基因组中。在无选择压力的PDA固体培养基上继代培养5次后仍检测到bFGF基因的存在,表明bFGF基因在转基因金针菇中稳定遗传。

Transformation of Flammulina velutipes with a basic fibroblast growth factor (bFGF) by Agrobacterium tumefaciens

A bFGF expression vector p139035S-bFGF was constructed and trasnsformed into Flammulina velutipes via Agrobacterium tumefaciens using a hygromycin phosphotransferase gene (Hyg) as a selective marker. The lowest inhibitory concentrations of hygromycin for F. velutipes were found to be 9μg/mL on PDA solid medium and 6μg/mL in liquid medium,respectively. The toxicity of cefotaxime to F. velutipes and the critical factors influencing transformation efficiency including the concentration of bacterium,infection time,the concentration of acetosyringone (AS),and the time of co-culture were tested. Our result showed that cefotaxime had no negative effect on F. velutipes growth and the best selective concentration was 400μg/mL. The maximum transformation efficiency was achieved under a condition when the bacterial growth reaches an OD600 value of 0.5,infection time was about 30min,AS concentration was 200μmol/L and co-cultivation time was 72h. The results of PCR and Southern blot indicated that bFGF was integrated into the F. velutipes genome. After five successive transfers on solid PDA medium without selection pressure,the bFGF gene was still detectable in transgenic F. velutipes strains,suggesting the stable integration of the exogenous gene.