Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine rich and metal binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the express ion of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C)of protoplasts is 6.745×106 /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01%. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome. SDS-PAGE, Western-blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6%-0.8%. Tested for metal resistance, the wild type mushroom growth was inhibited on the medium containing 1.0-1.2 mmol/L ZnSO 4. While the transgenic mushroom was inhibited on the medium containing 1.5-2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran∶sawdust =1∶3, and not in the medium of rice bran∶sawdust =1∶4.
应用电击法获得转MT基因平菇
张 竞 聂思惟 单 龙 茹炳根*
（北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室，北京100871）
摘要： 将MT基因用电击法转化平菇 (Pleurotusostreatus) ,MT基因表达蛋白与金属离子结合而形成络合物 ,用Zn诱导 ,转基因平菇能富集Zn ,可为缺Zn的人群补充Zn ,使平菇成为一种保健和治疗的食品或蔬菜。原生质体制备浓度为 6 .74 5× 10 6个 /mL。原生质体电击转化率为 0 .0 1%。PCR检测 ,2 0 0bp处有MT基因条带。蛋白检测 :转基因MT平菇ELISA检测阳性 ,表达率为 0 .6 %～ 0 .8%。SDS－PAGE显示有表达条带。Westernblot显示有阳性条带。抗ZnSO4结果 :野生型平菇抗ZnSO4浓度为 1.0mmol/L ,1.2mmol/L开始受抑制 ,转基因平菇抗ZnSO4浓度为 1.5mmol/L ,2 .0mmol/L开始受抑制。出菇试验结果表明 ,在米糠与锯沫比为 1∶3的培养基上生长 ,在米糠与锯沫比为1∶4的培养基上不生长。 2 4d菌丝可在广口瓶中长满 ,用用于子实体培养。
关键词： 电击法；原生质体；转基因平菇
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