双孢蘑菇萎锈灵抗性基因定点突变的载体构建与遗传转化

Construction of a vector containing a point-mutated carboxin-resistance gene and genetic transformation of Agaricus bisporus

为建立更为安全、有效的双孢蘑菇遗传转化体系,构建了双孢蘑菇琥珀酸脱氢酶的铁硫蛋白亚基Agsdi1突变(His突变为Leu)表达载体pAgsdi1,并通过农杆菌介导方法转化双孢蘑菇W192,经萎锈灵筛选以及PCR扩增和MnlⅠ酶切验证后获得了转化菌株。验证结果表明,点突变的铁硫蛋白亚基Agsdi1可以作为双孢蘑菇有效的抗性标记基因。因其并未引入新的外源基因,是一种比潮霉素抗性基因更为安全的筛选标记,将可用于双孢蘑菇等食用菌的遗传转化。

In order to establish a safe and efficient genetic transformation system for Agaricus bisporus,an expressed vector with mutant Agsdi1(a single amino-acid substitution,His to Leu)encoding iron-sulphur protein of succinate dehydrogenase was constructed.After transformation of A.bisporus strain W192 by Agrobacterium-mediated method,putative transformants were selected by carboxin and identified by PCR amplification and digestion with MnlⅠrestriction enzyme.The result showed that a point-mutated Agsdi1 can serve as an effective resistance marker for A.bisporus.Without introducing exogenous gene,point-mutated Agsdi1 gene is a safer selective marker than hygromycin resistance gene,and can be used for genetic transformation of edible mushroom such as A.bisporus.