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

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。

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

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 MnlⅠ restriction 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.