链格孢菌原生质体的制备与限制性内切酶介导整合(REMI)转化的致病性诱变

PROTOPLAST PREPARATION AND MUTATION FOR PATHOGENICITY OF ALTERNARIA ALTERNATA (FR.) KEISSLER BY RESTRICTION ENZYME MEDIATED INTEGRATION (REMI)

以链格孢菌Alternariaalternata菌株NEW为供试菌株,研究了菌龄、酶系统、酶解时间、酶解温度及稳渗剂对链格孢菌原生质体制备的影响。结果表明,制备链格孢菌原生质体比较适宜的条件为PD液体培养基培养20h,以0.7mol/LNaCl为稳渗剂、1%LysingEnzyme、1%Drislase和1%Snailase3种酶溶液混合使用,30℃酶解3h。对原生质体进行了限制性内切酶介导整合(REMI)转化,筛选到了链格孢菌弱致病突变株NEW001,为致病相关基因的标记和克隆打下了基础。

The strain NEW of Alternaria alternata was used for studying the effect of the mycelial age, enzyme system, digesting temperature and time and osmotic stabilizers on protoplast preparation. The results indicated that the optimum condition for preparing protoplasts was： the mycelia cultured in PD medium for 20 hours, enzyme mixture of 1% Driselase, 1 % Lysing enzyme and 1% Snailase used to digest the mycelia at 30℃ for 3 hours, and 0.7 mol/L NaCl used as the osmotic stabilizer. A mutant with weak pathogenicity was gained by restriction enzyme mediated integration（REMI）, which was useful for tagging and cloning genes related to pathogenicity.