以携带潮霉素磷酸转移酶基因的pBIG3C为转化载体,根癌农杆菌EHA105为转化介体,对樱桃干腐病菌LXS230101分生孢子进行转化。结果表明,樱桃干腐病菌的最优转化体系为:α型分生孢子浓度为106个/mL,培养基中添加200μmol/mL乙酰丁香酮(AS),共培养温度和时间分别为25℃和72 h,其转化效率为1×106个α型分生孢子产生686个转化子。随机选取转化子进行PCR和Southern blot鉴定,发现T-DNA已整合进樱桃干腐病菌基因组中;转化子在不含潮霉素的PDA培养基中连续培养5代后,仍表现出对潮霉素的抗性,表明外源基因能在樱桃干腐病菌中稳定遗传。
We developed an Agrobacterium- mediated transformation system for P．perniciosa by using the－ conidia of strain LXS230101 as transformation recipients，A． tumefacien strain EHA105 carring plasmid pBIG3C har- boring the hygromycin B phosphotransferase gene (hph)．Successful transformation of P．perniciosa was performord and the highest effiency reached on 686 transformants per 1 × 106spores．The optimal transformation conditions were that 1 × 106spores per milliliter of P．perniciosa－ conidia suspension were co- cultured with Agrobacterium cells at 25 ℃ for 72 h，in the presence of Co- culture medium containing acetosyringone (AS) at 200 mol /mL．The trans- formants were verified by PCR amplification and by Southern blot analysis with the hph primers and probe，respec- tively．The results showed that all the detected transformants could be amplified the target bands and the T- DNA was inserted into the genome of P．perniciosa． In addition，the transformants were stable when grown on PDA medium without hygromycin for five times．
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