农杆菌介导樱桃干腐病菌的遗传转化

摘要

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 α-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 ×106 spores.The optimal transformation conditions were that 1 ×106 spores per milliliter of P.perniciosa α-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 μmol/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 .