农杆菌介导樱桃干腐病菌的遗传转化  

Transformation of Phomopsis perniciosa Mediated by Agrobacterium tumefaciens

作　　者：王海艳[1] 李保华[1] 李桂舫[1] 王彩霞[1] 

机构地区：[1]青岛农业大学农学与植物保护学院,山东省植物病虫害综合防控重点实验室,山东青岛266109

出　　处：《华北农学报》2013年第4期218-222,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然基金项目(31000891;31272001);现代农业产业技术体系建设专项资金项目(CARS-28);山东省科技攻关计划项目(2010GNC10918);"泰山学者"建设工程专项经费项目

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 harboring 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 transformants were verified by PCR amplification and by Southern blot analysis with the hph primers and probe,respectively.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.

关 键 词：樱桃干腐病菌 遗传转化 转化效率 转化子鉴定 

分 类 号：S436.6[农业科学—农业昆虫与害虫防治]