农杆菌介导灰葡萄孢菌株RoseBc-3的遗传转化

Agrobacterium tumefaciens-mediated transformation of Botrytis cinerea strain RoseBc-3

观察了农杆菌(Agrobacterium tumefaciens)介导转化灰葡萄孢(Botrytis cinerea)菌株RoseBc-3的影响因素,并对遗传转化的有效性进行评估。结果表明:农杆菌与灰葡萄孢共培养的温度和灰葡萄孢分生孢子浓度均能影响转化效率,当共培养温度为22℃、灰葡萄孢分生孢子浓度为1×105个/mL时,转化效率最高,平均每平皿(直径9cm)转化子数量超过100个;通过特异性引物介导的PCR和特异性探针杂交方法,证明所获得的转化子是农杆菌中T-DNA插入造成的,且大多数(87.5%)T-DNA插入为单拷贝插入;从灰葡萄孢T-DNA插入体库筛选获得了6类突变体,即产孢显著减少或不产孢突变体(占3.8%)、菌丝生长速率减慢突变体(占3.5%)、菌丝稀疏突变体(占1.6%)、菌落颜色异常突变体(占5.7%)、致病力丧失突变体(占3.3%)和致病力增强突变体(占3.0%)。采用反向PCR技术和hi-TAIL PCR技术从14个灰葡萄孢突变体菌株中获得了T-DNA侧翼序列,且发现T-DNA在基因编码区和非编码区插入的频率是相同的。

In this paper,we reported results about characterization of the factors affecting Agrobacterium tumefaciens-mediated transformation（ATMT） of the plant pathogenic fungus Botrytis cinerea strain RoseBc-3 and about the validity of ATMT for the particular strain of B.cinerea.The results showed that the temperatures for co-incubation of A.tumefaciens and B.cinerea and the conidial concentration of B.cinerea in the co-incubation mixture greatly affected the transformation efficiency.The optimal co-incubation temperature was 22 ℃ and the optimal conidial concentration was 1×105 conidia/mL.Under the optimal co-incubation conditions,the numbers of transformant reached 100 per dish（9 cm diameter）.All the 16 randomly-selected transformants of B.cinerea were confirmed to harbor the T-DNA of A.tumefaciens based on the result of the specific PCR.The result of the Southern blotting further indicated the integration of the T-DNA in the genome of B.cinerea with the frequency of the single-copy insertion reaching 87.5%.Six types of mutants were identified from the transformants.They were sporulation deficiency mutants（3.8%）,mycelial growth-reduced mutants（3.5%）,sparse mycelia mutants（1.6%）,colony color mutants（5.7%）,pathogenicity decreasing mutants（3.3%） and pathogenicity enhancing mutants（3.0%）.DNA sequences flanking the T-DNA tag were obtained using the inverse PCR technique and the hi-TAIL PCR technique from 14 mutants of B.cinerea.The insertion sites in these mutants were located with the same frequency in the coding region and in the non-coding regions.