农杆菌介导球孢白僵菌转化体系的建立及突变体筛选

以球孢白僵菌(Beauveria bassiana)YB-06为受体菌株, 采用农杆菌介导的遗传转化方法,实现了农杆菌AGL1(pATMT1)介导的球孢白僵菌的遗传转化,并研究了转化介质和pH对转化效率的影响。结果表明:在采用28℃、200μmol/L的乙酰丁香酮(AS)、农杆菌浓度OD₆₀₀=0.8、孢子浓度为1×106个/ml、pH5.1~5.8时可实现T-DNA的插入转化。当转化体系pH5.3时,转化效率最高,达586个转化子/106分生孢子;不同共转化介质对转化效率影响明显, 玻璃纸和硝酸纤维素膜转化效率较高。对3000个转化子生长速度和产孢量等性状分析获得46个性状特异突变体。本文结果为进一步研究球孢白僵菌的生长发育、致病机理和毒力相关基因功能奠定了基础。

GENETIC TRANSFORMATION OF Beauveria bassiana USING Agrobacterium tumefaciens-MEDIATED TRANSFORMATION AND MUTANTS SCREENING WITH SPECIAL TRAITS

In this study, Beauveria bassiana YB-06 was successfully transformed using Agrobacterium tumefaciens-mediated transformation approach, and T-DNA insertion mutants were further obtained. The effects of pH and cultivation media on genetic transformation were studied under the condition of 28℃, 1×106 spores/ml, A. tumefaciens (AGL1) OD600=0.8, 200μmol/L acetosyringone and 48h co-cultivation in the presence of induction medium. The results showed that the transformants could be obtained in specific pH value ranging from 5.1 to 5.8 and the transformantion efficiency reached the highest level at pH5.3 with about 586 transformants per 106 spores. The results also revealed that transformation efficiency was higher on cellophane and NC millipore filter compared to filter paper. HPH+ resistance and PCR assay of the transformants showed that the T-DNA had been successfully integrated into the genome of the fungus. A few mutants were identified by colony growth, colony color, and conidium output of the 3,000 transformants. Altogether, this transformation system provided a basis for the study of development, pathogenicity mechanism and functional genetics study of the fungus.