农杆菌介导尖孢镰刀菌西瓜专化型转化体系的建立及突变体筛选

摘要

以尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum)FO-11-06菌株为受体,采用农杆菌介导的遗传转化方法,实现了农杆菌AGL1(含pATMT1)介导的西瓜枯萎病菌的转化,并研究了转化介质和pH值对转化效率的影响.结果表明:在25℃下,采用乙酰丁香酮(AS)浓度为200 μmol/L、农杆菌OD600=0.15、尖孢镰刀菌分生孢子含量为1×106个/mL、pH值为5.1～5.8时可实现T-DNA的插入转化.当转化体系pH值为5.3时,转化效率最高,达到每106个分生孢子产生553个转化子.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滤膜转化效率较高,每106个分生孢子获得551个和549个转化子,显著高于滤纸.通过转化,获得尖孢镰刀菌西瓜专化型转化子1 989个,对生长速度、菌落颜色和产孢量等性状进行分析,发现有46个性状特异的突变体.研究结果为进一步研究尖孢镰刀菌西瓜专化型的生长发育、致病机制和相关基因的功能奠定了基础.%In this study, Fusarium oxysporurn f. Sp. Niveumis FO-11-06 was successfully transformed mediated by Agrobacterium tumefaciens, and T-DNA insertion mutants were further screened. The effects of pH and cultivation media on genetic transformation were studied under the condition of 25 ℃ , 1 X 106 spores/mL,A. Tumefaciens (AGL1) OD600 =0. 15,200 /μmol/L of acetosyringone and 48 h of co-cultivation in the presence of induction medium. The results showed that the transformants could be obtained in specific pH value range from 5. 1 to 5. 8 and the transformation efficiency reached the highest level at pH 5. 3, with about 553 transformants per 106 spores. It also revealed that transformation efficiency was higher on cellophane (551 transformants per 106 spores) and NC millipore filter (549 transformants per 106 spores) compared to filter paper. Hygromycin resistance and PCR assay of the transformants showed that the T-DNA had been successfully integrated into the genome of F. Oxysporum f. Sp. Niveumis FO-11-06. Forty six mutants with specific traits were identified from 1 989 transformants by assay of colony growth, colony color,and conidia output. The transformation system and mutants provided a basis for the study of development,pathogenicity mechanism and functional gene of the fungus.