尖孢镰刀菌西瓜专化型遗传转化体系的优化

Optimization of Transformation System of Fusarium oxysporum f. sp. niveum

以尖镰孢霉西瓜专化型[FusariumoxysporumSchlechtendahlexFriesf.sp.niveum(E.F.Smith)SnyderetHansen]为试材,以农杆菌[Agrobacteriumtumefaciens(Smith&Townsend)Conn=Rhizobiumradiobacter(BeijerincketvanDelden)Youngetal]的T-DNA为介导,将抗潮霉素基因插入西瓜枯萎病菌基因组中,对遗传转化体系进行优化,并对5个突变体菌株和野生型镰刀菌的最适生长温度和培养基进行筛选。结果发现,乙酰丁香酮(AS)浓度为300～400μmol/L,农杆菌OD660为0.1～0.2,野生型镰刀菌分生孢子浓度为(0.1～5.0)×106个/mL时,转化效率最高,约为500个转化子/106个分生孢子;F3181、F3280、F2192、F2195、F3179突变菌株的最适生长温度为25～28℃。

Genetic transformation system for Fusarium oxysporum Schlechtendahl ex Fries f. sp. niveum （E.F. Smith） Snyder et Hansen was optimized, and the optimum temperatures and cultural medium for F. oxysporum and the 5 T-DNA insertion mutants were screened. The results showed that Agrobacterium tumefaciens （Smith et Townsend） incubated for 5- 6 hours in broth IM with 300-400 μmol/L of AS before co-culture resulted in the highest transformation efficiency. A. tumefaciens （OD660 0.15- 0.20） and F. oxysporum spore （0.1 × 10^6～5.0×10^6 conidia per mL） solution were mixed equally in volume, and then plated onto a 60 mm diameter nitrocellulose filter （0.45μm pore） on the surface of the IM solid medium, 150μL per filter, at 26℃ for 48 hours, which resulted in best transformation.