橡胶树尖孢炭疽菌绿色荧光蛋白(GFP)标记转化株的获得

Collectotrichum acutatum GFP Tagged transformants Generated from Agrobacterium tumefaciens-mediated Insertional Mutagenesis

建立了根癌农杆菌介导转化橡胶树尖孢炭疽菌(Collectotrichum acutatum)获得T-DNA插入突变体的体系:在尖孢炭疽菌孢子浓度106个/mL、农杆菌浓度OD600=0.15～0.2后在200μmol/mL的乙酰丁香酮(AS)诱导6h,共培养48h,转化效率可达150～300个/106个分生孢子。获得的转化子通过PCR检测绿色荧光蛋白基因、Southern杂交验证、分生孢子的荧光观察,结果表明:被测转化子基因组中确实整合了目的片段,成功获得了橡胶树尖孢炭疽菌菌株CHY-1绿色荧光蛋白标记转化子。

Colletotrichum acutatum isolate CHY-1 （the causal agent of anthracnose of rubber tree） was transformed by Agrobacterium tumefaciens-mediated transformation. The isolate CHY-1 with the concentration of 10^6 spores/mL were co-cultured with A. tumefaciens at OD600=0.15-0.2 for 48 hours in presence of acetosyringone（200μmol/mL） , and 150-300 transformants were generated per 10^6 conidia. The transformants were identified by PCR, southern blot analysis of GFP cassette, and green fluorescence observation with CLSM. All results confirmed that the target genes were successfully integrated into the genome of C. acutatum.