根癌农杆菌介导的白腐丝状真菌——黄孢原毛平革菌的转化

利用农杆菌介导的方法成功地对黄孢原毛平革菌（Phanerochaete chrysosporium）进行了遗传转化。将含有潮霉素磷酸转移酶融合基因的双元质粒pCH61300转入根癌农杆菌（Agrobacterium tumefaciens）208中，然后用该转化菌分别感染黄孢原毛平革菌的分生孢子和原生质体，获得16株可能的转化子，经复筛，共获得6株潮霉素抗性水平为100μg/mL的稳定转化子，分生孢子和原生质体的转化频率没有明显差别。PCR检测结果显示，抗性基因已导入黄孢原毛平革菌细胞中；Southern杂交表明，TDNA以单拷贝形式整合到黄孢原毛平革菌基因组中。其中的一个转化子菌落形态与原野生型菌株相比有所不同，菌丝稀薄，分生孢子较少。利用分生孢子转化更为简便易行，无需特殊的设备和制备原生质体，此方法为深入开展该菌的遗传转化研究奠定了基础。

Agrobacterium tumefaciens-mediated transformation of the white-rot basidiomycete,Phanerochaete chrysosporium

Agrobacterium tumefaciens has been used to transfer its T-DNA to filamentous fungus Phanerochaete chrysosporium. A DNA fragment containing recombinant hygromycin B phosphotransferase gene (rhyg(r)) which is controlled by a promoter from P. chrysosporium and the lignin peroxidase gene terminator was cut out from pCH6, and inserted into a plant binary plasmid pCAMB1300, resulted in the recombinant plasmid pCH6-1300. The plasmid pCH6-1300 was then transformed into Agrobacterium tumefaciens strain A208, which was used to infect conidia or protoplasts of P. chrysosporium. Sixteen hygromycin B-resistant P. chrysosporium colonies were obtained. The transformation frequency is about 8 - 9 transformants per 106 conidia or protoplasts, which had no significant difference with the values obtained from conidia or protoplasts. One of the transformants named AT18, whose colony morphology was different from the wild-type strain. The results from both PCR and Southern blot analysis of transformants demonstrated that the rHyg(r) gene was integrated into the host genome with single copy. This approach may not require any instrument and digestion of fungal cell walls. A . tumefaciens-facilitated transformation should be very useful for further studies of the molecular genetics of P. chrysosporium.