TUB2基因鉴定及其在毛壳菌中的转化

TUB2基因为植物病原菌抗苯并咪唑类杀菌剂基因 .以pRB12 9质粒为模板对该基因进行PCR扩增 ,得到一条长约 1 4kb的DNA片段 ,经酶切鉴定证明该DNA片段是TUB2基因 .利用PEG方法 ,将含有TUB2基因的pRB12 9质粒转化于毛壳菌原生质体中 ,并在高于毛壳菌敏感的多菌灵浓度 (10 μg mL)下筛选转化子 ,此质粒转化率为 3 (2× 10 5) .使得对多菌灵非常敏感的毛壳菌能够在 30 0 μg mL多菌灵的培养基上正常生长 ,其抗药性提高了30 0倍以上 ,而且转化子稳定性试验表明 ,其抗药性在非选择性培养基上连续培养 10代保持不变 .

Identification of TUB2 gene and its transformation into Chaetomium spp.

TUB2 gene is the resistance gene to benzamidazole in plant pathogenic fungi. An about 1 4 kb nucleotide sequence of the target gene fragment was yielded by PCR amplification with the plasmid pRB129. It was identified by restriction enzyme digestion that the PCR product was TUB2 gene. Then the target gene was transformed into the protoplast of Chaetomium spp. by PEG method. The transformants were selected on the PDA containing 10 μg/mL carbendazim. The transformation rate was 3/(2×10 5). The transformant could grow on the PDA containing 300 μg/mL carbendazim, which was 300 times concentration higher than that of the original Chaetomium spp. The resistance was stable after 10 times transfer on non selective medium.