摘要
根据苯并咪唑类杀菌剂抗性基因的核酸序列 ,在其阅读框架外的上、下游设计一对特异性引物 ,并在其 5′ 端分别加上PstI位点。以pRB12 9质粒为模板 ,PCR扩增得到一条长约 1 4kb的片段 ,经酶切鉴定证明是TUB2基因。将此PCR产物经消化后与同样酶切的pTA质粒连接 ,构建出 pTA TUB2质粒并转化大肠杆菌。再以pTA TUB2质粒为模板 ,PCR扩增该目的基因 ,通过对其核苷酸序列分析证明构建的质粒是含TUB2基因的 pTA TUB2质粒。利用PEG方法 ,将该质粒转化毛壳菌 ,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 30 0倍以上 ,且转化稳定性试验表明 ,其抗药性在非选择性培养基上连续培养 10代保持不变。结果表明质粒pTA TUB2对毛壳菌的转化率为 2 7/ (2× 10 5)。
According to nucleotide sequence of the resistance gene (TUB2 gene) to carbendazim of various fungi, a pair of oligonucleotide primers in the upper and the lower reaches besides its reading frame were designed and a PstI restriction site in its 5'-end was added. An about 1.4kb nucleotide sequence of the target gene fragment was yielded by PCR amplification with the plasmid pRB 129. It was identified by restriction enzyme digestion that the PCR product was TUB2 gene. The TUB2 gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TUB2 plasmid. The TUB2 gene was PCR amplified with the template pTA-TUB2 plasmid. The results proved the recombinant plasmid pTA-TUB2 contains TUB2 gene. Then the plasmid was transfomed into the protoplast of Chaetomium sp. by PEG method. The transformants were selected on the PDA containing 10 μg/ml carbendazim. The transformant can grow on the PDA containing 300μg/ml carbendazim, which is 300 times higher than the original Chaetomium sp. The resistance was stable after 10 times transfer on non-selective medium. The results showed the transformation rate was 27/(2×105).
出处
《高技术通讯》
EI
CAS
CSCD
2003年第2期34-40,共7页
Chinese High Technology Letters
基金
国家自然科学基金 ( 3940 0 0 87)资助项目。
