FMDV结构基因P1的克隆与植物双元表达载体的构建

Cloning of foot-and-mouth disease virus structural gene P1 and construction of binary expression vector in plants

通过RT-PCR法获得阿克苏(Akesu/O/58)FMDV结构基因P1,将该基因克隆到pGEM-T Easy载体进行核苷酸序列测定。将P1基因、Kozak序列等插入中间表达质粒pB in438,构建重组中间表达载体pB inP1。采用三亲融合法构建植物双元表达载体pB inFMDV-P1。结果表明:P1基因为2 208 bp。重组中间表达载体pB inP1经BamHⅠ/SalⅠ双酶切、PCR扩增和序列测定,表明P1基因、Kozak序列等已插入pB inP1。在含50mg/L卡那霉素、25 mg/L链霉素和50 mg/L利福平的YEB培养基上筛选及对融合农杆菌质粒进行P1基因的PCR检测,证明植物双元表达载体pB inFMDV-P1构建正确。

The RNA of foot-and-mouth disease virus strain Akesu/58 Serotype O was isolated and the structural gene P1 was obtained by reverse transeriptase-polymerase chain reaction （RT-PCR） procedures. P1 gene was cloned into pGEM-T Easy vector to analyze the nueleotide sequence. The recombinant Mini-expression vector pBinP1 containing the P1 gene and Kozak sequence was constructed and checked by PCR, restriction enzyme analysis with BamH/Sal I and nucleotide sequencing. The binary expression vector was constructed by triparental mating. These results showed that P1 gene was 2208 bp. P1 gene and Kozak sequence were cloned into the recombinant Mini-expression vector pBinP1 and the nucleotide sequence was the same as original sequence of P1 gene. Construction of the binary expression vector proved correct, when it was checked by PCR and the triparental mating Agrobacterium Tumefaciens was selected by culture medium containing 50 mg/L Kanamyein, 25 mg/L Streptomycin and 50 mg/L Rifampiein.