【摘要】 利用农杆菌侵染法获得抗软腐病转BrWRKY33基因大白菜,首先要构建植物表达载体。本实验根据BrWRKY33基因的序列及pROK2表达载体的酶切位点设计引物(FN/RN),获得BrWRKY33基因,然后将该基因连接到克隆载体pGEM-TEasy,重新构建了克隆载体T-WRKY。经测序及酶切验证,选取PCR反应中错配率最低的产物,构建了表达载体pROK2-WRKY,并将该表达载体转入到根癌农杆菌EHA105中,并对其转化子进行了鉴定。结果表明,PCR扩增获得目的基因BrWRKY33全长约1443bp,PCR及酶切结果鉴定表明克隆载体T-WRKY已经重新构建成功。表达载体的PCR及BamHⅠ和KpnⅠ双酶切鉴定表明有4个重组子表现阳性,表明目的基因已经插入到pROK2表达载体,表达载体构建成功。PCR鉴定表明表达载体pROK2-WRKY已经转入根癌农杆菌EHA105中。本实验结果将为进一步研究大白菜抗软腐病基因的转化奠定基础。更多还原

【Abstract】 In order to obtain transgenic Chinese cabbage of BrWRKY33 gene on resistance against soft rot by using Agrobacterium tumefacien infection process, plant expressing vector should be contructed first. In this research, we designed two primers (FN/RN) according to the gene BrWRKY33 sequence and the restriction enzyme sites of plant expression vector pROK2 to gain the gene BrWRKY33, which was connected to the clone vector pGEM-T Easy next, then reconstructed the clone vector T-WRKY. After the validation of sequence and digestion, we established the expression vector pROK2-WRKY by choosing the product with lowset mismatch rate in PCR amplification. Finally, we identified the transformants after this vector was transformed into the Agrobacterium tumefacien EHA105. The results showed that the full-length target gene BrWRKY33 was 1 443 bp by PCR amplification. The result of PCR and digestion identified that the clone vector T-WRKYwas reconstructed successfully. Further, the result of PCR validation and digestion with BamHⅠand KpnⅠtwo enzymes displayed that there were 4 positive recombinants, which indicated that the target fragment has been inserted into pROK2 expression vector, that was the expression vector has been constructed successfully. The identification of PCR proved that the expression vector pROK2-WRKY has been transformed into the Agrobacterium tumefacien EHA105. In conclusion, the results of this investigation would lie a foundation for further research on transformation of genes in Chinese cabbage resistance against soft rot.更多还原