论文部分内容阅读
以致病性嗜水气单胞菌(Aeromonas hydrophila)人工感染的中华鳖(Trionyx sinensis)心、肝、脾、肾为材料,应用SMART技术(switching mechanism at 5’end of RNA transcript)构建了嗜水气单胞菌感染组织的cDNA文库。将获得的cDNA片段连接到PGEM-T载体,然后电转化进入大肠杆菌DH5α感受态细胞中,PCR阳性检测结果显示插入片段在0.5~2 kb之间。该库含有4.3×105个重组子,重组效率为92%,本实验分析鉴定了164个≥1000bp的片段,39个序列在中华鳖中首次发现,鉴定的基因包括免疫基因11个;信号转导基因6个:催化类基因15个;转运相关基因3个;结构基因7个;未知基因9个。本实验文库的成功构建,不仅鉴定了中华鳖机体与细菌感染相关的免疫基因,同时为深入展开免疫学、系统发育和病原与机体的互作研究奠定了良好基础。
The heart, liver, spleen and kidney of artificially infected Trionyx sinensis infected with Aeromonas hydrophila were used as materials, and the SMART (switching mechanism at 5’end of RNA transcript) A DNA library of Aqueous Aeromonas infected tissues. The obtained cDNA fragment was ligated into the PGEM-T vector and then electroporated into E. coli DH5α competent cells. PCR positive results showed that the inserted fragment was between 0.5 and 2 kb. The library contained 4.3 × 105 recombinants and the recombination efficiency was 92%. 164 fragments ≥1000bp were identified in this experiment. 39 sequences were found for the first time in Chinese soft-shelled turtle. The identified genes included 11 immune genes and signal transduction 6 genes: 15 catalytic genes, 3 transport related genes, 7 structural genes and 9 unknown genes. The successful construction of the experimental library not only identifies the immune genes related to bacterial infection in the body of the Chinese soft-shelled turtle, but also lays a good foundation for further studies on immunology, phylogeny and interaction between the pathogen and the body.