贵州野生天麻抗真菌肽基因表达载体的构建

Construction of Antifungal Peptide Gene Expression Vector of Wild Gastrodia in Guizhou Province

为了构建凤冈野生天麻的抗真菌肽基因(gaf)的表达载体,经BamHI和HindIII酶切的pMD18-T-gaf和pET32a(+)质粒的纯化回收产物并进行连接,连接产物pET32a(+)-gaf转化大肠杆菌感受态细胞DH5α后,采用菌落PCR、酶切鉴定和阳性质粒测序分析。结果表明:构建的凤冈野生天麻抗真菌肽基因表达载体通过菌落PCR、酶切鉴定和阳性质粒测序分析,目的片段成功插入载体,目的片段与预期的gaf基因大小(545 bp)相符。试验成功构建了贵州野生天麻抗真菌肽基因表达载体,pET32a(+)-gaf可以用来制备抗真菌蛋白。

To build antifungal peptide gene（gaf） expression vector of wild Gastrodia in Fenggang county,the pMD18-T-GAF was digested with BamH I and Hind III and ligated into plasmid expression vector Pet32a（＋） which had also been digested with the same enzymes.The resultant plasmid Pet32a（＋）-gaf was transferred into E.coli strain DH5а.The expression vector Pet32a（＋）-gaf was constructed by colony PCR,restriction enzyme digestion and positive plasmid sequencing.The results showed that the target fragment was successfully inserted into the vector,Consistent with the expected size of 545 bp of gaf gene.The gaf expression vector of wild Gastrodia was successfully constructed,pET32a（＋）-gaf can be used to prepare for anti-fungal protein.