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以核盘菌菌株NGA4提取的总RNA为模版,利用RT-PCR技术扩增获得SsXYN基因的全长cDNA片段,将其克隆到pMD19-T载体上,菌落PCR、酶切鉴定和cDNA测序结果表明成功克隆了核盘菌SsXYN基因。从pMD19-T:SsXYN载体上,用BamH I和Sal I双酶切切下目的基因片段,将该基因片段连接到原核表达载体pET32a中。菌落PCR和酶切鉴定结果显示成功构建了表达载体pET32a:SsXYN。利用热击法将该重组表达载体导入大肠杆菌BL21,获得携带SsXYN基因的大肠杆菌菌株,为进一步研究激发子诱发植物抗病性机理奠定了基础。
The full-length cDNA of SsXYN gene was amplified by RT-PCR and cloned into pMD19-T vector. The results of colony PCR, restriction enzyme digestion and cDNA sequencing indicated that the full-length cDNA of SsXYN gene was amplified by RT- The S. sclerotiorum SsXYN gene was successfully cloned. The target gene fragment was cut from the pMD19-T: SsXYN vector by double enzyme digestion with BamH I and Sal I, and the gene fragment was ligated into the prokaryotic expression vector pET32a. Colony PCR and restriction enzyme digestion showed that the expression vector pET32a: SsXYN was successfully constructed. The recombinant plasmid was transformed into Escherichia coli BL21 by heat shock method to obtain the Escherichia coli strain carrying SsXYN gene, which lays the foundation for further study on the mechanism of elicitor induced plant disease resistance.