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根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5’端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5’端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5’端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区(-565bp),核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。
According to the MIPS gene sequence of Jatropha curcas, the specific nested primers were designed and the 5 ’flanking sequence of MIPS gene was obtained by two steps of TAIL-PCR. Sequence analysis showed that there are several stress response related elements such as ABRE, HSE, etc. . Based on this sequence, five 5 ’flanking fragments were amplified by PCR, inserted into the pBI221 vector to replace the CaMV35S promoter, respectively. The constructed expression vector was transformed into protoplasts of tobacco leaves for transient expression under PEG-mediated conditions. GUS reporter gene activity. The results of GUS assay showed that GUS reporter gene was activated by different deletion fragments at the 5 ’end of the flanking sequence of MIPS gene. The most active promoter was WQ1 (-565bp) and the core region was located at -565 ~ -449bp. The promoter activity was enhanced under the induction of 100μmol·L-1ABA, but the growth of different segments was different. The WQ1 area has the largest growth rate, increasing by 41.4% compared with the untreated area.