麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

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%。

Isolation of MIPS Gene Promoter from Jatropha curcas L.and Activity Analysis of Transient Expression in Tobacco Protoplast

Using nested primers specific for the sequences of Jatropha curcas MIPS gene,the MIPS gene 5＇flanking sequences were obtained by TAIL-PCR.Some stress response elements were found by sequence analysis,such as ABA-responsive element （ABRE） and heat stress responsive element （HSE）.Five different 5＇end deletion fragments of the MIPS gene 5＇-flanking sequences were amplified by PCR,and they were inserted into the plant transient expression vector pBI221 to replace CaMV 35S promoter respectively.The expression vectors were transferred into tobacco leaf protoplasts by PEG-mediated transient expression and the different promoter activities were quantitatively estimated using GUS report gene.The results demonstrated that the isolated 5＇-end different deletion fragments of MIPS gene could initiate GUS reporter gene expression in a tobacco leaf protoplast transient expression system.The GUS activity of WQ1 area （-565 bp） was the highest and the core area was located between-565 bp and-449 bp.The activity was increased by ABA （100 μmol·L-1） treatment,but the increase in GUS activity of different segments was not the same.GUS activity was increased by 41.4% in WQ1 area,when compared with the control.