罗勒烯信号传导途径各组分突变体高通量活体荧光筛选技术系统的建立

Establishment of a Living Fluorescence Detection System for High-throughput Screening Mutants of Different Components in the Ocimene Signaling Pathway

为了高效率地发现罗勒烯信号传导途径上的各信号传递成分,本研究拟采用正向遗传学的策略,即利用EMS诱变与荧光素酶-荧光素活体荧光成像检测技术体系相结合的方法筛选突变体。为此,构建了指示基因启动子PR1pro::Luciferase和PDF1.2pro::Luciferase质粒并转化拟南芥;通过抗性筛选与PCR检测,鉴定获得了T3代转基因纯合子植株。同时,购买了合适的高灵敏CCD相机、暗箱与软件,通过组装调试,成功制造了一台自制的、经济适用的活体荧光检测仪。转基因纯合植株经过茉莉酸、水杨酸或罗勒烯处理和喷施荧光底物后,放到荧光检测仪中,成功观察到了诱导后的转基因植株释放出高亮荧光,说明活体荧光成像检测系统构建成功。这些结果为进一步利用正向遗传学的方法高效筛选罗勒烯信号传导途径中各成分的突变体植株,以及阐明罗勒烯诱导的防御反应的作用机理奠定了良好基础。

In order to efficiently find various signal transduction components in ocimene signaling pathway,this study uses forward genetic strategy, namely the technology system combining EMS mutagenesis andluciferase- luciferin in vivo fluorescence imaging detection technology for screening mutant. To do this,PR1pro：：Luciferase expression cassette and PDF1.2pro：：Luciferase expression cassette driven by PR1 genepromoter and PDF1.2 gene promoter respectively were constructed and the recombinant plasmids weretransformed into Arabidopsis. By antibiotic selection and PCR verification, T3 transgenic homozygous plantsfrom several lines were obtained. Meanwhile, a suitable high sensitivity CCD camera, camera obscura andsoftware were purchased, through assembling and debugging, a self- made, affordable in vivo fluorescencedetector was successfully generated. After treated by jasmonic acid, salicylic acid or ocimene alone followed by fluorescent substrate spraying, homozygous transgenic plants were submitted to fluorescence detector, highintensity of induced-fluorescence signal in transgenic plants was detected, indicating that in vivo fluorescenceimaging detection system was successfully constructed. These results provided a powerful forward geneticmethodology and made it possible to use high-throughput selection of plants carrying mutations in ocimenesignaling pathway components, as well as shed light on the mechanism of defense responses induced byocimene.