植物microRNA功能瞬时验证体系的建立

Construction of the in vivo Validation Systems of Plant microRNA Functions

目的:建立植物microRNA(miRNA)功能的瞬时活体验证体系,并检验该体系的有效性。方法:选用双元表达载体pCAMBIA1200,并插入烟草花叶病毒双35S启动子,以驱动目标miRNA超表达;选用双元表达载体pFGC5941的绿色荧光蛋白(GFP)改造载体用于潜在的靶基因与GFP融合蛋白的超表达,以转入这2种载体的农杆菌侵染烟草叶片,观察GFP融合蛋白的荧光,作为验证miRNA对其潜在靶基因调控作用的瞬时验证体系。选取拟南芥已知功能的miR393及其靶基因AFB3,分别构建pCAMBIA1200-35S-miR393和pFGC5941-GFP-AFB3载体,利用农杆菌注射烟草叶片进行2个载体共转化,并以pFGC5941-GFP-AFB3单转化作为对照,激光共聚焦显微镜下观察融合蛋白的表达。结果:只将AFB3导入烟草表皮细胞,可观察到绿色荧光;而将miR393与AFB3同时导入烟草表皮细胞后,未能观察到绿色荧光。表明miR393抑制了AFB3的表达。结论:本瞬时表达体系可作为植物miRNA功能的活体瞬时验证体系,为miRNA调控靶基因表达功能提供简单、快速、有效的证据。

Objective： To constructing the in vivo transient tion and to confirm its effectiveness. Methods： The modified validation system of plant microRNA（miRNA） func binary expression vectors pCAMBIA1200 with insert ed CAMV double 35S promoter and pFGC5941 with inserted green fluorescence protein（GFP） coding sequences were adopted to transform the miRNA and potential target-GFP fusion protein respectively by agroinfiltration of the leaves of tobacco. Then the impact of the miRNA on the expression of the potential target could be validated by observing the fluorescence of the GFP fusion protein. The arabidopsis miR393 and its target AFB3, were adopt ed to construct pCAMBIA1200-35S-miR393 and pFGC5941-GFP-AFB3 vectors. The two vectors were co-trans- formed into tobacco leaf by agroinfihration and the pFGC5941-GFP-AFB3 were transformed alone as the control. Results： By a confocal laser scanning microscope, the green fluorescence could be observed in the nuclear of the control tobacco cells. However, in the co-transformed tobacco cells, no green fluorescence could be observed, dem onstrating that miR393 had inhibited the expression of AFB3. Conclusion： This transient expression system we constructed could be used for the in vivo validation of plant miRNA functions and as a simple and speedy sys tem, and could supply efficient evidence to the miRNA function.