基于烟草瞬时表达体系对amiRNA沉默效果快速有效的预验证

Rapid and Effective Pre-validation of amiRNA Silencing Strength by Transient Expression in Nicotiana benthamiana

amiRNA(artificial microRNA)作为一种诱导基因发生特异性沉默的技术已在多种植物中应用,但设计出的不同amiRNAs在所转化株系中的沉默效率难以预测,因此对amiRNA载体的沉默效率进行预验证是非常必要的。本实验以丹参(Salvia miltiorrhiza)的1个MYB类转录因子基因SmPAP1的mRNA序列为amiRNA作用对象,并挑选2个经在线软件WMD3(Web MicroRNA Designer)设计的amiRNAs,分别命名为amiRNA1-SmPAP1和amiRNA2-SmPAP1,然后通过农杆菌介导将构建的2个amiRNA载体和SmPAP1过表达植物载体在烟草叶片细胞中进行瞬时共表达。结果显示,amiRNA2的表达丰度约是amiRNA1的2倍;amiRNA2对靶标SmPAP1的沉默效率约是amiRNA1的2.5倍;SmPAP1在mRNA和蛋白水平上均与相应amiRNA的表达水平呈显著负相关。因此,amiRNA在烟草细胞中的瞬时表达可快速、有效地对不同amiRNA沉默效果进行预验证,从而为后续的植物遗传转化研究提供重要参考。

The utility of artificial microRNA（amiRNA）to induce specific gene silencing has been reported in many plant species,but silencing efficiency of differently designed amiRNA constructs in transgenic plants is less predictable.Thus,pre-validation of the silencing efficiency of designed amiRNA constructs is indispensable.In this study,to target the mRNA of SmPAP1,a R2R3-MYB transcription factor gene of Salvia miltiorrhiza,two amiRNAs were designed using WMD3（Web MicroRNA Designer）,designated as amiRNA1-SmPAP1 and amiRNA2-SmPAP1,respectively.The transient co-expressions of the two amiRNAs constructs combined with the 35S∶SmPAP1 plant overexpression vector were subsequently examined by Agrobacterium-mediated transformation into tobacco leaf cells,respectively.Results showed that the expression level of amiRNA2 was almost twice that of amiRNA1,and the silencing strength of SmPAP1 by amiRNA2 was 2.5 times higher than that by amiRNA1.The significant negative correlation between amiRNA abundance andexpression level of SmPAP1 at both the mRNA and protein level was observed in the transient agroinfiltration assays.Therefore,the assay for the transient expression of amiRNA in tobacco leaf cells can rapidly and effectively pre-validate silencing efficiency of diverse designed amiRNAs,and provide an important reference for subsequent genetic transformation in plants.