Small non-coding RNAs (sRNAs) are a large number of regulators and are significantly important in physiological processes in bacteria.However,few approaches are available for identifying their cellular targets in vivo.In this study,a two-plasmid system based on the initial vectors pMycVec2 and pMycVec1 was established to screen the targets of mycobacterial sRNA.The vector (pMycVec2-D) for cloning sRNA was derived from the vector pMycVec2 and contained the strong promoter rrnBp and an effective transcription terminator,which has been modified to enable tight regulation of the expression of sRNA.The vectors for cloning target genes,pMycVec1-lacZ and pMycVec1-GFP,were constructed from the pMycVec1,which stabilized the transcription with a weak promoter Pwk,and the translation level of target was detected through the reporter genes.The two-plasmid system was validated by detecting the interaction between sRNA and its regulatory target,MicC/ompC mRNA and MicF/ompF mRNA respectively.