Abstract：Mediator is a large, modular protein complex. Interactions among mediator subunits are crucial for mediator complex to play the important roles in regulating transcription in vivo. We cloned tobacco(Nicotiana tabacum)NtMed6、NtMed18 and NtMed21 by homology searching method. Sequencing results showed that the full-length coding sequences (CDS) of NtMed6, NtMed18 and NtMed21 span 753, 651 and 366 bp respectively, which share high similarities (69.46%, 76.91% and 77.01% respectively) with their corresponding homologues in Arabidopsis thaliana. By screening different infiltration buffers, we optimized the method for detecting the subcellular locations of these fused genes by transient transformation assays performed with Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves. At the same time, we constructed the expression vectors of these three genes fused with enhanced yellow fluorescent protein (EYFP) genes driven by 35S promoter, and analyzed the function of linker between NtMed8 protein and EYFP in subcellular locations. The results revealed that YFP expression showed higher fluorescence intensity and cell density which scattered the fluorescent protein infected with the infiltration buffer of YEB+100 mmol/L AS and AA+100 mmol/L AS, and the linker with five amino acids improved the truth of subcellular locations. The four subunits of mediator NtMed8, NtMed18, NtMed6 and NtMed21 were all located in both nucleus and plasma membrane, but mainly in nucleus. To further confirm whether NtMed8 belongs to tobacco mediator subunits as well as the interactions between NtMed8 and other tobacco mediator subunits, bimolecular fluorescence complementation (BiFC) vectors containing coding sequence of EYFP N-terminus from amino acids 1-173 (pNYE) and coding sequence of YFP N-terminus from amino acids 151-238 (pCYE) driven by 35 S promote were constructed to subclone NtMed8, NtMed6, NtMed18 and NtMed21 full length ORFs into the BiFC vectors respectively. Then the subcellular locations with different combinations of the vectors by transient cotransformation assays performed with Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves were also detected. And the results showed that interactions exist between NtMed8 and NtMed18, NtMed8 and NtMed6 as well as NtMed18 and NtMed6. On the basis of these results, we conclude that BiFC is a good method which can be used in analysis of interactions between subunits of mediator
