Construction Of A Set Of Plant BiFC Expression Vectors For Nimble Cloning

Nimble Cloning is a novel,nolecular Cloning technology.which is simple,versatile,and efficient.But Nimble Cloning requires supporting expression vectors.In this study,the series BiFC expression vectors of pDOE were reconstructed,by removing their intrinsic SfiI sites,and inserting NC Cloning frames into the vector to construct a set of plant BiFC expression vectors for Nimble Cloning,and they were used for protein interaction research.The series BiFC expression vectors of pDOE used pCambia vector as backbone.Their intrinsic Sfil sites should be removed first to become Nimble Cloning system vectors.In this study,five Sfil restriction sites in the pCambia were removed by base substitution,and the stability of the reconstructed vector in Agrobacterium and the expression in plants were analyzed.The results showed that the mutations of 5 Sfil sites had no negative effect on the stability and expression of the vector.The mutant vector backbone is suitable for constructing to Nimble Cloning system vector.The Sfil sites in the four integrated BiFC expression vectors of pDOE were removed,by using the fragment containing five Sfil sites mutation in Nimble Cloning reaction.Then the NC clone frame was inserted in the multiple cloning sites of the four vectors,and a series of integrated BiFC expression vectors with pCambia as the backbone were constructed.They are named as pNC-BiFC-VD series vectors.On the basis of the integrated vector,one of the expression cassette for fluorescence protein fragment expression frame was removed by enzyme digestion and ligation,and a series of BiFC expression vectors of traditional single ORF were constructed,which named as pNC-BiFC-V series vectors.In order to widely use this set of BiFC vectors,the pCambia backbone was replaced with pGreen backbone in this study,and a series BiFC expression vectors with pGreen backbone were constructed.The interactions of two pairs of proteins were examined by using the pNC-BiFC-V series vectors.The two pairs of proteins are papaya eIFiso4E and PRSV-vpg which occur interaction in the nucleus,and PLDMV-HCPro which occur self interaction in the cytoplasm.Genes of these proteins were cloned into the pNC-BiFC-V series vectors by using Nimble Cloning technology,then the plasmids were transformed into tobacco for transient expression by agrobacterium-mediated transformation.The expression of fluorescence was observed by laser confocal microscope.The results suggested that the tobacco leaves injected with papaya eIFiso4E and PRSV-vpg genes all showed fluorescence in the nucleus,while the tobacco leaves injected with PLDMV-HCPro showed different levels of fluorescence in the cytoplasm.The plant BiFC vectors constructed in this study for the Nimble Cloning system were simple and convenient,and they are expected to be widely used in protein interactions in plants.At the same time,this set of BiFC vectors will also enrich the supporting vectors of Nimble Cloning system,and promote the popularization and application of Nimble Cloning technology.