A versatile set of ligation-independent cloning vectors for functional studies in plants

Abstract

With plant molecular biology entering the omics era, there is a need for simple cloning strategies that allow high throughput to systematically study the expression and function of large numbers of genes. Such strategies would facilitate the analysis of gene (sub)families and/or sets of coexpressed genes identified by transcriptomics. Here, we provide a set of 34 ligation-independent cloning (LIC) binary vectors for expression analysis, protein localization studies, and misexpression that will be made freely available. This set of plant LIC vectors offers a fast alternative to standard cloning strategies involving ligase or recombination enzyme technology. We demonstrate the use of this strategy and our new vectors by analyzing the expression domains of genes belonging to two subclades of the basic helix-loop-helix transcription factor family. We show that neither the closest homologs of TARGET OF MONOPTEROS7 (TMO7/ATBS1) nor the members of the ATBS1 INTERACTING FACTOR subclade of putative TMO7 interactors are expressed in the embryo and that there is very limited coexpression in the primary root meristem. This suggests that these basic helix-loop-helix transcription factors are most likely not involved in TMO7-dependent root meristem initiation.

Figure 1.

LIC cloning procedure with modified LIC site. Vectors are first digested with HpaI restriction enzyme, and fragments are amplified by PCR with primers containing the LIC adapter sites. Overhangs are made by the 3′-5′ exonuclease activity of T4 DNA polymerase in excess of dCTP or dGTP for the vector and fragment, respectively. The sticky end overhangs that are created allow for easy annealing of vector and insert. LB, Left border; ori, origin of replication; RB, right border. [See online article for color version of this figure.]

Figure 2.

The linker introduced by LIC cloning. As an example, the linker is shown between a genomic fusion without stop codon and a fluorescent protein of choice (sYFP) by LIC cloning. AA, Amino acid. [See online article for color version of this figure.]

Figure 3.

Overview of LIC vectors. Indicated are left and right border (LB and RB), NOS promoter and terminator (pNOS and NOSt), resistance genes (B/K/H), nuclear localization signal (SV40), the LIC site (LIC), specific promoters for misexpression (p35S, pRPS5a, pMP, and upstream activating sequence), and respective fluorescent proteins (GFP, sCFP, sYFP, and tandemTomato). [See online article for color version of this figure.]

Figure 4.

Overview of promoter expression patterns of a subclade of TMO7-related bHLH transcription factors using the pGIIK-LIC-VP40-3GFP-NOSt vector. The phylogenetic tree shows a subclade of the bHLH transcription factors based on full-length protein sequences. Branch lengths indicate phylogenetic distances (see scale bar: fraction of deviations). Confocal images of primary root meristems were counterstained using FM4-64 dye (red). [See online article for color version of this figure.]