A series of TA-based and zero-background vectors for plant functional genomics

Abstract

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.

Figure 1. Construction of the TA-based expression vectors.

A, workflow for the direct cloning of PCR products using the TA-based expression vector system. TA-based expression vectors were digested by XcmI to produce 3′-T-overhangs. The PCR products were ligated with the XcmI-digested vector by T4 ligase. B, Left: Self-ligation of the XcmI-digested vector by T4 ligase. Right: Ligation of the PCR product of the GFP gene with the XcmI-digested vector by T4 ligase, followed by transformation into DH5α strains. C, Samples of restriction digestion analyses of randomly selected colonies from the ligation of the XcmI-digested pGreen vector with the product of the GFP gene using T4 ligase.

Figure 2. TA-based expression vectors for gene overexpression, protein subcellular localization, protein tagging, promoter analysis and inducible expression in Arabidopsis.

A, Schematic structures of TA-based transient expression vectors generated by XcmI digestion. B, Schematic structures of Agrobacterium-mediated stable transformation vectors generated by XcmI digestion. The dark boxes at the left and right ends signify the T-DNA LB and RB borders.

Figure 3. Subcellular protein localization using TA-based expression vectors.

A&B, Transient expression of 35S promoter lined with GFP-fused AtSOS2 protein in Arabidopsis mesophyll protoplasts(A), or Ubiquitin promoter linked GFP-fused OsICE1 protein in rice mesophyll protoplasts (B) visualized by fluorescence microscopy. Top: mesophyll protoplasts transformed with empty vectors, Bottom: mesophyll protoplasts transformed with the vector with AtSOS2-GFP (A) or OsICE1-GFP (B). C, Overexpression of the AtSOS2-GFP fusion protein in the root tips of transgenic Arabidopsis by the TA-based pGreen -35S-K expression vector. T3-1, T3-9 and T3-15 indicate three individual T2 transgenic lines overexpressing the AtSOS2-GFP fusion protein.

Figure 4. Overexpression analysis of the CBL5-HA protein by TA-based expression vectors.

A) Transgenic Arabidopsis overexpressing CBL5-HA showed more tolerance to salt stress. Two individual transgenic lines overexpressing CBL5-HA (indicated by CBL5-OX-3 and CBL5-OX-7) were treated with 100 mM and 200 mM NaCl, respectively, for two weeks, after which the pictures were taken. B) Real-time quantitative PCR confirmed a higher transcriptional level of the CBL5 gene in the individual transgenic lines CBL5-OX-3 and CBL5-OX-7. C) Western blot showing the high accumulation of CBL5-HA protein in the two individual transgenic lines CBL5-OX-3 and CBL5-OX-7.

Figure 5. Promoter analysis using TA-based cloning vectors.

A. GUS staining of an Arabidopsis line transformed with P_HOS1::GUS, in which the HOS1 promoter was cloned into the pGreen-GUS-K vector by TA cloning. Left: The control line without transformation, Right: The transgenic line with the Tubulin promoter fused to GUS. B, GFP fluorescence of Arabidopsis transformed with P_HOS1::3xGFP, in which the HOS1 promoter was cloned into the pGreen-3GFP-K vector by TA-based cloning. Top: The control lines without transformation. Bottom: The transgenic lines with the SOS2 promoter fused to 3xGFP.

Figure 6. Overexpression of an artificial miR319 by the pGreen TA-based cloning system.

A, Construction of an artificial miR319 by the TA-based pGreen-35S-HA-K vector. B, Detection of miR319 accumulation in five individual transgenic lines with overexpression of the artificial miR319 (indicated by L1, L3, L8, L11 and L15). C, the leaf phenotypes of the transgenic L8 line overexpressing miR319 and the control, wild type Arabidopsis. Left: The control, wild type Arabidopsis, Right: The transgenic line L8 overexpressing artificial miR319.

Figure 7. Induction of gene expression by the inducible TA-based pGreen vector system.

A, Construction of the inducible expression vector with ICE1-HA by the pGreen-XVE-K vector. Only the region between the right and left borders is shown (not to scale). PG10-90, a synthetic promoter controlling XVE transcription after beta-estradiol treatment. XVE: the DNA unit encoding a chimeric fragment containing the DNA-bind domain of LexA, the transcription activation domain of VP16 and the regulatory of the human estrogen receptor; OlexA: eight copies of the LexA operator sequence; −46: the −46 35 S minimal promoter; ICE-HA; the arabidopsis ICE1 gene linking the HA tag. B, Detection of ICE1-HA fusion protein accumulation in transgenic lines (ICE1-OX-1 and ICE1-OX-3) and wild type control lines. Two-week-old Arabidopsis were either not treated or treated with 10 µM beta-estradiol for 3 days, and the ICE1-HA protein was detected by the anti-HA antibody. –ER: without beta-estradiol treatment, +ER: with beta-estradiol treatment. C, Tests of the germination capabilities of seeds from the transgenic ICE1-OX-1 line and the wild type control line at either normal 22°C conditions or at a chilling treatment of 4°C. These tests were performed for 5 days, after which photos were taken. The experiments were repeated three times with similar results, and one represent experiment is shown.