A versatile zero background T-vector system for gene cloning and functional genomics

Abstract

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.

Figure 1.

Construction of the ZeBaTA system. A, Schematic representation of direct cloning of PCR product using the ZeBaTA vector system. The linker of the vector (in gray) is removed after XcmI digestion yielding a linearized T-vector. B, TA cloning tests of the ZeBaTA system. (1) Self-ligation of XcmI-digested pGXT using T4 DNA ligase from Promega. (2) Ligation of XcmI-digested pGXT with the PCR product of the rice blast fungus M. oryzae gene MGG_07986.5 using T4 DNA ligase from Promega. (3) Ligation of XcmI-digested pGXT with the PCR product of MGG_07986.5 using T4 DNA ligase from USB Corporation. C, Samples of restriction digestion analysis of the randomly selected colonies derived from ligation of XcmI-digested pGXT with the PCR product of MGG_07986.5 using T4 DNA ligase from Promega. pGXT contains two BamHI recognition sites outside the two XcmI recognition sites (Supplemental Fig. S1), and MGG_07986.5 contains one internal BamHI site. All samples (lanes 1–20) digested by BamHI released two bands as expected. M, 1-kb DNA ladder.

Figure 2.

Site-specific mutagenesis of the maize ubiqutin-1 promoter (A) and the backbone of the binary vector pCAMBIA1300 (B) in which three XcmI recognition sites were deleted. The nucleotides represented in lowercase italic letters are the positions where deletions or mutations were made. Kan, Kanamycin resistance gene; LB, T-DNA left border; RB, T-DNA right border. C, Comparison of the levels of GUS expression mediated by the original and modified maize ubiquitin-1 promoter in transiently transfected rice protoplasts. GUS activities are represented as a ratio of relative GUS/LUC. The experiment was repeated three times with similar results. 1, Protoplast sample transfected with pUbiGUS; 2, protoplast sample transfected with pXUN-GUS.

Figure 3.

ZeBaTA-based expression vectors for gene overexpression/silencing, protein tagging, protein subcellular localization, and promoter analysis in plants. A, Schematic structures of the transient expression vectors generated by XcmI digestion. B, Schematic structures of the Agrobacterium-mediated stable transformation vectors generated by XcmI digestion. LB, T-DNA left border; RB, T-DNA right border.

Figure 4.

Transient expression and protein-tagging detection of the ZeBaTA vectors in rice protoplasts. A, Fluorescence microscopy of the expression of HA-tagged GFP in rice protoplasts. B, Detection of HA-tagged GFP by western blot. Lane 1, Nontransfected control protoplast sample; lanes 2 to 4, independent protoplast samples transfected with pXUN-HA-GFP. [See online article for color version of this figure.]

Figure 5.

Schematic illustration of the construction of hpRNAi or amiRNA constructs by single-step cloning. A, Generation of hpRNAi constructs by overlapping PCR approach. The target gene fragment and the stuffer sequence fragment are amplified in the first-round PCR. Primers P2, P3, and P4 introduce complementary adapters (indicated by vertically lined boxes) to the amplified fragments. The two amplified fragments are fused together as an inverted-repeat cassette in the second-round PCR by using single P1 primer. The resulting fragment is then directly cloned into the plant expression T-vector. B, Generation of amiRNA constructs by overlapping PCR approach. C, Generation of amiRNA constructs for rice genes by single-step PCR. The expression vectors pXUN-osaMIR528 and pCXUN-osaMIR528 were preassembled with 5′ and 3′ stemloop backbone sequences of a rice miRNA precursor Osa-MIR-528 (Warthmann et al., 2008). Thus, making amiRNA constructs for rice target genes only requires an amiRNA-amiRNA* fragment generated from single-step PCR. The nucleotides represented in lowercase letters are the positions where mutations were made to introduce two XcmI recognition sites.

Figure 6.

Silencing of the PDS gene in Arabidopsis and rice by the ZeBaTA-based hpRNAi or amiRNA approaches. A, Arabidopsis plants transformed with the hpRNAi construct pCXSN-atPDS-RNAi showing the PDS silencing albino phenotype. (1) Control plant; (2 and 3) two examples of transgenic Arabidopsis plants. B, Rice plants transformed with the amiRNA vectors showing the albino phenotype. (1) Control plant; (2) example of pCXUN-amiPDS-transformed plants; and (3) example of pCXUN528-PDS-transformed plants. C, RT-PCR analysis of PDS suppression transgenic rice plants. Five independent primary plants (1, 2, 3, 4, and 5) transformed with pCXUN-amiPDS and five independent primary plants (6, 7, 8, 9, and 10) transformed with pCXUN528-PDS were selected for the analysis. CK, Wild-type Nipponbare plant used as the control.

Figure 7.

Protein subcellular localization and promoter activity analysis using the ZeBaTA vectors. A, Fluorescence microscopy of the coexpression of GFP and DsRed, or GFP-SPIN1 and DsRed-SPIN1 fusions in rice protoplasts. Scale bar = 20 μm. The RNA binding nuclear protein SPIN1 was used as a tester (Vega-Sánchez et al., 2008). B, GUS staining of Arabidopsis transformed with pCX-35S-GUS, where the 35S promoter was cloned into the vector pCXGUS-P to test the system. CK, Plant transformed with control vector pCAMBIA1300 (www.cambia.org); pCX-35S-GUS-1 and pCX-35S-GUS-2, two independent primary transgenic plants.