2010.11.20 酵母的FLP-FRT重组系统应用 [PBJ]

Site-specific gene integration in rice genome mediated by the FLP–FRT recombination system Soumen Nandy1 and Vibha Srivastava1,2,* 1Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA 2Department of Horticulture, University of Arkansas, Fayetteville, AR, USA Received 5 September 2010; revised 14 October 2010; accepted 18 October 2010. *Correspondence (Tel: 479 575 4872; fax: 479 575 7465; email vibhas@uark.edu) Keywords: FLP–FRT, Cre-lox, DNA recombination, site-specific gene integration, gene targeting, rice transformation. Summary Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombina- tion systems are versatile tools for precise genomic manipulations such as DNA exci- sion, inversion or integration. The yeast FLP–FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP–FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was intro- duced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene con- struct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the trans- genic events contained a precise integration and expressed the transgene. Interest- ingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expres- sion. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP–FRT system for site-specific gene integration in plants using rice as a model. Introduction Transgene integration mediated by site-specific recombina- tion (SSR) systems is an effective approach for generating precise single-copy integrations in higher plants (reviewed by Srivastava and Gidoni, 2010). Most plant species can be transformed by Agrobacterium or particle bombard- ment–mediated gene delivery; however, the rate of single- copy integrations in these protocols is fairly low (Dai et al., 2001; Travella et al., 2005; De Buck et al., 2009). More- over, as the introduced DNA integrates randomly into the genome, it is impossible to predict the structure of the transgene locus. Therefore, it is highly desirable to develop strategies for controlling the transgene integration pro- cess. These strategies will not only improve the rate of single-copy integrations; they will also allow rapid charac- terization of the locus, and precise full-length integration of large constructs containing multiple transgene units into a single site, a feature most desirable for engineering poly- genic traits. As precise single-copy loci tend to express at higher levels compared to complex locus and maintain expression levels (correlated with gene dosage) through successive generations (Day et al., 2000; Schubert et al., 2004; Chawla et al., 2006; De Buck et al., 2007; Nanto et al., 2009; Akbudak et al., 2010), site-specific inte- gration (SSI) is an attractive feature for transformation technologies. While homologous-recombination-mediated gene targeting is now possible in higher plants (Terada et al., 2002; Shukla et al., 2009; Townsend et al., 2009), well-characterized SSR systems continue to serve as excel- lent tools as they carry out a simple and accurate reaction in a variety of plant cells, without causing ‘off-target’ genetic aberrations (Coppoolse et al., 2003; Ream et al., 2005). Several SSR systems are functional in plants, among ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd 1 Plant Biotechnology Journal (2010), pp. 1–11 doi: 10.1111/j.1467-7652.2010.00577.x which P1 phage Cre-lox and yeast FLP–FRT are the two most widely used and extensively characterized systems (Luo and Kausch, 2002; Ow, 2002; Srivastava and Gidoni, 2010). A number of SSR systems, including Cre-lox and FLP–FRT, are able to efficiently excise DNA fragments from transgene locus via a recombination between two sites placed in the same orientation (Grønlund et al., 2007; Gidoni et al., 2008; Thomson et al., 2009, 2010). How- ever, only a few SSR systems have been successfully used for SSI of foreign gene, which involves a recombination between two sites in trans. Of these, Cre-lox has so far displayed a superior SSI efficiency in plant cells (Srivastava and Gidoni, 2010). Because Cre-mediated recombination between two identical lox sites is freely reversible, use of a combination of mutant sites is critical in stabilizing the integration structures (Albert et al., 1995). Developing alternative systems for SSI application is highly desirable to incorporate additional features in the transformation plat- form such as marker removal. Selection of SSI events involves incorporation of a selectable marker gene, which cannot be removed from the genome unless a removal- strategy is incorporated in the transformation vector. Cre-lox-mediated marker removal is a popular strategy owing to its high efficiency (Gilbertson, 2003; Gidoni et al., 2008). Based on which, a marker-free site-specific gene integration strategy has been proposed that involves the use of an alternative SSR system for site-specific gene integration followed by marker removal by highly efficient system such as Cre-lox (Srivastava and Ow, 2004; Darbani et al., 2007; Nanto and Ebinuma, 2008; Fladung and Bec- ker, 2010). Because a number of studies have shown that FLP–FRT recombination occurs precisely in a variety of plant species (Luo and Kausch, 2002; Gidoni et al., 2008), it is an obvious choice for developing a new application. However, initial attempts on using FLP–FRT for site-specific gene integration in plants were not successful (Kerbach et al., 2005; Srivastava and Gidoni, 2010). Two different strategies for site-specific gene integration have been developed, each producing the same end prod- uct, the vector-backbone-free precise integration of the defined DNA segment. The recombinase-mediated cas- sette exchange (RMCE) strategy is based on the dual recombination between a pair of recombination sites resulting in the exchange of DNA cassettes between the target locus and the donor DNA. To prevent the excision or inversion of the DNA cassettes, two incompatible recombination sites are used in the RMCE strategies, e.g. the use of a wild-type lox and a mutant lox site in Cre-lox-based RMCE strategy (Louwerse et al., 2007). The co-integration strategy, on the other hand, consists of a single recombination between two recombination sites in trans resulting in the integration of the circular DNA mole- cule into the genomic target. The circular molecule is gen- erated de novo in the cell via recombination between two directly oriented sites in the introduced DNA, separating the gene cassette from the vector-backbone (Srivastava and Ow, 2002). Recently, Li et al. (2009) showed FLP–FRT- mediated gene integration in soybean, and Fladung et al. (2010) presented a preliminary report on FLP–FRT-medi- ated gene integration in hybrid aspen. Both groups used the RMCE strategy based on the recombination between a pair of FRT sites, resulting in the exchange of DNA cas- settes between the target locus and the donor DNA. Here, we demonstrate the use of FLP–FRT system for the co-integration of a circular DNA into the genomic tar- get site via a single FRT · FRT recombination in rice gen- ome. In the research leading up to the present work, we found that the co-integration strategy based on the expression of recombinase gene from the target locus was highly successful with Cre-lox system but not with the FLP–FRT system. The expression of the FLP gene was severely down regulated when an FRT site was incorpo- rated between the promoter and the coding region of the FLP gene, a component of the original co-integration strat- egy developed using the Cre-lox system (Srivastava and Ow, 2002). By supplying strong FLP activity through co-bombardment of FLP gene, SSIs were obtained at workable efficiency in two different target lines used in the study. This work describes a robust platform for plant transformation employing the biolistic gene gun for DNA delivery and FLP–FRT recombination system for gene integration. Results Molecular strategy The target construct, pNS5, contains a target FRTL site between the promoter and the coding sequence of FLP gene (NCBI accession no. NC_001398), while the donor construct, pAM18, contains a cassette flanked by FRT and FRTR (Figure 1a, b). The FRTL and FRTR, each contain a sin- gle-base mutation at -10 position in the left or the right FLP-binding sequence as described by Senecoff et al. (1988). FLP-mediated recombination between FRT and FRTR is expected to occur rapidly resulting in the genera- tion of a vector-backbone-free donor-circle containing FRTR (Figure 1c). FLP-mediated integration of the donor ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 1–11 Soumen Nandy and Vibha Srivastava2 circle would then generate a precise co-integration struc- ture consisting of transcriptional fusion of the selectable marker gene (NPT) with the Ubi promoter in the genomic target site (Figure 1d). The direct integration of the pAM18 DNA into the target locus is highly unlikely as the inter-molecular FRTL · FRTR reaction is kinetically disfa- voured over the intra-molecular FRTR · FRT reaction (Huang et al., 1991). However, if in rare cases the integra- tion of pAM18 DNA occurred, the resulting SSI structure will not be stable as it will contain pAM18-backbone flanked by two FRT sites, which would rapidly recombine out as a result of the highly efficient FRT · FRT recombina- tion. The resulting SSI structure would be identical to that generated by the integration of the donor circle (Fig- ure 1d). Within this structure, there are distinct 5¢ and 3¢ junctions spanning FRTL+R and FRT sites. Formation of FRTL+R, a relatively inactive site, is expected to stabilize the integration locus. The target construct, pNS5, also contains a hygromycin resistance gene, hpt, as a transfor- mation selection marker, and a heat shock cre gene for the excision of the lox-flanked marker genes. To allow removal of the marker genes flanking the gene-of-interest (e.g. GUS), pNS5 construct is flanked by two loxP sites. Integration of the gene-of-interest that is also flanked by inversely oriented loxP sites into the pNS5 target will gen- erate the SSI structure in which the DNA fragment on either side of the gene-of-interest will be flanked by directly oriented loxP sites. Thus, all target locus-specific genes and the NPT marker gene could be deleted by heat- induction of Cre-lox recombination. Previous studies have demonstrated the efficacy of soybean heat shock 17.5E promoter, utilized in this construct, for effectively regulat- ing cre expression for DNA excision (Zhang et al., 2003; Wang et al., 2005). Thus, the long-term goal of pNS5- target lines is to develop the method of marker-free site- specific gene integration based on the strategy described UBI FLP35S::hpt HSP::Cre L+R RBLB NPT Ubi::GUS SSI locus 6.8 1.72.8 p1 p2 p3 p4 0.7 0.5 UBI FLP35S::hpt HSP::CrepNS5 L RBLB 3>0.5 (a) 3.5 >1.3 NPT Ubi::GUS R (b) (c) (d) (e) Ubi FLPe pAM18 Donor circle pUbiFLPe R R R R R R R E E7.8 E0.8 E0.9 E E3.5 E0.9 Probe 1 Probe 4 Probe 2Probe 3 R >1.3 Figure 1 Design of DNA constructs and molecular strategy of site-specific gene integration. (a) pNS5 target construct contains three transcription units flanked by loxP sites (open triangles): hygromycin phosphotransferase (hpt) gene driven by CaMV 35S promoter, cre gene driven by soybean heat shock 17.5 E promoter (HSP), and FLP gene driven by maize ubiquitin (Ubi) promoter. Target FRTL site (shaded triangle) is incorporated into the leader sequence of FLP gene. RB and LB represent T-DNA borders. The two loxP sites are designed to enable future marker excision using the strategy described earlier by Srivastava and Ow (2004). (b) Donor vector pAM18 contains a promoterless neomycin phosphotransferase II gene (NPT) and b-glucuronidase gene (GUS) driven by Ubi promoter between FRTR and FRT sites (shaded triangle) in the pBluescript SK backbone (grey line). (c) The structure of donor circle generated from pAM18 upon FLP-mediated FRTR · FRT recombination. The circle contains the gene construct and FRTR without the vector-backbone. (d) Structure of site-specific integration (SSI) locus. Integration of donor circle via FRTL · FRTR recombina- tion traps Ubi promoter to activate NPT gene and forms a unique FRTL+R and FRT sites (shaded triangles). EcoRI (E) and EcoRV (R) sites for each construct and sizes of intervening fragments are shown. Shaded bars represent the DNA probe fragments used for Southern hybridization. PCR primers (p1–p4) and the expected PCR product sizes are also indicated. (e) pUbiFLPe vector contains FLPe gene driven by Ubi promoter in a pBlue- script SK backbone (grey line). Each transcription unit contains a transcription termination signal of nopaline synthase gene (nos 3¢). ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 1–11 FLP–FRT-mediated gene integration in plants 3 earlier (Srivastava and Ow, 2004). In the future experi- ments, a modified donor construct, containing the GUS gene flanked by inversely oriented loxP sites, will be used to carry out marker excision from the SSI locus. In the pres- ent work, however, the efficiency of FLP–FRT-mediated gene integration was tested without any interference of Cre-lox recombination. Therefore, pAM18 lacks loxP sites. Development and characterization of target lines Thirty-nine transgenic events with target construct, pNS5, were obtained using Agrobacterium-mediated transforma- tion. EcoRV-digested genomic DNA of each line was sub- jected to Southern hybridization with hpt, cre and FLP probe fragments (see Figure 1a). Subsequently, PCR was carried out using p1 and p4 on selected lines, and the resulting 0.5-kb PCR product was sequenced. Seven of 39 NS5-lines were found to contain a single-copy of pNS5 T-DNA and the expected FRTL site in the PCR product (Figure S1; data not shown). Two of these lines, A and B, were used in the subsequent experiments, and FLP activity was supplied by co-transformation of FLPe gene as these target lines failed to express sufficient levels of FLP activity (described below). T1 seeds of these lines were used to generate callus on hygromycin-containing media. Use of hygromycin was intended to eliminate the wild-type segre- gants among T1 progeny. FLP expression analysis To explore the basis of sub-optimal FLP expression in the NS5-lines, two FLP constructs (pUbiFLP and pUbiFrtFLP) differing only in the presence of the FRT site, were com- pared (Figure 2a, b). Each contains a strong Ubi promoter. Co-bombardment of pUbiFLP and pRP9 on rice callus gen- erated strong GUS activity (Figure 2a), while single bom- bardment of pRP9 did not display any GUS activity. Thus, FLP expression originating from pUbiFLP catalysed a strong FRT · FRT reaction. However, co-bombardment of pUb- iFrtFLP and pRP9, on wild-type callus, did not generate any GUS activity (Figure 2b), suggesting that insertion of FRT between Ubi promoter and FLP coding region ham- pers gene expression. Next, two GUS constructs, differing only in the presence of the FRT site, were compared (Figure 2c, d). The FRT-containing vector, pUbiFrtGUS, displayed detectable GUS activity, although it appeared weaker than that of the FRT-lacking vector, pUbiGUS. Thus, insertion of FRT sequence downstream of promoter confers a general suppressive effect on the gene activity. However, in case of pUbiFrtFLP, a greater suppressive effect, presumably as a result of the tight binding of FLP protein to FRT site, was observed. This hypothesis is sup- ported by the analysis of the NS5-lines, which contain a barely detectable level of FLP transcript (as per RT-PCR, data not shown). The formation of the FLP–FRT complex on the NS-5 locus could severely suppress transcription resulting in the sub-optimal FLP activity in the target lines. Generation and characterization of SSI lines To supply strong FLP activity for site-specific gene integra- tion, FLP gene was co-bombarded with the donor con- struct, pAM18. An enhanced version of FLP recombinase, FLPe, that displays 3–5 times higher efficiency compared Ubi FLP pUbiFLP Ubi FLP pUbiFrtFLP Ubi GUS pUbiGUS Ubi GUS pUbiFrtGUS Ubi GUS pRP9 Block 30 – 50 + Ubi GUS pRP9 Block 0+ >50 10 – 15 (a) (b) (c) (d) Constructs Bombarded GUS activity (No. of blue spots) Figure 2 Effect of FRT site on gene expres- sion. The effect of the presence of FRT site in the leader sequence of FLP and GUS genes was studied by bombardment of plas- mid DNA into wild-type callus cells and staining them for GUS activity 48 h later. (a, b) Co-bombardment of FLP constructs differing only in the presence of FRT site (grey triangle) with the FLP recombination target construct, pRP9. (c, d) Single bom- bardment of GUS constructs differing only in the presence of FRT site. GUS activity for each construct is given as number of blue spots per bombarded plate. ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 1–11 Soumen Nandy and Vibha Srivastava4 to the wild-type FLP (wtFLP) (Buchholz et al., 1998), was used in the present experiment. FLPe was developed by cycling mutagenesis using DNA shuffling approach and therefore contains 95% sequence homology with the wtFLP. Callus derived from T1 seeds of target lines A and B was bombarded with an equimolar mixture of pAM18 and pUbiFLPe. Cultures of target lines were raised on hygromycin-containing media to avoid wild-type segre- gants within T1 seeds. The bombarded callus was selected on geneticin (100 mg ⁄ L) to isolate the putative SSI events. Ten geneticin-resistant events were obtained in two sepa- rate experiments using target line A (A1–A10), and three events using target line B (B1–B3) (Tables 1 and 2). Nine of these lines expressed GUS activity (Table 2). Genomic DNA from all 13 callus lines was analysed by PCR for the presence of SSI junctions and with Southern hybridization for the accuracy of the SSI structure. All but one line (B3) amplified the 5¢ junction fragment (0.6 kb) in a PCR using primers p1 and p2 (Figure 3a, Table 2), although A9 amplified a larger than expected fragment. Sequencing of this fragment from the selected lines (A1, A2, B11 and B12) revealed the presence of accurate recombination footprint consisting of FRTL+R sequence (data not shown). PCR on 3¢ junction, using primers p3 and p4, amplified the expected 0.5-kb fragment from only nine of the 13 lines (Figure 3a; Table 2). Sequencing of this fragment from the above-selected lines also revealed the presence of the accurate recombination footprint consisting of FRT sequence. Southern hybridizat