Vector Design Flanking Regions

Agrobacterium-mediated transformation utilizes universal vector systems (Lee and Gelvin 2008; see also Chapter 1) in which the transgene expression cassette is flanked by two rather short sequence stretches, termed left border (LB) and right border (RB). These sequences facilitate almost random insertion of the transgene cassette into the host genome, resulting in multiple individual lines differing in site and numbers of transgene integration. In absolutely contrast, insertion of foreign DNA into the chloroplast genome relies on targeted integration of transgenes by homologous recombination, facilitated by a bacterial recombination system inherited from the plastids cyanobacterial ancestors (Cerutti et al. 1992). Hence, a transgene could be targeted to virtually any site in the chloroplast genome by designing the flanking regions according to the desired location. This is not only a big advantage for the positioning of expression cassettes to defined locations but also enables the targeted inactivation of plastid genes for functional studies and gene knock-outs. For the former, preference is naturally given to intergenic regions to circumvent deleterious effects and interference with endogenous gene expression. For gene knock-out, the targeted sequence is mutated in vitro and reinserted into the plastome. In the case of tobacco, numerous studies describe the targeted knock-out of plastidal genes for functional studies (reviewed by Maliga 2004). Additionally, a total of 13 sites on the plastome has been utilized for the integration of an expression cassette (Fig. 2.2), demonstrating that modification and integration

Petunia Plastome

Fig. 2.2 Graphic map of the Nicotiana tabacum plastid genome (GeneBank accession number NC_001879), made with the web-based program OGDRAW (Lohse et al. 2007). Genes on the outside of the circle are transcribed counter-clockwise, those on the inside clockwise. IRA Inverted repeat A, IRB inverted repeat B, LSC large single copy region, SSC single copy region. Numbered arrows Transgene integration sites. Dashed arrows (numbers 8-13) Site of integration into the inverted repeat region; therefore integration sites are in duplicate. First published reports are given:

I Carrer and Maliga (1995), 2 Bock and Maliga (1995), 3 Huang et al. (2002), 4 Svab and Maliga (1993), 5 Huang et al. (2002), 6 Kuroda and Maliga (2003), 7 Suzuki and Maliga (2000) and Klaus et al. (2003), 8 Zoubenko et al. (1994), 9 Staub and Maliga (1993), 10 Svab et al. (1990),

II Muhlbauer et al. (2002), 12 Huang et al. (2002) and Zou et al. (2003), 13 Koop et al. (1996) and Eibl et al. (1999)

Fig. 2.2 Graphic map of the Nicotiana tabacum plastid genome (GeneBank accession number NC_001879), made with the web-based program OGDRAW (Lohse et al. 2007). Genes on the outside of the circle are transcribed counter-clockwise, those on the inside clockwise. IRA Inverted repeat A, IRB inverted repeat B, LSC large single copy region, SSC single copy region. Numbered arrows Transgene integration sites. Dashed arrows (numbers 8-13) Site of integration into the inverted repeat region; therefore integration sites are in duplicate. First published reports are given:

I Carrer and Maliga (1995), 2 Bock and Maliga (1995), 3 Huang et al. (2002), 4 Svab and Maliga (1993), 5 Huang et al. (2002), 6 Kuroda and Maliga (2003), 7 Suzuki and Maliga (2000) and Klaus et al. (2003), 8 Zoubenko et al. (1994), 9 Staub and Maliga (1993), 10 Svab et al. (1990),

II Muhlbauer et al. (2002), 12 Huang et al. (2002) and Zou et al. (2003), 13 Koop et al. (1996) and Eibl et al. (1999)

of heterologous genes can be performed at many given sites in the circular plastid genome.

One of the unique features of the chloroplast genome is the presence of two large inverted repeat regions (IRA and IRB in Fig. 2.2). Integration of the transgene into this particular region leads under selection to a doubling of the gene by a process called copy correction. Especially, sites in the rrn operon have been frequently chosen for transgene integration and gene expression from these sites has proved to be high in many cases (Verma et al. 2008).

To facilitate efficient recombination, flanking regions of about 1-2 kb endogenous DNA should frame the sequence to be inserted. The question whether a transformation vector for a certain plant species requires strain-specific flanking regions was clearly negated by Lutz et al. (2007). This is due to the sufficiently high sequence homology of plastid genomes between species to facilitate homologous recombination. In fact, vectors designed for transgene integration into the tobacco plastome could be also used for transformation of tomato (Ruf et al. 2001; Chapter 25), potato (Sidorov et al. 1999; Chapter 20), and petunia (Zubko et al. 2004; Chapter 19). Two recent papers describe convenient vector systems designed for chloroplast transformation (Lutz et al. 2007; Verma and Daniell 2007).

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