A schematic diagram of the CLX vector and β-estradiol-induced DNA excision. a Structural features of CLX vector pX6-AtIpk2β. Three transcription units are located within the two loxP sites: XVE consists of the coding sequence of the XVE hybrid transactivator terminated by the rbcs E9 polyA addition sequence and is activated by the G10-90 promoter upstream of the loxP site; npt-II consists of the nopaline synthase (NOS) gene promoter, the coding sequence of the neomycin transferase II (NPT-II) and the NOS polyadenylation sequence; cre-int consists of eight copies of the LexA operator sequence fused to the −46 CaMV 35S promoter, with the coding sequence of Cre interrupted by an intron and terminated by the NOS polyadenylation sequence. Downstream of the second loxP site, the AtIpk2β cDNA was terminated by the rbcs 3A polyA addition sequence. Arrows inside squares indicate the direction of transcription. P1–P5 denote primers used for PCR analysis shown in Fig. . b Putative products of the β-estradiol-induced site-specific DNA recombination. c Schematic map of marker containing construct pGI2300-AtIpk2β. Expression of AtIpk2β is also driven by the G10-90 promoter
The resultant vector, pX6-AtIpk2β, consists of three transcription
units (Fig. 1 a).
Upon induction by β-estradiol, sequences encoding the selectable
marker NPT-II, Cre and XVE between the two loxP sites were
excised from the tomato genome, leading to expression of the
downstream AtIpk2β (Fig. 1 b).
The selectable marker gene ( npt-II ) was controlled by
the cauliflower mosaic virus 35S (CaMV 35S) promoter (Fig. 1
To test the β-estradiol-induced DNA recombination in the
pX6-AtIpk2β transgenic tomato, we performed PCR analysis with
primers specific for the excised sequences and flanking nonexcised
sequences (Fig. 1 a, b).
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