|Artlip, Timothy - Tim|
Submitted to: Acta Horticulturae
Publication Type: Proceedings
Publication Acceptance Date: 10/14/2005
Publication Date: 3/1/2007
Citation: Norelli, J.L., Bassett, C.L., Artlip, T.S., Aldwinckle, H.S., Borejsza-Wysocka, E., Gidoni, D., Flaishman, M. 2007. Inducible dna promoters for use in apple. Acta Horticulturae. 738:329-332.
Technical Abstract: A chemically inducible and a light inducible DNA promoter are being developed and evaluated for their ability to regulate gene expression in transgenic apple. The estradiol-induced XVE gene expression system (Zou et al. 2000 Plant J. 24:265-273) is regulated by a chimeric transcription factor containing the DNA-binding domain of LexA, an activation domain and the regulatory region of the human estrogen receptor, and a corresponding DNA promoter containing multiple copies of the LexA operator fused upstream to a minimal CaMV 35S promoter. These elements were cloned into a binary vector (pBinPlusARS) compatible for use in Agrobacterium-mediated transformation of apple. A GUS marker gene was cloned into pBinPlusARS.XVE using 4 different technologies including PCR cloning, TOPO (Invitrogen) cloning in 2 different reading frames, and GATEWAY (Invitrogen) cloning. When evaluated in tobacco under non-inducing conditions, GUS expression from the GUS containing XVE constructs was indistinguishable from that of the negative control. However, in the presence of estradiol, the PCR and GATEWAY derived vectors had significantly higher levels of GUS expression than either of the TOPO cloned vectors or the negative control (824, 1377, 7.2, 1.12, and 0.03 nM MU/min/ug protein, respectively; 35S control = 138). The low level of expression in the TOPO cloned vectors was probably due to an initiation codon carried over from the TOPO cloning intermediate (pCR2.1-TOPO). To evaluate the activity of the light inducible promoter of the peach chlorophyll a/b binding protein and XVE in transgenic apple, binary vectors were constructed to allow comparison of test promoter activity with 35S activity. Using pCAMBIA2301, vectors were constructed with test promoters driving expression of green fluorescent protein (gfp94) and 35S driving expression of GUS. Test vectors were transformed into apple, and the promoters are currently being evaluated in apple.