Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.46

ISOLATION AND FUNCTIONAL DISSECTION OF THREE CHS PROMOTERS IN VITIS VINIFERA

A. SCIALPI, A. ROSATI, M. MASIERI, G. MERSI, M. BUIATTI, P. BOGANI

Dipartimento di Biologia animale e Genetica “Leo Pardi”, Università di Firenze

Vitis vinifera, Chs, promoters, cis-acting elements, gene regulation, functional markers

Chalcone synthase (CHS) is the key enzyme in the first step of the biosynthetic pathway of anthocyanin and flavonoid compounds (Holton and Cornish, 1955). Genes encoding CHS form a multigene family the copy number of which varies among different species, from the single copy in Arabidopsis (Koch et al., 2001), to eigtheen gene isoforms characterized in Asteraceae (Yang et al., 2002).

In grapevine, up to the present Chss genes have been estimated at three to four (Sparvoli et al., 1994) and three have been sequenced and deposited in the GenBank DNA database (Goto-Yamamoto et al., 2002). Expression analysis, based on RT-PCR, suggested the genes to be under a different transcriptional control.

As to our knowledge, Chs grapevine promoters, have not been yet investigated, in this work we report the isolation of upstream regions of Chs1, Chs2 and Chs3 coding sequences, with the aim to identify conserved cis-acting elements conferring functional patterns of gene regulation. For this purpose, based on the Chs coding sequences, reverse primers were designed which annealed to the first exon, while forward ones were supplied by “Genome walker kit” (Clontech). PCR amplification products from DNA of Vitis vinifera cv Cabernet Sauvignon, were then cloned and sequenced by the dideoxy chain termination method (Sanger et al., 1977) with the DYEnamic™ ET Terminator cycle sequencing kit (Amersham Biosciences), using the ABI Prism 310 automated sequencer (Applied Biosystem). The obtained sequences were successively analyzed with PLACE (Higo et al., 1999), PlantCARE (Lescot et al., 2002), TRANSFAC (Wingender et al., 2000) softwares for the search of putatively conserved motifs related to the control of specific gene regulation. Moreover, we analyzed the amplified regions with the “STRETCHES” algorithm, developed in our laboratories for the search of hypervariable homogeneous stretches in coding and non coding regulatory regions and the detection of polymorphisms, to be used as functional markers in the assessment of genetic variability.

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