Isolation and characterization of snakin-1 gene promoter.

ALMASIA, NATALIA INÉS; BAZZINI, ARIEL ALEJANDRO; HOPP, H. ESTEBAN; VAZQUEZ-ROVERE, CECILIA

Ischia (Italy)

Workshop; 2nd SOLANACEAE GENOME WORKSHOP.; 2005

Snakin-1 (SN1) is a newly characterized antimicrobial peptide isolated from potato tubers that has been found to be active in-vitro against bacterial and fungal pathogens from potato and other plant species. Gene expression has been detected in tubers, stems, axillary buds, and young floral buds suggesting that protein SN1 may be a component of constitutive defense barriers, especially those of storage and reproductive plant organs (Segura et al., 1999). Its regulatory region has not been studied until now. We report here the cloning, sequencing and analyis of the promoter region placed upstream of Solanum tuberosum snakin-1 gene. A 671 pb fragment was amplified by IPCR using potato Kennebec germplasm as template. A probableTATA and CAAT motives were identified as well as potentials cis-acting regulatory elements in the sequence. Although binding sites detection is important, it is generally not sufficient to elucidate promoter function.  GUS expression was detected in onion and stem apex, tubers and axillary buds of potato in transient expression assays using two versions of the cloned sequence fused to the reporter gene betaglucuronidase. In order to study in- vivo this region we obtained transgenic potato plants with the two constructions.  Further studies will clarify the influence of differents growth regulators in order to better understand its physiological functions. On the other hand, using the cloned sequence as a probe we have identified ten positives clones from a commercial library of Solanum bulbocastanum. Two fragments of 4000 and 6000 pb were identified in Southern blot assays of restricted DNA from two clones and they are being subcloned in order to perform a deeper study