SUBCELLULAR LOCALIZATION OF ASPARTIC PEPTIDASES FROM MILK THISTLE BY TRANSIENT EXPRESSION IN NICOTIANA

COLOMBO, MA.L.; VAIRO CAVALLI, S.; TORNERO, P.

Buenos Aires

Congreso; Molecular mechanisms in cell signaling and gene expression SAIB 2013; 2013

SAIBBM

Typical plant aspartic peptidases (APs) (E.C.3.4.23) from thistle flowers are expressed as zymogens. APs are not randomly distributed within plants. Moreover, in each part of the cell, proteolytic activity is performed by a separate protease specifically targeted to particularsubcellular compartments. This cellular anatomical specificity of plant APs has suggested a huge number and wide diversity of these enzymes. The aim of this work was to establish an eukaryoticexpression system for the study of silpepsin 1 and 2, two Aps from flowers of Silybum marianum (milk thistle) and to analyse its subcellular localization by confocal laser scanning microscopy.Transient expression in Nicotiana benthamiana leaves was accomplished by Agrobacterium- mediated DNA transfer. Both preprosilpepsins were cloned using GATEWAY BP reaction inpDORN222 and, subsequently subcloned in the binary vector pB7FWG2 by LR recombination reaction. The coding sequences were cloned in-frame with fusion region coding for Egfp under thecontrol of the CAMV 35S promoter. Recombinant clones were selected with spectinomycin. Positive clones were confirmed by PCR. Four-week-old plants grown at 25°C in long-day condition were agroinfiltrated. Results obtained would indicate that silpesin 1 and 2 direct GFP extracellularly in accordance with the expected localization in the secretory pathway.