The family of subtilisin-like serine proteases in barley and Brachypodium distachyon

SONIA A. WIRTH; MARIA ISABEL RODRIGUEZ; IRMA N. ROBERTS

Parana

Congreso; LIV Reunión Anual SAIB; 2018

SAIB

Proteases play a crucial role in nitrogen remobilization during plant senescence. Free amino acids produced by protein degradation in senescent tissues are translocated to new developing leaves and reproductive organs allowing an efficient nutrient recycling. Among other protease families, subtilisins (family S8A) have been strongly associated to leaf senescence in wheat but are still scarcely studied in other plant species. The aim of this work was to identify the members of subtilisin family in barley and the model plant Brachypodium distachyon, and perform the phylogenetic analysis in both species. By using conserved sequences of wheat subtilisins, we were able to identified 40 complete genes in barley and 60 in B. distachyon databases. Phylogenetic trees were constructed and the identification of conserved protein domains was performed. Although all sequences contain the typical domain PA (PA_subtilisin_like), a few of them lack domain I9 thought to act both as internal chaperon and protease inhibitor in the zymogen. Previous studies allowed us to identified sequence AK365933 as one of the highest expressed subtilisin gene in senescent barley leaves. Here, cloning of the gene and expression of the recombinant protease in Pichia pastoris was intended. Two constructions were tested for cloning, one complete and one without domain I9. Primers were designed and both sequences were amplified by PCR, ligated to pGEMT-easy cloning vector and used for E. coli transformation for sequence amplification prior to subcloning in P. pastoris expression vector pPIC9. Construction containing I9 domain did not gave positive clones in E. coli, possibly due to lethality of basal expression of active subtilisin. Construction without domain I9 was subcloned in P. pastoris expression vector and used for transformation of GS115 yeast strain. Preliminary analysis of transformants showed production of the recombinant protein but no protease activity was observed. Currently, work is in progress in order to clone the complete version containing the I9 activation domain in P. pastoris for the subsequent characterization of the recombinant protein.