Molecular Cloning of the Gene Encoding the L-Asparaginase Gene of Arabidopsis thaliana

Abstract

In plants, ammonium is first assimilated into the amino acids L-Gln and L-glutamate and then channeled into L-aspartate and L-Asn. These amino acids serve as nitrogen donors in biosynthetic reactions, as well as compounds for intercellular nitrogen transport. In amide-transporting plants, Asn can act as the sole nitrogen source for the growth of plant tissues. In higher plants the catabolism of Asn is thought to occur mainly through two routes. The first pathway involves the hydrolysis of Asn, releasing ammonia and aspartate by asparaginase activity. The second pathway proceeds via transamination in a reaction catalyzed by the asparaginase:2oxoacid transaminase (Lea et al., 1990). L-Asparaginase is a key enzyme for Asn utilization by plants that plays an important role in the nitrogen metabolism of developing plant tissues (Sieciechowicz et al., 1988). The ammonia released from the hydrolysis of the translocated Asn is utilized in the synthesis of all nitrogen-containing compounds of the cell and in particular in the synthesis of the amino acids needed for protein synthesis. Recently, the genomic clone of asparaginase from Lupinus angustifolius seeds was cloned and characterized (Dickson et al., 1992). The gene consisted of four exons and three introns (Dickson et al., 1992). Also, a cDNA corresponding to the clone that codes for the asparaginase from Lupinus arboreus was isolated (Lough et al., 1992). Southern blot experiments have shown that this gene is a single-copy gene expressed only in seeds (Lough et al., 1992). A genomic clone for L-asparaginase has been isolated from an Arabidopsis tkaliana cv Landsberg erecta AFIX genomic library by heterologous screening with an L. arboreus cDNA probe. The clone, which contains a 14-kb insert, has been studied by restriction and Southern blot analysis (Table I). The L-asparaginase gene was included within two 2.5-kb HindIII fragments. Both fragments were exonuclease I11 deleted, and the corresponding clones were sequenced. Sequences were compared with the genomic sequence of L-asparaginase of L. angustifolius. The results show that both Arabidopsis and Lupinus genes have a similar molecular organization and consist of three introns