3.3 Transgene expression and biuret decomposing activity in transgenic rice plants
To examine the effect of biuret hydrolysis in plant cells on the biuret tolerance of rice plants, we generated transgenic rice plants overexpressing biuret hydrolase that were cloned fromRhizobium sp. KaB01. The inserted biuret hydrolase encoded a protein consisting of 238 amino acid residues (Figure S2), whose amino acid sequence showed 92% similarity to a known biuret hydrolase ofR. leguminosarum bv. viciae 3841 (WP_011654379.1; Cameron et al., 2011). In addition, purified recombinant maltose-binding protein fusion proteins expressed in Escherichia coli showed the biuret decomposing activity (Figure S3). Signal peptides were not detected via the SignalP-5.0 program (Almagro Armenteros et al., 2019), and the protein was predicted to be cytoplasmic localized, using the program PSORTb v.3.0 (Yu et al., 2010).
The biuret hydrolase transgene was expressed in varying levels in regenerated T0 rice leaves, while its expression was not detected in wild-type Nipponbare (Figure 2). Self-pollinated progenies of two independent transgenic lines, B2-3-3 and B3-9-1, which showed high expression levels, were used for further examination.
Crude extracts prepared from 19-day-old seedlings of B2-3-3-3 (T2) or 16-day-old seedlings of B3-9-1 (T1) showed the biuret decomposing activity. In both lines, the amount of ammonia released from biuret increased linearly with time (Figure 3). The line B3-9-1 showed a higher specific activity than B2-3-3-3. The specific activities of extracts from the B3-9-1 and B2-3-3-3 plants were 8.6 and 0.67 nmol min-1mg-1 protein, respectively. Wild-type Nipponbare plants did not exhibit ammonia-releasing activity (Figure 3). Extracts heated at 100ÂșC for 5 min were inactive. In addition, extracts prepared from a null segregant of B3-9-1 did not exhibit ammonia-releasing activity. These results indicated that the transgenic rice lines were conferred with biuret decomposing ability by the exogenous biuret hydrolase .