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 .