Yf. Liu et al., Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed, PLANT PHYSL, 122(4), 2000, pp. 1015-1024
A small cysteine-rich protein with antimicrobial activity was isolated from
pokeweed (Phytolacca americana) seeds and purified to homogeneity. The pro
tein inhibits the growth of several filamentous fungi and gram-positive bac
teria. The protein was highly basic, with a pi higher than 10. The entire a
mino acid sequence of the protein was determined to be homologous to antimi
crobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. ameri
cana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and
sequenced. The deduced amino acid sequence shows the presence of a signal
peptide at the amino terminus, suggesting that the protein is synthesized a
s a preprotein and secreted outside the cells. The chromosomal gene shows t
he presence of an intron located within the region encoding the signal pept
ide. Southern hybridization showed that there was small gene family encodin
g Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, an
d was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted
into the environment of the seeds during germination, and may create an in
hibitory zone against soil-borne microorganisms. The disulfide bridges of P
a-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicat
es that the protein has a small cystine-knot folding, a positive patch, and
a hydrophobic patch.