Plant Physiol, April 2000, Vol. 122, pp. 1015-1024
Purification, Characterization, and Molecular Cloning of the
Gene of a Seed-Specific Antimicrobial Protein from
Pokeweed1
Yingfang
Liu,
Jingchu
Luo,
Chunyu
Xu,
Fucheng
Ren,
Cheng
Peng,
Guangyao
Wu, and
Jindong
Zhao*
The National Laboratory of Membrane Biology, College of Life
Sciences, Peking University, Beijing 100871, China
A small cysteine-rich protein with
antimicrobial activity was isolated from pokeweed (Phytolacca
americana) seeds and purified to homogeneity. The protein
inhibits the growth of several filamentous fungi and gram-positive
bacteria. The protein was highly basic, with a pI higher than 10. The
entire amino acid sequence of the protein was determined to be
homologous to antimicrobial protein (AMP) from Mirabilis
jalapa. The cDNA encoding the P. americana 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 as a preprotein and secreted outside the cells. The
chromosomal gene shows the presence of an intron located within the
region encoding the signal peptide. Southern hybridization showed that
there was small gene family encoding Pa-AMP. Immunoblotting showed that
Pa-AMP-1 was only present in seeds, and 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 inhibitory zone against
soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were
identified. The three-dimensional modeling of Pa-AMP-1 indicates that
the protein has a small cystine-knot folding, a positive patch, and a
hydrophobic patch.
1
This research was supported by the Chinese
National Natural Science Foundation (grant no. 39535002) and by the
Department of Science and Technology (grant no. J99-A-032).
*
Corresponding author; e-mail jzhao{at}pku.edu.cn; fax
86-10-6275-6421.
© 2000 American Society of Plant Physiologists
