title: Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.
abstract: Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application.
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China.
authors.forename: Huihui
authors.initials: H
authors.lastname: Cao
authors.affiliation: Department of Life Science and Technology, Nanyang Normal University, Wolong Road, Nanyang 473061, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Ke
authors.affiliation: Zhejiang Academy of Agricultural Sciences, 198 Shiqiao Road, Hangzhou, 310021, P. R. China.
authors.forename: Renhu
authors.initials: R
authors.lastname: Liu
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China.
authors.forename: Jingyin
authors.initials: J
authors.lastname: Yu
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China; Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan 430062, China.
authors.forename: Caihua
authors.initials: C
authors.lastname: Dong
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China.
authors.forename: Mingxing
authors.initials: M
authors.lastname: Cheng
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China; Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan 430062, China.
authors.forename: Junyan
authors.initials: J
authors.lastname: Huang
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China; Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan 430062, China.
authors.forename: Shengyi
authors.initials: S
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D029688:Brassica napus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology*; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010802:Phylogeny; D010935:Plant Diseases / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011392:Proline