Two Foreign Antimicrobial Peptides Expressed in the Chloroplast of Porphyridium purpureum Possessed Antibacterial Properties

Abstract

To solve the problem of antibiotic abuse in aquaculture and to utilize the application potential of antimicrobial peptides (AMPs), a chloroplast transformation system of Porphyridium purpureum was successfully constructed for effectively expressing two exogenous AMPs. The endogenous fragments of 16S rDNA/trnA-23S rDNA were used as flanking fragments for the homologous recombination in the chloroplast genome. Two AMPs encoded by the transformation vector were controlled by the native promoter psbB in a polycistron. The plasmids were transferred into P. purpureum via particle bombardment and the transformation vectors were screened using phosphinothricin (bar), a dominant selection marker under the control of the psbA promoter. Subsequently, in the positive transformed colonies, the exogenous fragments were found to be inserted in the flanking fragments directionally as expected and two foreign AMPs were successfully obtained. Finally, two exogenous peptides with antibacterial properties were obtained from the transformed strain. The two AMPs expressed by the transformed strain were shown to have similar inhibitory effects to antibiotics by inhibition tests. This suggested that AMPs can be introduced into aquaculture using baited microalgae, providing new ideas and ways to solve a series of aquaculture diseases caused by bacteria.

Keywords: NZ2114; Piscidin-4; Porphyridium purpureum; antimicrobial peptides; chloroplast transformation system; inhibitory effect; microparticle bombardment.

Figure 1

The construction of pPs/ch/bar-anti vector.

Figure 2

The confirmation of M strain. (a) Identification of bar gene in M strain using PCR; (b) the detection of complete transformed plasmids in M strain using PCR; (c) identification of bar, ant1, and ant2 genes in M strain using Southern blotting; (d) identification of NZ2114 (ant1) and piscidin-4 (ant2) proteins in M strain using Western blotting. Marker, DNA maker; W, wild P. purpureum.

Figure 3

Bacterial inhibitory effect of protein crude extracts containing two AMPs on bacteria compared with antibiotics. (a) Antibacterial effect on S. aureus; (b) antibacterial effect on V. parahaemolyticus. For 1–3, 2 μL ampicillin was added; 4–6, 2 μL crude protein extract of the M strain was added; and 7–9, 2 μL sterile water was added.