Modification Of Antimicrobial Peptide Genes And The Construction Of Plant Expression Vectors Harboring These Genes

Cationic antibacterial peptides are found in insect, animal and plant. They are grouped into four classes including cecropins, magainins, melittins and defensins. CEMA consists of eight amino acid from N-terminus of cecropin A and C-terminus of modified melittin. It maintains potent antibacterial activity and deprives of the hemolytic activity. With its broad-spectrum and non-specific potent antibacterial activity, cationic antibacterial peptide has the potential to be widely used in plant disease resistance genetic engineering. However, CEMA was refrained in plant genetic engineering application due to its toxic to plant cells. The modification of CEMA will facilitate its wide application. In this paper, signal peptide was added before the N-terminus of CEMA with the intention to reduce its toxic to plant cells while maintaining the strong antibacterial activity.Plant defensins are cystein-rich antibacterial peptides. Plant defensin disrupts the cell membrane of phytopathogen differently from CEMA that contacts directly with the cell membrane. Plant defensins are thought to interact with specific receptor harboring in the cell membrane. They inhibits the growth of fungi (filamentous fungi especially) while are non-toxic to plant cells. The main results were as follows:1. Obtaining of SPCEMA(signal peptide modified CEMA)SPCEMA(187bp) was amplified with two long complementary primers (P2 and P3) and two primers (Pl and P4) containing restriction enzyme recognition site. Plant expression vector pESPCEMA was constructed after subcloning in pUC121 and other vectors.2. Obtaining of MCEMA(modified CEMA) and plant defensin AFP geneMCEMA (141bp) was amplified with pUCSPCEMA plasmid as template and P5, P4 as primers, and plant expression vector pEMCEMA was constructed.Plant defensin AFP(238bp) was amplified with six long complementary primers after tworounds of PCR amplification and plant expression vector pEAFP was constructed.3. Construction of plant binary expression vector pEASPIn consideration of the different cell membrane disruption mechanism of plant defensin and CEMA, plant binary expression vector pEASP were constructed. Simultaneous expression of CEMA and AFP may synergistically inhibit the growth of phytopathogens that are the main cause agent of agricultural loss.Armed with modified antibacterial peptide (SPCEMA and MCEMA) and plant defensin AFP, transgenic plants with broad-spectrum and strong disease resistance may be obtained.