Bioinformatics Analysis Of Antibacterial Peptide Gene From Rape EST And Validation Of BnLTP2 Activity

Antimicrobial peptides (AMPs) play an important role in biological natural defense system, which is composed of 10-50 amino acids. Most AMPs are cationic peptide alkaline, with 2 to 7 positive charges. In order to mining plant AMP genes,1185911 EST sequences of Brassica napus were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/nucest) to the local server. After screening for low-quality DNA and trimming the vector sequences, the EST sequence were clustered and produced 201200 unigenes. Unigenes were annotated using blastx and tblastn, GO, Interproscan, Clustalw and other tools. All the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLAST program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated. After masking of the repeat sequence,605 peptides that were orthologous.27 matched to different AMP families were found.606 potential AMPs were aligned via ClustalW to construct the unrooted phylogenetic tree. Six groups of known AMP families were found, including the most abundant types LTP(237), defensin-like peptides (DLPs,106), snakin (extracted from gastl genes,91), hipposin (histone-derived AMPs, HDAPs,79), hevein (extracted from endochitinase genes,32), and thionin(15).An predicted AMP gene called were selected from LTP family numbers. Its protein sequence consists of 27 amino acids. The synthetic BnLTP2 AMP gene was cloned into pET30a-EDDIE-GFP expression vector by overlapping extension PCR. After in vitro renaturation of inclusion body fusion protein which expressed in Escherichia coli, BnLTP2 without any excess amino acids were obtained by self shearing and purification of fusion protein EDDIE. SDS-PAGE electrophoresis results showed that there was an antibacterial peptide band at 5 kD. Using Micrococo luteus as gram positive bacteria, Escherichia coli as the representative of gram negative bacteria,Pichia pastoris GS 115 as the representative of the fungal and cylindekplate method as the detection of antibacterial activity of AMP BnLTP2 standard. The results show that BnLTP2 has strong antibacterial activity against Escherichia coli, Pichia pastoris and Micrococo luteus. The antifungal activity of AMP BnLTP2 against filamentous fungi was detected by using Sclerotinia sclerotiorum as the representative of filamentous fungi. The results showed that BnLTP2 also had strong antibacterial activity to Sclerotinia sclerotiorum. The purified BnLTP2 were dissolved in PBS,25%TFE,50%TFE and 75%TFE solutions respectively, circular dichroism(CD) results showed that the peak of chromatography, a positive peak at 194 nm, a negative peak at 208 and 222 nm, which indicated that the AMP has the typical structure of the alpha helix, but the peak is not high, that the antibacterial peptide BnLTP2 in spiral low content. LTP family members composed of 91 to 95 amino acids, its biggest feature is the molecular composition containing eight highly conserved cysteine with the lack of tryptophan, a hydrophobic cavity in the molecule, fatty acid molecules can be accommodated by the hydrophobic cavity, to form a stable the structure consists of four two disulfide linked four alpha helix. The protein sequence of BnLTP2 consists of 27 amino acids, containing four cysteine, KICCPRTIDRNIYNACRLTGASMTNCA, but the highest homology with LTP family, presumably part of the complete LTP protein structure.