EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Tao Ke,Caihua Dong,Jingyin Yu,Qiuhong Niu,Han Mao,Shengyi Liu,Junyan Huang,Fan Hu,Xiangdong Ma,Huihui Cao,Jin Huang,Fengli Hui,Xi Wang

doi:10.1186/s12864-015-1849-x

Abstract

BackgroundBrassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.ResultsIn total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.ConclusionThe study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.

Highlights

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm
expressed sequence tags (ESTs)-based search for discovery of novel B. napus antimicrobial peptides (AMPs) In this study, a general search aiming to identify AMPs was performed on three different B. napus EST databases including: 136,202 ESTs generated from immature seeds of two rapeseed lines [7]; 35,788 ESTs generated from an cDNA library which was constructed from mixed mRNAs of B. napus leaves inoculated with S. sclerotiorum or treated with chemicals benzothiadiazole (BTH), methyl jasmonate (MeJA), or oxalic acid (OA, a toxin and pathogenicity factor produced by S. sclerotiorum); 643,944 ESTs downloaded from the EST database of PlantGDB
A total of 237 unigenes from B. napus leaves that were matched to the known AMP genes had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset (Table 1)

Summary

Introduction

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). We used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression. Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, and are essential lines of host defence against pathogens. These peptides are less susceptible to bacterial resistance than traditional antibiotics, and could form the basis for a new class of therapeutic agents [1]. Plant AMPs provide novel strategies in therapeutic use, but can potentially increase agricultural yields through phytopathogen or pest control [3]

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Results

Conclusion