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


RESULTS
In 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.


CONCLUSIONS
The 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.
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. ketao2@hotmail.com.|College of Life Science and Technology, Nanyang Normal University, Nanyang, 473061, China. ketao2@hotmail.com.
Tao
T
Ke
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. perfect_ch@sina.cn.
Huihui
H
Cao
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. huangjy@oilcrops.cn.|Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. huangjy@oilcrops.cn.
Junyan
J
Huang
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. fhu2008@qq.com.
Fan
F
Hu
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. 543000863@qq.com.
Jin
J
Huang
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. dongch@oilcrops.cn.|Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. dongch@oilcrops.cn.
Caihua
C
Dong
Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. mabo1978@163.com.
Xiangdong
X
Ma
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. yujyinfor@gmail.com.
Jingyin
J
Yu
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. maohan1976@hotmail.com.
Han
H
Mao
College of Life Science and Technology, Nanyang Normal University, Nanyang, 473061, China. 253800854@qq.com.
Xi
X
Wang
College of Life Science and Technology, Nanyang Normal University, Nanyang, 473061, China. qiuhongniu@163.com.
Qiuhong
Q
Niu
College of Life Science and Technology, Nanyang Normal University, Nanyang, 473061, China. huifl@126.com.
Fengli
F
Hui
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of CAAS, Wuhan, 430062, P. R. China. Liusy@oilcrops.cn.|Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, 430062, P. R. China. Liusy@oilcrops.cn.
Shengyi
S
Liu
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D001203:Ascomycota / Q000187:drug effects; D029688:Brassica napus / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects; D020224:Expressed Sequence Tags; D017343:Genes, Plant; D008826:Microbial Sensitivity Tests; D016982:Micrococcus luteus / Q000187:drug effects; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins