De Novo Assembly Of Alternanthera Sessilis Transcriptome And Antimicrobial Peptides Discovery

Alternanthera sessilis(L.)DC is an important traditional medicine and is used all over Asia and Africa as a natural home remedy,to treat a variety of disorders.A.sessilis was first recorded by Ayurvedic Medicine in India,called Matyakshika,which is one of the three sources of the Ayurvedic drug Lokina,commonly used in the treatment of flatulence,cough,bronchitis and diabetes.In Senegal and India,people use grinding powder of A.sessilis for treatment of snake bites;in Taiwan,A.sessilis for local medicine,usually mixed with other medicinal plants to cure hepatitis,chest tightness,bronchitis,asthma and other lung diseases;tribes in the Bakar region of India with this plant to treat bloody dysentery,ulcers and stab wounds.A.sessilis has a strong antibiotic activity,and rich in antimicrobial peptides.Currently,there are few reports related to A.sessilis.As a non-model organism,the genome and transcriptome of A.sessilis have not yet been sequenced,and there are very few A.sessilis nucleic acid and protein sequences available in the NCBI database.Antimicrobial peptides(AMPs)are small molecular peptides which is produced by organisms themselves and is used for defending against the invasion of pathogens.AMPs are widely distributed in animals and plants,which is an important component of the organism innate immune system.Plant AMPs are part of the plant defense systems,they have been isolated from roots,seeds,flowers,stems,and leaves of a wide variety of species and have activities towards phytopathogens,as well as against bacteria pathogenic to humans.In recent years,due to the unreasonable use of antibiotics in clinical and production practice,a large number of pathogenic microorganisms have caused drug resistance.However,the plant AMPs as a natural,powerful,and broad-spectrum antimicrobial compounds,is a promising alternative of antibiotics.Plant AMPs are different from other organisms',they are usually rich in cysteine residues that form several disulfide bonds.These structural properties make plant AMPs highly chemically,thermally and proteolytically stable.Cysteine-rich plant AMPs are usually classified into families based on their sequence similarity and cysteine motifs,including thionins,defensins,hevein-like peptides,knottins,lipids transfer proteins,snakins,and a-hairpinins.In order to study the antimicrobial peptide sequence in A.sessilis,we sequenced all messenger RNAs of A.sessilis with Illumina Hiseq 2000 high throughput sequencing platform.There are 182462 transcripts and 94439 unigenes obtained from de novo assembly by using BinPacker software,and the N50 reached 2256bp.We did function annotation for these unigenes by searching homologous sequence of unigenes in the databases including NR,Swissprot,trEMBL,COG,KEGG,and PFAM and accessing to the corresponding gene function information.We found homologous sequence in these databases for 32109(30%)unigenes.In the NR database search results,the most of homologous sequences of A.sessilis unigenes are derived from Beta Vulgaris and Spinacia Oleracea,while Beta Vulgaris and Spinacia Oleracea both belong to Amaranthaceae family with A.sessilis as a family member,indicating that the most closely related species of A.sessilis were deep sequenced and annotated only in family level.We then used our in-house PERL scripts to identify the putative AMPs sequences in the A.sessilis assembled transcripts by combining methods of sequence homology searching and the cysteine motif pattern searching.A total of 192 AMPs were obtained,and these AMPs from six different families,include 75 lipoproteins,66 plant defensins,22 thionine proteins,17 snakins,5 hevein-like peptides,and 7 a-hairpin structural protein.For the first time we sequenced A.sessilis transcriptome and de novo assembled the transcripts.The antimicrobial peptides contained in A.sessilis were also analyzed in the whole transcriptome.The results show that transcriptome sequencing is a cost-effective means of studying small molecules in the organisms which reference genomes are unavailable.Studying plant AMPs at the level of transcriptome is a simple and efficient way to effectively reduce the experimental workload and speed up the research process.