Identification and bioinformatics analysis of defensin antimicrobial genes from six lentil genotypes

Document Type : Research Paper

Authors

1 Assistant Professor, Department of Agronomy and Plant Breeding, Khorramabad Branch, Islamic Azad University, Khorramabad, Iran

2 Researcher, Young Researchers and Elite Club, Khorramabad branch, Islamic Azad University, Khorramabad, Iran

3 Assistant Professor Department of Agronomy and Plant Breeding, Kangavar Branch, Islamic Azad University, Iran

Abstract

Plant defensins are a large family of cysteine-rich antimicrobial peptides. These peptides are small molecules with molecular mass between 5 and 7kD and eight conserved cysteines that are involved in disulfide bond formation. In the present study, some members of defensin gene family were isolated and characterized from lentil genotypes. All coding sequences of the defensin genes of dicotyledon plants were retrieved from GenBank. The retrieved coding sequences were aligned against lentil EST library and the resulted sequences were pooled and assembled. The resulted contigs and singletons were aligned against GenBank nr database using BLASTn tool. The contigs and singletons with the full open reading frame were used to primer design. In the next step, DNA extraction was performed from Gachsaran, Mahali, Filip2003-2L, Filip2003-9L, Filip2005-2L and Filip2006-10L genotypes. Finally, by using PCR, genomic sequence of defensins were amplified, cloned into pTZ57R/T plasmid and sequenced. The results showed complete identity among the isolated defensins from all genotypes. The identified genes also contain conserved introns with variable length that comprised of regulatory elements for response to different factors and conditions. For first time in this study, genomic sequences of three members of defensin gene family were isolated from lentil genotypes and structures and features of them were determined.

Keywords

Main Subjects

References

Ast G (2004) How did alternative splicing evolve? Nature Reviews Genetics. 5 (10): 773-782.
Bao R, Huang L, Andrade J, Tan W, Kibbe WA, Jiang H, Feng G (2014) Review of current methods, applications, and data management for the bioinformatics analysis of whole exome sequencing. Cancer informatics. Sup 2: 67-83.
Chang W-C, Lee T-Y, Huang H-D, Huang H-Y, Pan R-L (2008) PlantPAN: Plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC genomics. 9 (1): 561.
Cools TL, Struyfs C, Cammue BP, Thevissen K (2017) Antifungal plant defensins: increased insight in their mode of action as a basis for their use to combat fungal infections. Future Microbiology. 12 (5): 441-454.
De Beer A, Vivier MA (2008) Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity. BMC plant biology. 8 (1): 75.
Doyle JJ (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull Bot Soc Am. 19: 11-15.
Elsbach P (2003) What is the real role of antimicrobial polypeptides that can mediate several other inflammatory responses? Journal of Clinical Investigation. 111 (11): 1643.
Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic acids research. 44 (D1): D279-D285.
Flavia Cancado Viana J, Campos Dias S, Luiz Franco O, Lacorte C (2013) Heterologous production of peptides in plants: Fusion proteins and beyond. Current Protein and Peptide Science. 14 (7): 568-579.
Gill RW, Sanseau P (2000) Rapid in silico cloning of genes using expressed sequence tags (ESTs). Biotechnology annual review. 5: 25-44.
Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM (2005) Defensin gene family in Medicago truncatula: structure, expression and induction by signal molecules. Plant molecular biology. 58 (3): 385-399.
Holaskova E, Galuszka P, Frebort I, Oz MT (2015) Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnology advances. 33 (6): 1005-1023.
Hoskin DW, Ramamoorthy A (2008) Studies on anticancer activities of antimicrobial peptides. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1778 (2): 357-375.
Janssen BJ, Schirra HJ, Lay FT, Anderson MA, Craik DJ (2003) Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds. Biochemistry. 42 (27): 8214-8222.
Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics. 30 (9): 1236-1240.
Kumar S, Rajendran K, Kumar J, Hamwieh A, Baum M (2015) Current knowledge in lentil genomics and its application for crop improvement. Frontiers in plant science. 6: 78.
Lacerda AF, Vasconcelos ÉA, Pelegrini PB, de Sa MFG (2014) Antifungal defensins and their role in plant defense. Frontiers in microbiology. 5: 1-10.
Laverty G, Gorman SP, Gilmore BF (2011) The potential of antimicrobial peptides as biocides. International journal of molecular sciences. 12 (10): 6566-6596.
Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic acids research. 30 (1): 325-327.
Lico C, Santi L, Twyman RM, Pezzotti M, Avesani L (2012) The use of plants for the production of therapeutic human peptides. Plant cell reports. 31 (3): 439-451.
Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI (2014) CDD: NCBI's conserved domain database. Nucleic acids research. 43 (D1): D222-D226.
Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR (2010) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic acids research. 39 (suppl_1): D225-D229.
Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: towards a cellular code. Nature reviews Molecular cell biology. 6 (5): 386-398.
Muehlbauer FJ, Cho S, Sarker A, McPhee KE, Coyne CJ, Rajesh P, Ford R (2006) Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress. Euphytica. 147 (1-2): 149-165.
Nielsen H (2017) Predicting Secretory Proteins with SignalP. Protein Function Prediction: Methods and Protocols. 59-73.
Pelegrini PB, Franco OL (2005) Plant γ-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins. The international journal of biochemistry & cell biology. 37 (11): 2239-2253.
Periwal V, Scaria V (2014) Bioinformatics Review. Bioinformatics. 45: 1-9.
Sagaram US, Kaur J, Shah D (2012) Antifungal plant defensins: structure-activity relationships, modes of action, and biotech applications. In:  Small Wonders: Peptides for disease control. ACS Publications, pp 317-336
Silverstein KA, Graham MA, Paape TD, VandenBosch KA (2005) Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant physiology. 138 (2): 600-610.
Solovyev V, Kosarev P, Seledsov I, Vorobyev D (2006) Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome biology. 7 (1): S10.
Srivastava R, Vasishtha H (2012) Saponins and lectins of Indian chickpeas (Cicer arietinum) and lentils Lens culinaris. Indian Journal of Agriculture and Biochemistry. 25 (1): 44-47.
Thomma BP, Cammue BP, Thevissen K (2002) Plant defensins. Planta. 216 (2): 193-202.
Yadav SS, McNeil DL, Stevenson PC (2007) Lentil: An ancient crop for modern times. 1 edn. Springer Netherlands, Netherlands
Yu C-S, Cheng C-W, Su W-C, Chang K-C, Huang S-W, Hwang J-K, Lu C-H (2014) CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation. PLoS One. 9 (6): e99368.

Files

Share

How to cite

Statistics