Genome-level identification, gene expression, and comparative analysis of porcine ß-defensin genes

Abstract

Background: Beta-defensins (β-defensins) are innate immune peptides with evolutionary conservation across a wide range of species and has been suggested to play important roles in innate immune reactions against pathogens. However, the complete β-defensin repertoire in the pig has not been fully addressed.

Result: A BLAST analysis was performed against the available pig genomic sequence in the NCBI database to identify β-defensin-related sequences using previously reported β-defensin sequences of pigs, humans, and cattle. The porcine β-defensin gene clusters were mapped to chromosomes 7, 14, 15 and 17. The gene expression analysis of 17 newly annotated porcine β-defensin genes across 15 tissues using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) showed differences in their tissue distribution, with the kidney and testis having the largest pBD expression repertoire. We also analyzed single nucleotide polymorphisms (SNPs) in the mature peptide region of pBD genes from 35 pigs of 7 breeds. We found 8 cSNPs in 7 pBDs.

Conclusion: We identified 29 porcine β-defensin (pBD) gene-like sequences, including 17 unreported pBDs in the porcine genome. Comparative analysis of β-defensin genes in the pig genome with those in human and cattle genomes showed structural conservation of β-defensin syntenic regions among these species.

Figure 1

Comparison of amino acid sequences among 29 porcine β-defensin genes. Amino acid sequences were predicted from cDNA sequences and aligned using ClustralW2 with minor manual manipulations to maximize sequence alignment. The 6-cysteine motifs are shaded. The 9 significantly conserved sites, including the 6-cysteine motif, are indicated at the bottom.

Figure 2

A phylogenetic analysis of β-defensin genes among humans, cattle, and pigs. 36 β-defensin genes from humans, 48 from cattle, and 29 from pigs were analyzed using the neighbor-Joining method. The bootstrap consensus tree inferred from 1,000 replicates and branches corresponding to less than 50 % bootstrap replicates were collapsed. The analysis involved 113 amino acid sequences of β-defensin prepropeptides. pBD, β-defensins; BBD, bovine β-defensins; DEFB, human β-defensins. The “ψ” symbol of pBD117ψ and -127ψ indicates Pseudogenes or partial genes. Sequences of human and cattle β-defensin genes [47] were obtained from NCBI.

Figure 3

Comparison of β-defensin-containing chromosomal regions among human, pig, and cattle genomes. The evolutionarily conserved flanking markers and the clustered β-defensin genes are shown. The genes with orthologous relationships are indicated by lines among maps of different species. The names of the β-defensin genes are indicated with only numbers without species-specific symbols (DEFB for human, BBD for cattle, and pBD for pigs). Pseudogenes or partial genes identified in the pig genome sequencing results at NCBI are shown as dotted lines. Information from genome build 37.2, Sscrofa10.2, and Btau5.2 were used for humans, pigs, and cattle, respectively. ¹Some of the cattle β-defensin genes have less typical names, including TAP, LAP, and EBD.

Figure 4

Analysis of the tissue expression profiles of 16 porcine β-defensins from 15 tissues using semi-quantitative RT-PCR.GAPDH was used as a control to control for the amount of cDNA and the level of gene expression. (A) The gene expression analysis of β-defensins from a 2-week-old pig. (B) Temporal changes in the gene expression of pBD115 in testes between 2-week-old and 5-month-old pigs. 1, small intestine; 2, tongue; 3, eye; 4, cerebrum; 5, spleen; 6, kidney; 7, liver; 8, lung; 9, stomach; 10, testis; 11, muscle; 12, skin; 13, rectum; 14, trachea; 15, thymus.