| Being an important member of the antimicrobial peptide family,pig betadefensin(PBD)has the same basic biological functions as antimicrobial peptides: low toxicity,drug resistance,broad-spectrum antibacterial activity,etc,and plays an important role in the immune system.In this research,we use the sequences of Sus Scrofa defensin gene from NCBI as the template to amplify the CDS region from Large white pig,Min pig,and Wild pig crossbreed populations by RT-PCR.The sequences were analyzed by some bioinformatic software and the relative expression level in blood,spleen and kidney from three groups.The serum content of the PBD-2 and PBD-110 were detected by ELISA,and the single nucleotide polymorphisms(SNP)in 95 pigs of the three groups were screened and analyzed in combination with the serum content;Finally,the function of PBD gene was identified by bacteriostatic and antiviral tests.According to the results,PBD-2,PBD-110,PBD-119,and PBD-121 were successfully cloned.The PBD-119 gene encodes 83 amino acids,with a molecular weight of 9.6ku,a theoretical isoelectric point(PI)of 9.38,a stability coefficient of 59.43,and a fat coefficient of 9.38;PBD-121 gene encodes 60 amino acids,with a molecular weight of 6.8ku,a theoretical isoelectric point(PI)of 8.9,a stability coefficient of 35.48,and a fat coefficient of 107.33.It is speculated to be a stable alkaline fat-soluble protein;Protein analysis results show that both PBD-119 and PBD-121 have three kinds of structures(α-helix,β-sheet,and random coil),phosphorylation sites and they are secreted proteins.What’s more,the four target genes conform to the law of species evolution,expressed in different tissues of different populations as the m RNA expression results showed among Large white pig,Min pig,and Wild pig crossbreed.In addition,PBD-2 and PBD-110 possess SNP loci in intron 1.The T214 C had three genotypes TT,TC and CC in the PBD-2.Besides,TC was the dominant genotype as we can say from the polypeptide analysis of the proportions in 3 different groups of pigs :16.13%,54.84% and 29.03% in Large white pigs,25%,56.25% and 18.75% in Min pigs,28.13%,46.87%,and 25% in Wild pigs crossbreed.What ‘more,the serum levels in the peripheral blood of the three groups all showed the trend of being the highest in Min pigs,the lowest in Wild pigs crossbreed,and the middle in large white pigs;interesting,the T214 C of PBD-110 also has three genotypes TT,TC and CC,but TC was the dominant according to the frequencies data: 51.61%,38.71% and 9.68% in Large white pigs,78.13%,18.74% and 3.13% in Min pigs,75%,21.88% and 3.12% in Wild pigs crossbreed,the serum levels in the peripheral blood of the PBD-2: highest in Min pigs,the lowest in Wild pigs crossbreed,and the middle in Large white pigs.The results of functional identifications show that: the chemically synthesized protein PBD-121 showed broad-spectrum antibacterial activity against Escherichia coli DH5α,Escherichia coli K88,Staphylococcus aureus,Salmonella,Shigella and Pseudomonas aeruginosa by presenting the inhibitory circles.The minimum inhibitory concentrations were 18.38 um,20.06 μm,29.4 μm,58.8 μm,66.18 μm and 73.5 μm;PBD-121 protein also showed a certain inhibitory effect on virus infection and replication,but the effect was negligible.In order to further explore the antiviral function of PBD-121,NBL-PBD-121 transgenic cells were constructed,which also showed a weak inhibitory effect on antiviral infection and replication.This study provides a theoretical basis for the basic biological functions of defensin gene,and also provides a reference for defensin gene as an ideal candidate gene for antibiotics. |
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