SNP Analysis Of Exon 3 And Exon 4 Of Bactericidal/Permeability-Increasing Protein Gene In Four Swine Breeds

Bactericidal/permeability-increasing protein(BPI)is a highly cationic protein which is located mainly in the primary granules of polymorphonuclear leucocyte(PMN).BPI has a high affinity for LPS of Gram-negative bacteria.Binding of BPI to susceptible bacteria is followed by increasing outer membrane permeability,inhibition of growth and ultimately,irreversible loss of viability.In addition,BPI also possesses an opsonic function.Because of these important functions,BPI plays an important role in host initial defense.At present,the full-length cDNA sequences of BPI gene has been elucidated in several species,including human,bovine,brown rat,mouse,and pig of rongchang. In addition,several polymorphic sites were found in exons 3 and 4 of human BPI gene.It has been shown that the restriction fragment length polymorphism(RFLP)sites in exons 4 and 10 of porcine BPI gene are related to the susceptibility of Salmonella cholerasuis in several pig breeds.The porcine BPI gene was considered as a candidate gene of breeding for disease resistance.we have found several polymorphic sites in exons 3 and 4 of rongchang pig in our investigation we have done.However,Are different of polymorphism of exons 3 and 4 between Rongchang pig and other strain pigs(native,import,crossbreed pigs)BPI gene,and are the differens obvious is unclear.In this study,the pig breeds be investigated are rongchang,A,B and Cstrain pigs which were pure breeds elected by four generations.the SNP sites in exons 3 and 4 of this gene were detected by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)analysis.Then. the PCR products showing different patterns were purified and cloned for sequencing.Finally,the frequencies of difference genotypes were accounted and the allelic genes frequencies were calculated according to the former result.Further more,the SNP sites in exons 3 and 4 were found by sequence alignment of different allelic genes.Frequencies of the bases with higher appearance frequency in these SNP sites were calculated according to the distribution of SNP sites in all the allelic genes.In the exon 3 fragment,There was two novel SNPs(A376C,A397G)detected in the exon 3 of Rongchang pig BPI gene by PCR-SSCP.The A376C made Gln94Pro substitution,the hydrophobicity increase from 0.28 to 0.60.the A397G mutation made Arg101 Gin substitution,two novel SNPs(T371C,A445G)were detected in the exon 3 of B strain pigs BPI gene by PCR-SSCP,the T371C mutation was synonymous substitutions.the A445G mutation made Lys 117Arg substitution,and the hydrophobicity drop from -0.89 to -1.05.There was one novel SNP(A435G) detected in the exon 3 of Cstrain pigs BPI gene by PCR-SSCP,it made Lle114Val substitution,the hydrophobicity drop from 0.83 to 0.78.there was no novel SNP be found in A lineage pig.In the exon 4 fragment,six novel(T512C,G551T,C563T,T573C,G599A,T607C)of SNPs were detected,four of them(T512C,G551T,C563T,G599A)were synonymous substitutions.The G551T novel made a restriction endonuclease Msc I orBal I(TGG/CCA)disappear.the novel(T573C) made Ser160Pro substitution,and the hydrophobicity drop from 0.6 to 0.46.the novel(T607C)made Val171Ala substitution,hydrophobicity drop from -1.21 to -1.61.Three SNPs which were same whith three of the rongchang pig's SNPs were found in B strain of pigs,there were T512C, G551T,G599A.SNPs were not found in A and C strain pigs.The affluent polymorphism of exon 4 indicate its huge potentiality in genetic selection.Some of the SNP sites detected in this study may be important molecular markers of porcine disease resistance,specifically resistant to Gram-negative bacterias and its caused diseseses.