| 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 and mouse, but not in pig. 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. However, the polymorphism of exons 3 and 4 in Rongchang pig BPI gene, a stress preserving pig breed by Food and Agriculture Organization of United Nations, is unclear.In this study, the full-length cDNA of Rongchang pig BPI gene was cloned and the SNP sites in exons 3 and 4 of this gene were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. There results will offer some basic data to the function studying of porcine BPI, the association research between BPI gene polymorphism and the disease resistance of Rongchang pig and the breeding and preservation of Rongchang pig. Some useful molecular markers of breeding for disease resistance are also expected to find in the possible SNP sites.The main technical route and methods of this work were carried out as follows:(1) Cloning and analysis of the full-length cDNA of BPI gene in Rongchang pig:Firstly, about 5 ml anticoagulant peripheral blood was gained from a 100d healthy Rongchang pig, the total RNA from this blood was extracted using the RNA extraction kit. Then the RNA was taken to convert mRNAs into cDNA. Following, the coding sequence, 3'-UTR and 5'-UTR of Rongchang pig BPI were cloned using homology cloning approach combined with 3' and 5' RACE. Finally, the full-length cDNA of Rongchang pig BPI gene was obtained by overlapping the three sequences. Assessment and analysis of the full-length sequence were done by bioinformatics methods.(2) SNP analysis of the exons 3 and 4 of Rongchang pig BPI gene:Firstly, the genomic DNA was extracted from 120 healthy Rongchang pigs' ear tissues. And the exons 3 and 4 fragments were amplified using the genomic DNA templates. Then these PCR products were detected by polyacrylamide gel electrophoresis (PAGE) at 120 V for 14 h at 4℃, and gels were developed with silver staining. The PCR products showing different patterns were purified and cloned into pMD18T vector 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.The results and conclusions of this study were list as follows:(1) The full-length cDNA of Rongchang pig BPI gene was cloned for the first time in this study and it was also the first time reporting the full-length cDNA of porcine BPI gene. Its GeneBank accession number is EF436278. The structure and function of Rongchang pig BPI were similar to those of human, bovine and other species' BPI by sequences alignment and structure and function prediction.(2) There was one novel SNP (G397A) detected in the exon 3 of Rongchang pig BPI gene by PCR-SSCP. This mutation made Arg101Gln substitution and the base with higher appearance frequency in this SNP site is base G. Its appearance frequency is 0.7417. In the exon 4 fragment, three novel (T512C, G551T, C563T) of four SNPs (T512C, G551T, C563T, G599A) were detected and all of the SNPs were synonymous substitutions. The bases with higher appearance frequency in these SNP sites were base C, G, C and G and their appearance frequency were 0.6334, 0.725, 0.725 and 0.7501 respectively. In addition, another novel SNP (T479C) in exon 4 was found by aligning exon 4 sequence with porcine ESTs. 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.
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