| As a greatly effective pathway of protein degradation, ubiquitin-proteasome pathway can degrade selectively intra cellular protein; And make various proteins to be precisely degraded. It is very important for maintaining much important physiological function, such as control of cell cycle, angtigen presentation, transcriptional control, apoptosis, and signal transduction. However, ubiquitin-protein ligases (E3s) can especially recognize target protein, with a role of linking the target protein and ubiquitin-conjugating enzymes (E2s).In this study, the bioinformatics and molecular biology technology was applied to isolate and clone the partial genomic fragments and cDNA of four genes in FBXL sub family belonging to F-box proteins which play a role as the recognizing components for subtrate protein in ubiquitin-protein ligases components. The tissue expression, SNP detection and the association analysis with immune traits were conducted as followings:1. The human homologous gene mRNAs were searched and used to obtain porcine new gene ESTs. The first and sixth intron of porcine SKP2 gene, the second intron of porcine FBXL4 gene, the second intron of FBXL8 gene and the eighteenth, nineteenth and twentieth intron of porcine FBXL11 gene were isolated. As the same time, the cDNAs and CDSs of porcine SKP2 and FBXL4 gene were obtained, cDNA of porcine FBXL11 gene also was searched and analyzed.2. Real Time-PCR was carried out in analysis on the tissue expression for porcine SKP2 and FBXL4 mRNA in ten important tissues. In conclusion, porcine SKP2 mRNA expresses fully and evidently in these tissues, but the expression levels fluctuated in a large range. The highest level was expressed in thymus, and the lowest level was found in longissimus dorsi muscle. As similarly, porcine FBXL4 mRNA expresses visibly in all of tissues with great difference levels, the highest level was expressed in lung, and the lowest level was located in heart.3. SNP detections in these introns related to porcine SKP2, FBXL4, FBXL11 and FBXL8 gene were completed, the following results was got:the SNP-Cfr42I (T/C) was located in the 819th in the first intron of porcine SKP2 gene; the second intron of porcine FBXL4 gene contains eight SNPs, including the 49th (C/T),157th (A/T),187th (A/G),227th(G/T),321st (G/A),371st (G/A),381st (C/T) and 569th (C/A). Interestingly, the fore and post four SNPs belong to a kind of linkage mutations in the same intron. Therefore, two SNPs representing respectively the fore and post four SNPs which were the 49th SNP-Tail and the 381st SNP-BsaJI were conducted to detection and analysis; SNP-TspEI (A/G) was found at the 377th in the eighteenth intron of porcine FBXL11 gene; Furthermore, there were three mutations with G/A,C/T and A/G at the 325th,361st and 493rd in the second intron of porcine FBXL8 gene respectively.4. PCR-RFLP technique was applied in the population genetics analysis of porcine SKP2, FBXL4 and FBXL11 gene. According to porcine SKP2 Cfr42I-RFLP analysis on Bama, Laiwu, Guizhou, Wuzhishan four kinds of unrelated Chinese indigenous miniature pig breeds and pure Landrace pig breed:Allele A was in dominance absolutely in all detected pig breeds. Four kinds of unrelated Chinese indigenous miniature pig breeds almost belong to homozygous AA genotype, while introduced pure Landrace pig breed has two different genotypes containing homozygous AA and heterozygous AG genotype.For porcine FBXL4 gene, according to the results on Tail-RFLP, homozygous TT genotype owned absolute dominance in introduced Landrace pig breed. Simultaneously, for unrelated three kinds of Chinese miniature pig breeds, homozygous TT genotype was also more than two other genotypes in Guizhou and Wuzhishan breeds, but homozygous CC genotype was in absolute dominance in Laiwu breed. Meanwhile, in another genotyping analysis on BsaJI-RFLP, homozygous CC genotype was obviously more than other genotypes in two kinds of Chinese miniature pig breeds including Guizhou and Wuzhishan and introduced Landrace pig breed.The analysis results on TspEI-RFLP of porcine FBXL11 gene in pure Landrace pig resources shows that allele G was in dominance absolutely and introduced pure Landrace pig breed had two different genotypes including heterozygous AG and homozygous GG genotype.5. The preliminary association analysis with immune traits was conducted for porcine SKP2 and FBXL4 gene. According to the results on porcine SKP2 gene, SNP-Cfr42I in intron 1 was significantly associated with RDW of neonate piglets at 0 day. RDW of the newborn piglets with homozygous AA genotype was obviously higher than that of newborn piglets with heterozygous AG genotype (P=0.027).With regard to SNP-Tail analysis of porcine FBXL4, it shows that lymphocyte percentage of neonate piglets at 0 day with homozygous CC genotype was obviously lower than those with other two genotypes (P=0.0166). while another results of SNP-BsaJI revealed that neutrophil percentage of neonate piglets at 0 day with homozygous TT genotype was visibly higher than those with other two genotypes (P=0.0005). Lymphocyte percentage of individuals at 32-day with homozygous TT genotype was notably lower than those with other two genotypes (P=0.0351). However, absolute lymphocyte count of neonate piglets at 0 day with homozygous CC genotype was obviously higher than those with other two genotypes (P=0.0458). The mixed cells of neonate piglets at 0 day with heterozygous TC genotype were markedly higher than those with other two genotypes (P=0.0010).6. The clustalW2 software online was applied to construct and analyse the phylogenetic trees for porcine SKP2,FBXL4 and FBXL11 genes. The analyses on the phylogenetic tree show that these genes share the quite conserved molecular characteristic among these species. Meanwhile, compared with human and other various species, only Sus Scrofa and Bos Taurus always belong to the same group according to these trees, as shows that they have most relative relation characteristic in their evolution.7. The detection and analysis on motif of the putative proteins of porcine SKP2 and FBXL4 gene were employed by motif scan online. The results on porcine SKP2 gene show that a conserved motif of F-box protein was found at position between the 90th and 140th amino acid, as indicated that the putative protein belongs to F-box proteins; By comparison, it was predictable that porcine SKP2 protein has probably other similar 8 leucine-rich repeats.While another results on porcine FBXL4 gene similarly revealed that this putative protein has a conserved motif of F-box protein with 56 amino acids, which located between the 277th and 332nd amino acid, as demonstrated that it also belongs to a member of F-box proteins.
|
PDF Full Text Request