| The major histocompatibility complex( MHC) is a group of closely-linked gene, Which code the major histocompatibility antigen and play a critical role in immune response in vertebrate animals. The MHC genes are highly polymorphic and the research hot point in the origin and evolution of species and the disease resistant breeding. In this study, we succeed in getting the exon 2-3 fragement ofSLA-DRA and SLA-DQA gene in Swine MHC class II region using the method of comparative genome. The nucleotide sequence of the second exons of SLA-DRA, DQA, DRB and DQB genes of Swine MHC class II in 15 Chinese and introduced pig breeds are demonstrated by direct DNA sequencing. By the analysis of SNPs and amino acid sequence, we found some novel alleles. We study the genetic diversity of SLA-DRA, DQA, DRB and DQB genes in 15 pig breeds and the relationships between breeds. The polymorphisms of the second exon of SLA-DQB and SLA-DRB in the populations of Tongcheng, Large White ×(Swedish LandracexTongcheng), Swedish Landrace × (Large White × Tongcheng) pigs are detected by PCR-RFLP. The association of gene with immune traits is analysed. The results are as follows:1. We succeed in designing the primer pairs of DRApl67, DRAp428 and DQApl61 , DQAp443 for amplifying the SLA-DRA exon2-3 and SLA-DQA exon2-3,respectively. The 705bp fragment of SLA-DRA and 731bp fragment of SLA-DQA including completed intron 2, the near completed of exon 2 and partial exon 3 were obtained by PCR. The nucleotide sequences of the exon2-3 of SLA-DRA and SLA-DQA are known with clone and direct sequencing and the gene structure of partly segement of the two genes is also analysed. The 85 and 87 amino acid residues for al domain of SLA-DR and SLA-DQ a chain are deduced, respectively.2. The sequences of SLA-DRA, DQA , DRB, DQB exon2 and SLA-DRA , DQA intron2 in 15 pig breeds are analysed with direct sequencing of PCR products. We found that: there are three SNPs sites in 231bp of SLA-DRA exon 2, such as 190(G/A),363(C/A),374(C/T), and the SNPs frequency is 1.3%; there are six SNPs in the 443bp of SLA-DRA intron 2, such as 114(C/T), 127(C/T), 228(C/G), 346(G/T), 414(G/A), 428(G/A) and SNPs frequency is 1.4%. 26 SNPs and 2 mutation sites which include one three-alleles and one four-alleles are detected in the 251bp segement of the exon 2 in SLA-DQA and the SNPs frequency is 12%,all the SNPs are as follows: 198(C/T), 203(C/A), 211(A/T), 230(C/T),, 237(G/C), 245(C/T), 256(A/G), 276(C/A), 277(A/G), 293(C/T,C/A)s 294(A/G), 301(C/G), 314(G/A), 332(C/T), 339(A/G), 340(CA, 342(GH, 343(C/T,C/G), 348(C/T), 349(A/T), 352(A/C), 353(T/C), 362(C/T), 363(G/C,G/A,G/T), 367(T/C), 374(T/C). The insertion and delection mutations are detected in intron 2 by the units of six and nine nucleotides.Twenty-nine SNPs including 6 three-alleles and one four-alleles are found in the 174bp of the exon 2 of SLA-DRB gene ,allthe SNPs are as follows: 112 (T/G), 113 (T/A), 114 (T/C), 115 (C/A), 118 (G/T), 119 (G/T, G/A), 124 (T/G), 125 (T/C, T/A), 144 (T/C), 155 (A/G), 165 (A/G), 166 (T/A), 169 (G/C), 171 (G/C), 173 (G/A), 177 (A/C, A/G, A/T), 196 (T/C, T/A), 198 (T/A), 199 (G/T), 210 (C/T), 225 (G/A), 240 (C/T), 255 (A/T, A/G), 256 (G/T), 257 (A/C, A/T), 265 (A/T, A/G), 269 (A/G), 270 (A/G), 278 (A/G) . We also deteced 5 mutation sites and 3 SNPs in the 204 fragment of the second exon of SLA-DQB gene, it contains two three-allele and the mutation frequency is 15.7%, the distribution of SNPs is as follows: 243 (A/G), 245 (A/G), 246 (A/G), 247 (A/G), 257(A/G), 265 (T/C), 266 (A/T), 267 (T/C), 268 (G/T), 269 (C/T), 283 (G/A), 286 (G/A), 294 (G/A), 296 (A/T), 312 (T/G), 325 (G/A), 326 (C/A), 327 (C/G), 335 (A/C), 338 (G/T), 343 (A/G)→, 354 (C/G), 355 (A/G, A/T), 356 (T/C), 357(C/T), 368 (C/A, C/T), 375 (A/C), 379 (C/G), 386 (C/G), 387 (G/T), 418 (G/A) .3. A group of new alleles are found by the sequence alignment with the specific gene in GenBank... |
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