Studies On The Genes Associated With ETEC F4 Receptor And Screening The Related SNPs

The adhesive phonetype of Yorkshire and Shaziling piglets to 3 ETECF4 antigens were detected and its genetis differences between two breedswere analysed. Then the genes associaed with F4 receptor were screenedusing Delta DDRT-PCR method, the related genes were further studied toscreen the SNPs associated with F4 receptor, the main results were showedas follows:1. the adhesive test result showed the F4 ad antigen adhesive to 2 breedsintesively, however the ad antigen was no adhesive to all 2 breeds. Therewere high frequency of F4abR+-acR- phonetype. The ad antigen wasadhesive to Shaziling more intesively(0.400) than that toYorkshire(0.242). these results also indicated there were linkage betweenF4 ab receptor and ad receptor, the ad receptor has no linkage with otherreceptors.2. 67 differential segments were detected by Delta DDRT-PCR method. 22differential segments of F4 ab receptor were detected including 13segments expressed in adhesive samples especially and 9 segmentsexpressed in no-adhesive samples especially; 21 differential segments ofF4 ac receptor were detected including 13 segments expressed in adhesivesamples especially and 8 segments expressed in no-adhesive samplesespecially; 24 differential segments of F4 ad receptor were detectedincluding 13 segments expressed in adhesive samples especially and 11segments expressed in no-adhesive samples especially.3. among 67 differential segments, 7 segments were not re-amplified anddid not get further atudied. Northern blot result showed 7 segmentswere false postive and 53 postive segments were identified, the postiveratio was 88.3％(53/66)。 4. 30 cloned vector were sequenced and the sequenses were alignmentedby BLAST of NCBI website, the results showed 7 segments had highsimilarity(＞85％) and the homologous length no less then 60 nucleotides.12 segments did not showed the 85％similarity or the homologous lengthwas too short, the rest 11 were segments of novel genes.20 ESTs were deposited into GenBank and their accesion No wereDQ217772, EC268686, DW286736-2867939, EB739624-739627 and EE392468-392477.The BLAST results indicated the No. 13 differential segments of F4 abreceptor had 90％(111/123) similarity with agouti signaling protein(ASIP)gene; the No. 3 differential segments of F4 ac receptor had 91％(239/260)similarity with human ATPase, Ca++ transporting, plasma membrane 1 (PMCA1) gene; No. 9 differential segments of F4 ad receptor had 91％(216/237)similarity with glycosylinositolphohatidyl anchor 1 (GPI 1) gene.5. Four pair of primers were 3 genes were designed separately to screenthe SNPs associated with F4 receptors. The results indicated:5. 1 In ASIP gene, the segment amplified by primer 1 had SNPs associatedwith F4ab receptor, the nucleotide of all adhesive samples was G and thatof all no-adhesive samples was A at position 485 nucleotide; thenucleotide of all adhesive samples was A and that of all no-adhesivesamples was T at position 619 nucleotide.5.2 In PMCA 1 gene, the segment amplified by primer 2 had SNP associatedwith F4ac receptor, the nucleotide of all adhesive samples was A and thatof all no-adhesive samples was T at position 142 nucleotide.5.3 In GPI 1 gene, the segment amplified by primer 2 had SNPs associatedwith F4ad receptor, the nucleotides of all adhesive samples was AT andthat of all no-adhesive samples was TG at position 242,243 nucleotides.5.4 Some SNPs were also found in other loci of the three genes, however,these SNPs were not associated with F4 receptors.