Association Analysis Of Two Genotypes Of MiR-155 In Pigs

Micro RNA is an important subgroup in non-coding RNA, it takes part in the cell proliferation, development and apoptosis. Mi R-155 plays an important role in development and regulation of immune system, the mutations that at key sites on its host gene may lead to changes of mi R-155 expression, which may cause variation of the downstream immune genes. According to the information of SNP that located in the mi R-155 host genes BIC, we identified the different genotypes from cultivating breeds by PCR-RFLP method. Then we separated the Treg of two genotypes by flow cytometry. Meanwhile, association anaylsis is carried out to research the relevance between a key SNP and immune indices of Duroc × Er hualian F2 population. The specific results are followed:(1) Identification of two genotypes pigs According to the information of SNP that located in the mi R-155 host genes BIC, we see the first base as +1, so the 305 th base in the upstream of pre-mi R-155 is-305 bp A/C. Based on this SNP, we identified specific genotypes individuals from three breeds like Suzhong pigs(55 individuals), E’tong liangtouwu(300 individuals) and Hubei White pig(250 individuals). Though PCR-RFLP method, we found four AA genotypes and four CC genotypes of E’tong liangtouwu, two AA genotypes and two CC genotypes of Hubei White pig. In the E’tong liangtouwu population, the AA genotype frequency is 0.0348, the AC genotype frequency is 0.4609 and the CC genotype frequency is 0.5043. The gene frequency of A is 0.2653, and that of C is 0.7347. Meanwhile, in the Hubei White pig, the AA genotype frequency is 0. 8279, the AC genotype frequency is 0.1680 and the CC genotype frequency is 0.0041. The gene frequency of A is 0.9119, and that of C is 0. 3455. However, we fail to found CC haplotyoe in Suzhong pigs, so the AA genotype frequency is 0. 6545, the AC genotype frequency is 0. 3455. The gene frequency of A is 0. 8273, and that of C is 0. 1727.(2) Variation analysis of blood parameters in two genotypes pigs We checked the blood parameters of four AA and four CC genotypes individuals in E’tong liangtouwu. The result shows that MCV and RDW between two genotypes are different significantly(P<0.01).(3) Association anaylsis of-305 bp A/C SNP and immunity index of F2 population We got the genotypes datas in Duroc×Er Hualian F2 pigs according to the-305 bp A/C SNP, then the association anaylsis were carried out. The results shows: In this F2 population, the AA genotype frequency is 0.2507, the AC genotype frequency is 0.5171 and the CC genotype frequency is 0.2321. The gene frequency of A is 0.5092, and that of C is 0.4908. There are significant associations between this SNP and the percentage of CD4-CD8+CD3-,CD4-CD8+CD3+ in 20-day-old pigs(P<0.05). The SNP were correlated with RBC,HCT,HGB in 33-day-old pigs(P<0.05) and significantly associate with PDW and MPV(P<0.01). In 35-day-old pigs, the SNP were correlated with HGB, HCT(P<0.05) and significantly associate with PDW(P<0.01). There are associations between this SNP and the EO and the percentage of EO in 80-day-old pigs(P<0.05).(4) Separating of Regulatory T cell in two genotypes pigs We separated the Regulatory T cell by flow cytometry. At first, the lymphocytes were separate by the size et al of cell. Then we chose the CD3+ T cell before separating the CD4+ T cell. Finally, we got the Treg by chose the CD3+CD4+CD25+ T cell. The percentage of Treg in PBMCs is about 1%.(5)Vitro experiments research the target genes of mi R-155 Transfecting mimic155 to pig macrophages and researching the role of mi R-155 to four target genes. The result showed that there were no significant between two groups, which may suggest that mi R-155 do not affect the expression of m RNA of the target genes.This preliminary study demonstrated this SNP mutation that localed in the mi R-155 hose geng BIC are associated with some immune indices, and we found the different mutatuins in the pre-mi R-155 can effect the function of mi R-155.Thus, we provide some theoretical basis for revealing the immunity difference between two genotypes pigs, and provide the molecular marker for correlated character.