Cloning, Mapping, Expression Profile Of Porcine Pane1 Gene And Association Analysis Of Pane1 Gene And Longer Non-coding Rna In Pigs

The shortage of human organs is a rigorous problem always troubles us,unless we can find out perfect heterogeneous organ donors which can replace human.Swine have been used as a model for many human conditions because of their phenotypic similarities to humans including:cardiovascular anatomy and function,metabolism,lipoprotein profile,size,tendency to obesity,and omnivorous habits.So swine is an ideal choice in heterogeneous organ transplantation.But there are many problems such as graft-versus-leukemia(GVL) and graft-versus-host disease(GVHD) we cannot deal with. We are used to pay attention to MHC,especially HLA and H-2 antigen.However,many other investigations indicate that except MHC,there are some outcome of coding area which so-called minor histocompatibility antigens(mHAgs) also can beget immunological response.They are limited by MHC and recognized by CTL and TH. Some investigations also indicate that allogeneic transplantation in homogeneous different individuals having same MHC with different mHAgs also can beget GVL.In another word,mHAgs are able to induce T-cell response between MHC-matched,marrow transplanted donor and recipient.The PANE1(proliferation associated nuclear element 1) gene encodes a new mHAg—HLA-A~*0301.In some report,the gene highly express in resting CD19+ cell and B-lineage chronic lymphocytic leukemia(B-CLL) cell,so it can be seen as a potential therapeutic target.TncRNA is a long ncRNA that was isolated from our LongSAGE libraries.It was up-regulated along myogenesis and differentially expressed in fetal skeletal muscle at 90 days of gestation between Tongcheng and Landrace pigs.Previous studies on other animal species showed that TncRNA was expressed differentially in several expression libraries prepared from virus-infected and developmental tissues.But its basic structure and biological function remains unclear.In this study,we considered PANE1 gene and TncRNA as a candidate gene through cloning,chromosomal localization,polymorphism identification and association with production and immunity traits,providing a molecular genetic foundation on its application in the molecular marker assisted selection(MAS) in the future.The main results are showed as follows:1.The full sequence of coding region sequence of the porcine PANE1 gene was cloned using the pig specific primers which were designed from the pig EST contig obtained by the method of bioinformatics and comparative genomics according to the corresponding human cDNA sequence. 2.The INRA-University of Minnesota porcine radiation hybrid(IMpRH) panel was applied for detecting the precise localization of the PANE1 gene.The data of PCR results was analyzed using the mapping analysis software available on the web server.The result of the data analysis revealed that pig PANE1 gene is closely linked with microsatellite marker ACO2 on chromosome 5,with LOD scores 4.91.3.Gene tissue distribution was analyzed by the semi-quantitative RT-PCR in seven different tissues of skeletal muscle,liver,heart,kidney,spleen,lung and lymph node from a mature Chinese indigenous Tongcheng pig(Hubei Province,China).The result of RT-PCR displayed that PANE1 gene was expressed in all seven detected tissues.In the lymph node,spleen and skeletal muscle,it was expressed relatively higher.4.The polymorphism for the deletion mutation was analyzed by PCR-RFLP among the different pig groups,and allele frequencies and the allele distribution were determined among these populations.5.The polymorphism for PANE1 gene was analyzed in the Tongcheng population which constructed by our lab,including Tongcheng,Landrace♂×(Large White×Tongcheng)♀and Large White♂×(Landrace×Tongcheng)♀.The linear model was used to carry out association analysis.The preliminary results indicated that the PANE1 gene is showed a significant association with loin-muscle width and lumbosacral joint backfat thickness of swine.6.The resource population used in the association analyses is composed of 355 pure breed Landrace pigs,including parents(sire:17 and dam:36) and offspring(302).The linear model was used to carry out association analysis.The preliminary results indicated that the PANE1 gene is showed a significant association with RBW of 0-day-old pig, LYM of 17-day-old pig,MCV and LYMPH 32-day-old pig.6.The resource population used in the association analyses is composed of 355 pure breed Landrace pigs,including parents(sire:17 and dam:36) and offspring(302).The linear model was used to carry out association analysis.The preliminary results indicated that TncRNA gene is showed a significant association with MCV of 0-day-old pig (P＜0.05),with MPV and P_L of 32-day-old pig(P＜0.05) and with MCHC,PDW of of 32-day-old pig(P＜0.01).