Cloning, SNP Detection And The Association Analysis Of The Three Porcine RNASE, D6 And Its Partial Ligand Genes

PartⅠCloning,SNP detection and the association with haematological parameters of the three genes of the procine RNase A superfamilyBlindly pursuit for production performance improvement may low the disease-resistance capability,and almost all the diseases existing are of genetic correlation,thus,it is of essence,by the genetic point,to improve the disease-resistance capability.Heretofore, swine disease resistance breeding have achieved a certain progress,but still finite on its depth and identified relative genes.The thirteen members of the RNase A superfamily conclude a catalytic triad structure, resulting in the degradation of RNA.Previous studies reported that some members have special biological activities,including antibacterial,antimicrobial,antiviral and angiogenic properties.Combining of the bioinformatics,population genetics and the biotechnology, The RNASE1,ANG and RNASE6 genes were analyzed deeply.The main results are listed as follows:(1) Utilizing the porcine EST database information,the partial cDNA of the porcine RNASE1,ANG and RNASE6 genes were obtained.The sequences had been deposited in GenBank database,the accession numbers were:EU814500～EU814502.Then the deduced amino acid sequences,homologous gene sequences among different species and phylogenetic relationship were analyzed,respectively.(2) The SNP detection of these three genes was carried out and three SNPs were identified for further genotype by PCR-RFLP,these loci are as follows:g. 296A＞G polymorphism in the porcine RNASE1 gene,g.149G＞T polymorphism in the porcine ANG gene and g.389C＞T polymorphism in the porcine RNASE6 gene.(3) PCR-RFLP was performed to detect the genotype and gene frequences of the porcine RNASE6 gene in different porcine breeds.(4) Association analyses revealed the significant associations(P＜0.05) between the porcine ANG g.149G＞T polymorphism and mean corpuscular volume(MCV), mean corpuscular hemoglobin(MCH),mean platelet volume(MPV) and platelet-large cell ratio(P-LCR) measured on 0-day-old pigs and MCV measured at 32 days after birth.The porcine RNASE6 g.389C＞T polymorphism was significantly associated(P＜0.05) with MCV,MCH and neutrophil percentage (NEI%) measured on 0-day-old pigs,respectively. PartⅡCloning,initial detection of expression and SNP screening of the porcine D6 and its partial ligandsWith the influence from multi-factors,the genetic improvement is not available by the traditional breeding method to litter size traits.Therefore,molecular breeding method is employed to achieve genetic improvement of litter size,which may promote fast development of swine production again.In gestation,the maternal-fetal immune tolerance acts as a significant role to avoid the rejection to embryo and its appendage.Thus,the fetal can develop in the uterus until the successful delivery.Chemokine and its receptors play important roles in the immune tolerance of maternal-fetal interface.The chemokine decoy receptor,D6,[also known as Chemokine-binding Protein 2(CCBP2)]can not only recognize and bind to the active inflammatory CC chemokines(CCL2-5,7,8,11-14,17 and 22),but also prevent excessive leukocyte caused by the infiltration of abortion.Combining of the bioinformatics and the biotechnology,the porcine D6 and its partial ligands were analyzed.The main results are listed as follows:(1) Utilizing the porcine EST database information,the partia cDNA of the porcine D6, CCL8,CCL11,CCL14,CCL17 and CCL22 genes were obtained.The sequences had been deposited in GenBank database,the accession numbers were:FJ156751～FJ156756.Then the deduced amino acid sequences,homologous gene sequences among different species and phylogenetic relationship were analyzed,respectively.(2) Semi-quantitative RT-PCR was performed to determine the expression profile of the these genes in seven different tissues from the porcine endometrium from a healthy attachment site and arresting attachment site,chorion and amnion of abnormal pregnancy and normal pregnancy at gestation day of 65.(3) The results of SNP detection showed that there may are five mutations in the porcine CCL8 gene and four mutations in the porcine CCL11 gene.