Identification, Transcription Regulations And Association Analysis Of The Procine APC Gene

The anaphase-promoting complex (APC) is a multiprotein complex with ubiquitin ligase activity, which is required for the ubiquitination of securin and cyclinB.APC promotes metaphase/anaphase transition by ubiquitizing and degrading securin, an inhibitor of separase that participates in the degradation of the chromatic cohesion complex. APC also ubiquitinates cyclinB and accelerates its degradation during the late mitotic to the G1 phase, which results in mitotic exit. Moreover, the mitotic spindle checkpoint is activated if APC activation is prevented, suggesting that the dysregulation of APC may result in chromosome instability. In addition, several APC-targeting molecules such as securin, polo-like kinase, aurora kinase, and SnoN have been reported to be oncogenes.Therefore, dysregulation of APC may be associated with tumorigenesis.Subsequent work revealed that the vertebrate APC is a multiprotein complex consisting of at least 11 core subunits. In this study, we cloned and sequenced partial cDNA, introns and 5'-flanking sequences of porcine APC7,APC10 and APC13.the amino acid sequences of different species corresponding to the porcine APC7,APC10 and APC13 gene were collected through blast on the National Center for Biotechnology Information (NCBI) in order to conduct multiple sequence alignment and the phylogenetic analysis for estimation of evolutionary relationships among different organisms.Tissue distribution of porcine APC7,APC10 and APC13 were analyzed by real-time PCR. Several single nucleotide polymorphisms (SNPs) and insertion-deletion length polymorphisms (InDels) in the APC7 and APC13 were identified by sequencing PCR products. Association analysis of the relationship between genotypes and immune traits were performed in our resource population. Additionly, procine APC7 and APC10 gene promoter activity were identifyed and the interaction between APC7/APC10/APC13 proximal promoter and Sp1,as well as APC13 intronl and Spl,were detected. The knowledge gained in this study will contribute useful information for further research of APC and its application in the animal breeding to improve pork quality and quantity. The main results are as follows:1.Human mRNA sequences of APC7, APC10 and APC13 were compared to all sequences available in the pig in the express tag (EST) databases using the Blast algorithm. The porcine ESTs that shared more than 80% sequence identity to the corresponding human cDNA were selected in order to assemble the porcine gene.Primers were designed for amplifying coding DNA sequence (CDS) and partial genomic sequence of procine APC7,APC10 and APC13.With the purpose of ascertaining the evolutional properties of various animal species, comparison analyses of the deduced porcine APC7,APC10 and APC13 proteins with those of other species were carry out. The results suggested that APC7,APC10 and APC13 are evolutionarily conserved proteins.2.The expression of APC7,APC10 and APC13 in a number of porcine organs, including heart, liver, spleen, lung, lymph node, thymus, kidney and skeletal muscle, were detected by real-time PCR analyses and results revealed that mRNA expression levels of porcine APC7,APC10 and APC13 fluctuated within a small different range in detected tissues.3.The PCR restriction fragment length polymorphism (PCR-RFLP) and allele specific PCR(AS-PCR) methods were employed to genotype some polymorphic sites, including two SNPs and two Indels in porcine APC7, one Indel in porcine APC13. Allele frequencies were analyzed by genotyping the site among three or four unrelated pig breeds. Genotyping results showed apparent variation in allele frequencies among different breeds.4. Association analysis of the relationship between genotypes and some immune and growth traits were performed in our resource population. The results showed that the SNP (A/G) in intron 4 of porcine APC7 was significantly associated with the platelet count(PLT) of neonate piglets at 0 day (P=0.0427);the SNP (T/G) in intron 5 of porcine APC7 was significantly associated with platelet distribution width(PDW), mean platelet volume(MPV) and platelet-large cell ratio(P-LCR) (P=0.0175,0.0383 and 0.0213 respectively) at 32day; the 45bp InDel in promoter region of porcine APC7 was significantly associated with WB(P=0.0228) and WW(P=0.0096). The InDel Fnu4H1 in intronl of porcine APC13 is significantly associated with the absolute lymphocyte count (ALC) of neonate piglets at 17 day (P=0.0457), and mean corpuscular hemoglobin concentration (MCHC) of pigs at 32 days (P=0.0455).5.The essential 5'-flanking regions of porcine APC7,APC10 and APC13 were obtained by TAIL-PCR method. Potential transcription factor-binding sites on the region were predicted using online software (http://www.fruitfly.org/seq_tools/promoter.html, http://www.cbil.upenn.edu/cgi-bin/tess/tess).According these information, some 5'-deletion mutants were cloned and ligated into pGL3 basic vectors, then the plasmids were isolated and transiently transfected into PK15 cells.Promoter activity was measured relative to the activity of cells transfected with promoterless pGL2.6.Yeast one-hybrid assays were performed to examine the interaction of proximal promoters of porcine APC7,APC10 and APC13 and specificity protein 1(Sp1) transcription factor. A yeast one-hybrid assay also revealed Sp1 can interact with intron 1 of porcine APC13.