Study On PRNP, SPRN And 37/67-kDa LRP/LR Gene In Chinese Indigenous Goats

Scrapie is a transmissible spongiform Encephalopathy, caused by conformation conversion of normal host cellular prion protein and aggregation incentral nervous system. So we attempt to investigate three genes related with development and progression of scrapie: PRNP, SPRN and 37/67-kDa LRP/LR. The results should to provide theoretical foundation of scrapie-resistance breeding in goats and guide to control scrapie as viewed from genetics.A total number of 937 PRNP gene sequences belonging to 83 species in 56 genera of 26 families were analyzed in order to investigate its evolution and differentiation among Species. The length of PRNP for all species analyzed varied from 567bp to 825bp,which was mainly because of insertion or deletion in repeat region within or among species. TGA was the primary stop codon in PRNP gene. Bos taurus had the smallest average number of nucleotide differences (0.811) and nucleotide diversity (0.0011) and smaller nonsynonymous nucleotide diversity (0.0002) among all the ruminants. Delphinidae had the smallest number of total mutations (3), silent mutations (2), nonsynonymous mutations (1), average number of nucleotide differences (2.000), synonymous nucleotide diversity (π(s), 0.00739) and nonsynonymous nucleotide diversity (π(a), 0.00113). Muridae had the largest number of total mutations (349), silent mutations (94), nonsynonymous mutations (253), average number of nucleotide differences (150) and nucleotide diversity (0.19617). Muridae and Bovidae had higher ratio ofπ(a)/π(s) (0.673 and 1.209). The reconstructed phylogenetic tree of PRNP gene of families and species was basically consistent with the taxonomy of NCBI except for Felidae (Felis catus), which was firstly clustered with Moschidae rather than Mustelidae or Canidae.To determine the variability of the PRNP gene in goats in Chinese indigenous goat breeds. We isolated blood/kidney samples from 337 goats representing the eleven mail local goat breeds in eight provinces of our country to identify the PRNP polymorphisms and to determine whether these breeds are at risk for developing scrapie. In total , ten amino acid mutations (102, 127, 143, 146, 154, 211, 218, 219, 222 and 240)and three symnoumous mutations(42, 125 and 138) were detected,Two novel amino acid polymorphisms (R211G and T219I) and a novel silent mutation at codon 125 as well as nine previously reported polymorphisms were observed. Twenty-eight alleles and forty-nine different genotypes were obtained. The codon 142M associated with resistance of goat scrapie was not found in this study. The codon 146S, 154H and 127S associated with resistance or period of incubation was found in one goat respectively. The codon 211Q associated with protection towards natural scrapie infection was found in twenty-two goats. The codon 222K, a valuable candidate site to select for scrapie resistance, was also rare in Chinese main indigenous goats. Differences in allele distribution were found between Northern and Southern goats, in particular regarding K222 allele, possibly associated to scrapie resistance. These results could provide some useful data for assessing the risk of scrapie in Chinese indigenous goats.To analyze the character and variation of noncoding sequence in caprine PRNP gene, the fragments containing 5'UTR, intron 1 and 3'UTR were amplified by PCR technique. Two CpG islands were found in caprine PRNP gene. Fifteen transcription factor binding sites were predicted in putative promoter core region(4648-5169bp). The four conserved motif sequence is predicted for transcription factor YY1,E4BP4,Foxp3 and COE1 binding sites respectively. The promoter activity was found in intron 1. The total 52 SNPs were found in 5'flanking region and 35 SNPs in 3'UTR region. The polymorphism analysis result of position 2140 resulting to delete the transcription factor C/EBP alpha binding sites showed that allele A was preponderant alleles. The genotype frequency of allele B in different goat populations is 0.1452-0.4000. The genotype of allele B was very low and no homozygous genotype was detected. The variation at position 2140 could repress the promoter activity of caprine PRNP gene using luciferase reporter gene vector.To investigate the expression of cellular prion protein (PrPc) in taihang goat tissues and organs, the western blot technique was used. The PrPc was expressed in cerebellum, brain stem, thalamus, liver, spleen, kidney and muscle, the higher expressin was shown in cerebellum, brain stem, and thalamus. PrPc was not detected in lung. To investigate the expression location of PrPc, immunohistogram chemistry was used. The expression of PrPc is detected in molecular layer, granular layer and Purkinje's cells in cerebellum, neuron cell body in thalamus and brain stem. The expression of PrPc was also found in glomerular and proximal convoluted tubules in kidney, liver cells, and myeloid dendritic cells.We cloned and analyzed caprine SPRN gene, we isolated the genomic sequence including full-length coding sequence and analyzed the mutations in the caprine SPRN gene. The genomic length of caprine SPRN gene acquired is 4237 bp, the full-length of open reading frame is 441bp encoding 146 amino acids. Some transcription factors binding sites Sp1, AP-2 and YY1, no TATA box or CCAAT box, were predited in 5'UTR of SPRN gene. The key transcriptional regulation factors were located in 323-1329bp and the repressor factors binding sites were predicted in 111-323bp by luciferase reporter gene vector. In total, thirty-six mutations were found: ten in the promoter region, three in intron 1, seven in the coding sequence (five nonsymnonous mutations) and fifteen in the 3'untranslated region. The tissue expression profiles showed that SPRN mRNA is highly expressed in cerebrum and cerebellum, low levels in testis, mesentic lymph node, and lung and no mRNA was detected in other tissues.We cloned and analyzed genomic sequence and full-length coding sequence of caprine 37/67-kDa LRP/LR gene. The length of genomic sequence isolated is 13587bp composed of seven exons and six introns. The full-length coding sequence is 888bp, encoding 295 amino acids. Putative transcription factor binding sites Sp1, AP-1 and AP-2, TATA box and CCAAT box were predicted in 5'flanking region of caprine 37/67-kDa LRP/LR gene.The putative amino acid has high similarity with that of other mammals. The caprine amino acid of 37/67-kDa LRP/LR is shown identical amino acid with species susceptible to scrapie in 241,272 and 291. RT-PCR results of eleven tissues indicated that 37/67-kDa LRP/LR mRNA is expressed in all selected caprine tissues and highly expressed in cerebrum, cerebellum, thalamus and hippocampus.