Genetic Variations Analysis and Characterization of the Fifth Intron of Porcine NRAMP1 Gene *

: The natural resistance-associated macrophage protein 1 (NRAMP1) gene was identified as a candidate gene controlling the resistance and susceptibility to a number of intracellular parasites in pigs. The genetic variations in a 1.6 kb region spanning exon 1 and exon 3 of the porcine NRAMP1 gene were investigated by PCR-Kzn/ I-RFLP in samples of 1347 individuals from 21 Chinese indigenous pig populations and 3 western pig breeds. Three alleles ( A , B, C ) and four genotypes ( AA , BB, AB, BC) were detected. Significant differences in genotype and allele frequencies were observed between Chinese indigenous pig populations and exotic pig breeds, while in general the differences in genotype and allele frequencies among Chinese indigenous pig populations were not significant. The allele C was detected only in Duroc, Leping Spotted and Dongxiang Spotted pig, and the two Chinese pig populations showed similar genotype and allele frequencies. Four Chinese Tibetan pig populations displayed genetic differentiation at the NRAMP1 gene locus. In addition, intron 5 of the NRAMP1 gene was isolated and characterized by directly sequencing the PCR products encompassing intron 5. The alignment of intron 5 of the porcine, human, equine and ovine NRAMP1 gene showed a similarity of 45.38% between pig and human, 52.55% between pig and horse, 63.47% between pig and sheep, respectively. (Asian-Aust. J. Anim Sci. 2004. Vol 17, No. 9 : 1183-1187)


INTRODUCTION
The natural resistance-associated macrophage protein 1 (Nramp1), the macrophage specific protein encoded by the NRAMP1 gene, acts in the early preimmune phase of infection.NRAMP1 induces lysosomes to fuse with the bacterial phagosome, eventually leading to a mature, acidified and fully bactericidal phagolysosome.On one hand, the hydrolase hydrolyses pathogens in the phagolysosome.The NRAMP1 protein is a divalent-metal transporter and completes for the acquisition of essential divalent metals from the phagolysosome lumen, ultimately resulting in pathogen death for lack of ions and decompensation (Vidal, et al., 1993;Malo et al., 1994;Blackwell et al., 2000;Fo^es et al., 2001).The NRAMP1 gene was therefore identified as a candidate gene controlling the resistance and susceptibility to a number of intracellular parasites such as Salmonella (Sun et al., 1998;Bellamy, 1999).
Porcine NRAMP1 cDNA was isolated (Tuggle et al., 1997) and its chromosomal localization was assigned to SSC15q23-26 and linked with S0088, S0149 and S0284 STS-markers (LOD>3) (Sun et al., 1998).The NRAMP1 gene expressed specifically in reticuloendothelial cells (Cellier et al., 1994), its function was suggested to be associated with resistance to Salmonella (Tuggle et al., 1997) and many other kinds of intracellular pathogens in pigs (Sun et al., 1998).The objective of the present study is to detect genetic variations at the NRAMP1 locus in Chinese indigenous pig populations and exotic pig breeds by PCR-RFLP, and to isolate intron 5 of the NRAMP1 gene in order to study the breed characteristics of Chinese indigenous pig breeds and to take advantage of the valuable genetic resource of Chinese indigenous pigs for pig breeding.

Anim이s
The ear notches of 1,347 individuals were collected from 3 western commercial pig breeds (Landrace, Large White and Duroc) and 21 Chinese indigenous pig populations (Figure 1).Samples were collected from at least 40 individuals per breed from pig breed conservation farms.Care was taken to avoid sampling full-sib animals and to ensure as board a sampling area as possible in cases where no conservation farm exists.The sampling locations and the sample sizes are shown in Table 1 and Figure 1.Genomic DNA was extracted from the ear notches according to a modified phenol and chloroform method (Strauss, 1991;Wang et al., 2002).

Genotyping
Primer pair 1 (forward: 5'-ACC CAG CAC ACC ACT  Shanghai) with the supplemented 10xbuffer R+ and incubated at 37°C for 3-5 h or overnight.The restriction fragments were separated by 2.5% agarose gel in 1xTAE at a constant current of 50 mA.The gels were stained with ethidium bromide and visualized using an UV transilluminator.
PCR amplifications were performed in a final volume of 25 卩 l containing 100 ng of genomic DNA, 0.25 卩M of each primer, 0.2 卩M of dNTPs, 1 U Taq DNA polymerase with the provided 10xPCR buffer (MBI, Canada).PCR profiles were 34 cycles of 95°C for 45 s, 60°C for 45 s, 72°C for 45 s with an initial denaturation at 95°C for 3 min and a final extension at 72°C for 8 min.
PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced directly with two PCR primers using the ABI PRISM® BigDye™ Terminators v3.0 Cycle Sequencing Kit in an ABI PRISM® 3,100 Genetic Analyzer (Applied Biosystems, Foster City, USA) according to standard protocols.The sequence alignments were performed by using the SeqMan software from the DNAStar software package (http://www.lynnon.com)and the homologous sequences were analyzed by using BLAST (http://www.ncbi.nih.gov) and the identity of intron 5 of the NRAMP1 gene among the different species was calculated by the ClustalX software (downloaded from ftp://ftpigbmc.u-strasbg.fr).

SNPs screening in the isolated intron 5
Two DNA pools were constructed for screening single nucleotide polymorphisms (SNPs) in the isolated intron 5.Each of the DNA pools comprised of 5 individuals from each of the ten different breeds, namely the Duroc, Landrace, Min, Erhualian, Leping Spotted, Bama Xiang, Lingao, Wuzhishan, Yushan Black and Xizang Tibetan pig.PCR amplifications were performed with primer pair 2 using the two DNA pools as template, the resulting fragment was sequenced directly with two tagged primers to identify the putative SNPs.

Statistics
Significance between genotypes was assessed by the standard t-test using SAS system (1989)

Genotyping
The 1.6 kb PCR products amplified by primer pair 1 were digested by HinJI generating allele A (600 bp fragment), allele B (440-bp fragment) and allele C (230 bp fragment) as shown in Figure 2.

Allele and genotype frequencies
The genotype and allele frequencies of the NRAMP1 gene in 24 pig breeds are presented in Table 1.Three alleles (A, B, C) and four genotypes (AA, BB, AB, BC) were detected in 1347 animals from 24 pig breeds.The genotype for the polymorphisms of the porcine NRAMP1 gene, and investigated the genetic variations at the NRAMP1 gene locus among 24 Chinese and Western pig breeds.In addition, intron 5 of the porcine NRAMP1 gene was isolated and characterized.The present study provided a base for further studies on the association of the NRAMP1 gene with disease resistance traits in pigs.