ГЕНЕТИКА, 2015, том 51, № 3, с. 379-383
ГЕНЕТИКА ЖИВОТНЫХ =
DIFFERENTIAL EXPRESSION OF PORCINE TAP1 GENE
IN THE POPULATIONS OF PIGS © 2015 S. P. Zhu, X. M. Yin, L. Sun, S. Y. Sun, W. B. Bao, and S. L. Wu
Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou Jiangsu, 225009 China e-mail:email@example.com, firstname.lastname@example.org Received May 22, 2014
Transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum (ER) for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. TAP is consisted of two subunits: TAPl and TAP2. Using Real-time PCR technology, this study detected tissue expression profile and analyzed the differential expression of TAP1 gene in Sutai Escherichia coli-resistant group, Yorkshire and Meishan pigs. Tissue expression profile revealed that TAP1 gene expressed in all tissues we detected, and the expression levels were high in lung, immune tissues and intestines. Through the comparation of gene expression differention in different populations, TAP1 expression level of Sutai E. coli-resistant group was significantly higher than that of Yorkshire and Meishan populations in liver, spleen, lung, kidney, thymus, lymph, duodenum and jejunum (P < 0.05). Meanwhile TAP1 gene was more highly expressed in Sutai E. coli-resistant group than that of Meishan population in stomach (P < 0.05). In conclusion, the upregulation of TAP1 expression level in E. coli-resistant group could be related to E. coli F18 infection. In addition, Chinese local pigs may have special immune response and genetic mechanism in resisting E. coli F18 infection which is differing from MHC I moleculars.
Transporter associated with antigen processing (TAP) is a heterodimer composed of the TAP1 and TAP2 subunits, and both of them have an N-terminal membrane-spanning domain and a C-terminal nucleotide binding domain. The major function of TAP is endogenous antigen presentation . For immune system, TAP carries antigenic peptides, which are degraded by proteosome from the cytosol into the lumen of the endoplasmic reticular for subsequent loading onto major histocompatibility complex (MHC) class I molecules. After that, TAP transports the peptides to the cell surface to induce the specific recognition of CD8+ T cell . Therefore, TAP was related to autoimmune disease closely, and was taken as an effective candidate gene for porcine breeding for disease resistance in some reports .
Up to now, the study of diarrhea and edema disease has made some improvements. Escherichia coli F18 is one of the primary causative agents of these diseases. To be specific, with fimbriae, E. coli F18 can adhere to the receptor of enterocytes. Then, the bacterium settles, reproduces, and produces enterotoxin, which causes disease in piglets . Vogeli et al.  proposed that the alpha (1,2)-fucosyltransferase (FUT1) gene on M307 G/A could be an effective candidate gene for the E. coli F18-resistance. However, there is no polymorphism in Chinese local breeds, which demonstrates that there are some genetic differences in resisting E. coli F18 between local breeds and foreign breeds . Based on the paired full-sib individuals selected
from established resource of Sutai pig (Duroc x Meishan) that were characterized as resistant or sensitive to E. coli F18, our research team identified the expression profiles of some genes, including TAP1, in duodenum. The results showed that TAP1 played a vital role in resisting E. coli F18 infection .
At present, the reports about TAP1 are rare. Besides, the gene expression level in different breeds is not clear. Hence, this article analyzed the tissue expression profile among Sutai pig E. coli-resistant group, foreign breed Yorkshire and local breed Meishan in order to investigate the correlation between TAP1 gene and diarrhea and edema disease, and provide a theoretical foundation for distinguishing the molecular mechanism of E. coli F18 susceptibility between local and foreign pigs.
MATERIALS AND METHODS
Sutai pig, which was generated by crossing Duroc pigs with Taihu pigs, is a high-quality and lean-meat breed. And previous studies have shown that a-(1,2) fucosyltransferase gene (FUT1) is a candidate gene of controlling E. coli F18 strain adhesion [5, 8]. In previous study, our team detected a small number of FUT1 AG genotype individuals in Sutai pig populations that were adequate for selective breeding, and by breeding these homozygotes, we obtained the valuable E. coli
Table 1. Real-time PCR primers
Gene Sequence Expected length (bp)
TAP1 5'-CCACTGCTTTTCCTTCTGCCT-3' Reverse primer: 5'-ACAGAACCTCAATGGCCACCT-3' 109
GAPDH 5'-ACATCATCCCTGCTTCTACTGG-3' Reverse primer: 5'-CTCGGACGCCTGCTTCAC-3' 187
F18-resistant group in Sutai pigs. At the same time, our group also developed a technology, combined with receptor binding experiments, in which functional ad-hesins are displayed by the type V secretion system to analyse and verify the ETEC F18 resistance/sensitivity of each piglet with genotypes AA, AG and GG of the FUT1 gene .
Sutai piglets were from Suzhou Sutai Pig Breeding Centre, China; Yorkshire piglets were from Engineering Research Centre for Molecular Breeding of Pig in Changzhou City of Jiangsu Province, China; Meishan piglets were collected from Meishan Pigs Conservation Breeding Company, Jiangsu China. Selecting 8 individuals from each group, piglets were sacrificed after weaning (35 days) when the piglets are particularly prone to infect E. coli F18. After sacrifice, the heart, liver, spleen, lung, kidney, stomach, muscle, thymus, lymph, duodenum and jejunum, were collected and stored immediately in liquid nitrogen, and then placed in a low-temperature freezer (—80°C) for further study.
Real-time PCRprimer design
Using Primer Express 2.0 software, TAP1 primers were designed based on the sequence of NM001044581 (http://www.ncbi.nlm.nih.gov/) in GenBank and synthesized by Takara Biotechnology Dalian Co., Ltd. (China). GAPDH was used as an internal control to normalize all ofthe threshold cycle (Ct) values of other tissue products. Detail informations are listed in Table 1.
Total RNA extraction and real-time PCR
Total RNA was extracted from homogenized tissues (50—100 mg) using Trizol reagent (TaKaRa Biotechnology Dalian Co., Ltd., China) according to the manufacturer's instructions. Precipitated RNA was dissolved in 20 ^L RNase-free H2O and stored at —80°C. The RNA quality and quantity were assessed by agarose gel electrophoresis and Nanodrop-1000 spectrophotometer, respectively.
The 10-^L reaction mixture for cDNA synthesis contained 2 ^L 5x PrimerScript buffer, 0.5 ^L Prim-
erScript RT Enzyme Mix I, 0.5 |L Oligo dT, 0.5 |L random hexamers, 500 ng total RNA, and RNase-free H2O to make up the final volume of 10 |L. The reaction was carried out at 37° C for 15 min and then at 85°C for 5 s.
Real-time PCR amplification was performed in a 20-|L reaction mixture containing 1 |L cDNA (100— 500 ng), 0.4 |L each forward and reverse primer (10 |M), 0.4 |L 50x ROX Reference Dye II, 10 |L 2x SYBR Green real-time PCR Master Mix, and 7.8 |L ddH2O. The PCR conditions were as follows: 95°C for 15 s, followed by 40 cycles of 95°C for 5 s and 62°C for 34 s. The dissociation curve was analyzed after amplification. A peak melting temperature (Tm) of 85 ± ± 0.8°C on the dissociation curve was used to determine the specificity of the PCR amplification. The Tm value for each sample was the average of the real-time PCR data for triplicate samples.
Data processing and analysis
The 2-AACt method was used to process the realtime PCR results [10, 11]. Statistical analyses were carried out using SPSS 16.0 software (SPSS Inc., USA). The t-test was used to analyze the significance of differences in mRNA expression among different groups.
The purity of total RNA and the specificity of real-time PCR
Total RNA samples were assayed using 2.2% denatured agarose gel electrophoresis. Three bands, representing 28S, 18S, and 5S were observed with no significant band associated with degradation or DNA contamination, indicating that the extracted total RNA was highly pure. The RNA purity was also evaluated via Nanodrop-1000 spectrophotometer. The A260/A280 ratios of the samples were 1.8—1.9, which indicated that the extracted RNA was of high quality and could be used for subsequent tests. There was only one specific peak in real-time PCR, indicating that none of primer dimer and non-specific products existed.
DIFFERENTIAL EXPRESSION OF PORCINE TAP1 GENE 381
Table 2. Differentiation of TAP1 mRNA expression among different pig populations
Tissue Sutai E. coli-resistant Yorkshire Meishan
Heart 6.94 ± 2.46 1.92 ± 0.63 1.49 ± 0.59
Liver 51.35 ± 10.69a 8.00 ± 3.34b 4.98 ± 1.54b
Spleen 153.37 ± 39.36a 58.47 ± 7.87b 33.48 ± 8.61b
Lung 242.57 ± 24.48a 76.60 ± 2.95b 47.46 ± 21.40b
Kidney 46.99 ± 7.33a 14.57 ± 5.82b 17.58 ± 9.46b
Stomach 100.73 ± 16.62a 40.19 ± 3.01ab 18.26 ± 10.21b
Muscle 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00
Thymus 163.85 ± 20.49a 26.17 ± 13.58b 26.97 ± 0.80b
Lymph 254.31 ± 16.57a 61.81 ± 15.24b 31.31 ± 0.85b
Duodenum 236.69 ± 6.72a 49.74 ± 9.14b 47.45 ± 9.03b
Jejunum 397.82 ± 59.74a 61.22 ± 7.44b 29.40 ± 6.33b
Note: Values between columns with small letters indicate P < 0.05.
Tissue expression profile of TAP1 gene
The average expression level of TAP1 in the muscle tissues was defined as 1.0 so that the expression levels of this gene in other tissues could be quantified (Table 2). The results showed that the tissue expression profile of TAP1 in Sutai E. coli-resistant group presented the same trend as that in Yorkshire and Meishan pigs. The average expression level of TA
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