| The TCF3 gene, synonyms (E2A; immunoglobulin enhancer-binding factor E12/E47; ITF1) contributes to transcription regulation in many cell lineage and codes for two proteins, E12 and E47, that are members of basic helix-loop-helix (bHLH) family involved in regulating growth and development in many tissues. To study the relative expression level of TCF3 in different tissues and screen the gene sequence for the presence of single nucleotide polymorphisms (SNPs) and their association with some phenotypic characters, two new chicken breeds, Jinghai yellow chicken and Suqin yellow chicken were examined. Thirty mixed-sex individuals were selected randomly from each breed at 16 weeks of age. Genomic DNAs were extracted from the whole blood, while RNAs were extracted from kidney, lung and spleen tissues for two chicken breeds. DNA and RNA purity and integrity were assessed photometerically and electrophoretically.Reverse transcriptase polymerase chain reaction (RT-PCR) results showed that the expression pattern of the splice variants E12 and E47 on serial diluted cDNA were very similar. Co-amplification of candidate TCF3 gene andβ-actin as internal control with two primer combinations in a duplex RT-PCR was performed. Calibration values (T/C) of relative level of amplification product of TCF3 gene (T) overβ-actin (C) obtained as volume (μg) using ImageQuant software showed that the relative expression level of TCF3 was greater in Jinghai yellow chicken breed (6.07±1.84, 5.39±3.27 and 7.51±6.06) than Suqin yellow chicken breed (5.12±3.57, 3.91±2.48 and 4.52±3.12) for kidney, lung and spleen tissues, respectively. The increasing relative expression level account 18.55%, 37.85% and 66.15% for kidney, lung and spleen tissues, respectively. The result showed that the degree of expression divergence in lung and spleen tissues between two breeds was found to be significant (p<0.05). Analysis of sex effect upon TCF3 expression level revealed substantially higher expression level in males than females for both two breeds. However, the trend was occurred in all tissues, differences were significant (p<0.05) only for the expression level in kidney and lung tissues. The results showed that sex affected TCF3 relative expression level insignificantly.Single strand conformation polymorphisms (SSCP) analysis was conducted to scan for single nucleotide polymorphisms (SNPs) in TCF3 gene different sequences of chicken breeds. The results showed that SNPs were not detectable for Jinghai yellow chicken breed . However, for Suqin yellow chicken breed SNPs were existed and confirmed by SSCP-PCR product automated sequencing. The automated sequencing of the SSCP-PCR product indicated that single nucleotide loci mutations were C→T at 139 nt. position of code sequence for primer Ex1401-Ex1402 .Hardy-Weinberg Equilibrium (HWE) analysis was conducted to calculate genotype and gene frequencies of primer Ex1401-Ex1402 for Suqin yellow chicken breed. Gene frequencies for allele A and B for primer Ex1401-Ex1402 were 0.80 and 0.20 .Chi-square test (X2) showed no significant differences (p>0.05) for the distribution of the genotypes of primer Ex1401-Ex1402 of Suqin yellow chicken breed, indicating that the populations were in Hardy–Weinberg Equilibrium situation according to primer Ex1401-Ex1402-SNP loci distribution.The correlation between TCF3 relative expression level in different tissues and genotypes resultant from single nucleotide polymorphisms of primer Ex1401-Ex1402 showed that the expression level of AA was greater than AB for different tissues of Suqin chicken breed. The expression levels of genotype AA were 5.762±4.004, 4.290±2.212 and 5.059±3.249 for kidney, lung and spleen tissues, respectively, while the relative expression level of the genotype AB account 4.272±2.883, 3.435±2.867 and 3.106±2.506 for kidney, lung and spleen tissues, respectively. Significant correlation (P<0.01) was found only for the relative expression level of spleen tissues for genotype AA and AB.The Least Square Differences (LSD) analysis of live body weight and relative expression level of TCF3 gene in kidney, lung and spleen tissues in chicken breeds showed insignificant differences; however, the relative expression level of AA pooled-sex individuals was higher than AB polled-sex individuals. Regardless genotype differentiation, sex significant effect (p<0.05) upon the relative expression level was occurred for kidney and lung tissues. The relative expression level of the AA males was substantially higher than that of the AA females. On the contrary, the relative expression level of AB females was higher than that of the males of same genotype.Monitoring gene expression differences by measuring mRNA levels in different tissues and generate a measurable signals to quantify, semi-quantitative RT-PCR assay can be a sensitive method to detect subtle changes in gene expression, these changes may reflect true changes in expression rather than RNA loading, Furthermore, to correlate the relative expression changes of specific gene with genetic make-up of an individual and other phenotypic characters, single nucleotide polymorphisms (SNPs), the most common type of segregating DNA sequence variation, could be a reliable genotyping method to identify allelic association with gene relative expression level. |
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