| 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 indigenous duck breeds, Gaoyou (GY) duck breed and Kunshan (KS) duck breed and two improved chicken breeds, Jinghai yellow chicken (JYC) breed and Suqin yellow chicken (SYC) breed were examined. Thirty mixed-sex individuals were selected randomly from each breed at 16 weeks of age. Genomic DNAs were isolated from the whole blood, while RNAs were extracted from kidney and lung tissues for duck breeds and kidney, lung and spleen tissues for 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 (JYC) chicken breed (6.07±1.84, 5.39±3.27 and 7.51±6.06) than (SYC) 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 influence 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 relative expression level in kidney and lung tissues of Gaoyou (GY) duck breed was 3.406±2.611 and 2.562±2.058, respectively, while it was 2.535±2.402 and 1.594±1.398 for Kunshan (KS) duck breed. The higher relative expression level of (GY) duck breed account 34.35% and 60.70% over that of (KS) duck breed for kidney and lung tissues, respectively. Significant differences (p<0.05) were obtained for the relative expression level of kidney and lung tissues within (GY) duck breed and for the relative expression level of lung tissues between the two breeds. The results showed that sex affected TCF3 relative expression level insignificantly. The average of the relative expression level of TCF3 in kidney and lung tissues of chicken breeds was almost twice its relative expression level in correspondence tissues of duck breeds (189.33% and 223.77%, respectively).Single strand conformation polymorphisms (SSCP) analysis was conducted to scan for single nucleotide polymorphisms (SNPs) in TCF3 gene different sequences of improved chicken breeds and homologous sequences of indigenous duck breeds. The results showed that SNPs were not detectable for the indigenous duck breeds. However, for the improved chicken breeds (JYC and SYC) SNPs were existed and confirmed by SSCP-PCR product automated sequencing.The results of SSCP-PCR with two primers (TCF-P4 and TCF3-P7) showed the presence of different genotypes of TCF3 gene; however for TCF3-P4 primer no polymorphism was detected in (JYC) chicken breed. The combination of the two primers (TCF3-P4 and TCF3-P7) showed the presence of the three genotypes AA, AB and BB, but all expected genotypes were not achieved by single primer set; neither TCF3-P4 nor TCF3-P7.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 TCF3-P4 and G A at 110 nt. site of code sequence for primer TCF3-P7.Hardy-Weinberg Equilibrium (HWE) analysis was conducted to calculate genotype and gene frequencies of TCF3-P4 and TCF3-P7 for (JYC) chicken breed, (SYC) chicken breed and pooled individuals of the two breeds. Gene frequencies for allele A and B for TCF3-P4 were 0.80 and 0.20 and 0.90 and 0.10 for (SYC) chicken breed and pooled individuals, respectively. While for TCF3-P7 the gene frequencies were 0.483 and 0.517 for (JYC) chicken breed, (SYC) chicken breed and pooled individuals. Chi-square test (X2) showed significant differences (p<0.01) for the distribution of the genotypes of TCF3-P7 of the two breeds and pooled individuals, indicating that the populations were not in Hardy–Weinberg Equilibrium situation according to P7-SNP loci distribution. The TCF3-P4 genotypes were distributed insignificantly among the populations, revealing that the P4-SNP loci were in Hardy-Weinberg Equilibrium status.The correlation between TCF3 relative expression level in different tissues and genotypes resultant from single nucleotide polymorphisms of TCF3-P4 showed that the expression level of AA was greater than AB for different tissues of Suqin chicken breed. The relative 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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