Different Transcriptions Cloning, Expression And SNPs Analysis Of IGF-I Gene In Swine

IGF-I plays a fundamental role in postnatal mammalian growth, development, and metabolism, and is the primary mediator of the growth-promoting activity of growth hormone. Despite the relative simplicity of its protein structure, but due to alternative splicing, and the use of multiple polyadenylation signals and different promoters, the IGF-I gene and its mRNAs are complicated. Recent studies demonstrated some high degree of polymorphism in the IGF-I gene in pigs. Therefore, the present study were to establish the structure of Songliao black pig IGF-I gene, to identify the Songliao black pig class 1 and class 2 IGF-I mRNAs by RACE, to compare their expression in different tissues of Songliao black pig, to compare their expression in livers and muscles among five different pig breeds using real-time RT-PCR technique, and to analyze the genetic correlations between mRNAs levels and performance traits. We detected the polymorphisms of the IGF-I genes by PCR-SSCP (single-strand conformation polymorphism) in the population of Songliao black pig, and analyzed the genetic correlation between the polymorphisms and growth and carcass traits by SPSS.To identify the IGF-I mRNA transcripts expressed in Songliao black pig liver, RLM-RACE was carried out. To distinguish whether Songliao black pig alternative usage of exon 5 and exon 6 for IGF-I expression, a 3'-RACE was performed on Songliao black pig liver mRNA. The 3'-RACE generated two major cDNA products. As revealed by sequencing, two cDNA bands contained 76 bp of IGF-I exon 4 at the 5'end and 245 and 175 bp of IGF-I exon 6, respectively, at the 3'end, but no exon 5 sequence was seen. The two cDNA bands resulted primarily from the use of different polyadenylation signals in exon 6. The results suggest that liver IGF-I mRNA of Songliao black pig contains only exon 6 to encode IGF-IA.To determine whether IGF-I gene is expressed as class 1 and class 2 IGF-I mRNAs in Songliao black pig, a 5'-RACE was performed on liver mRNA. The 5'-RACE generated f major cDNA products. As revealed by sequencing, four cDNA products contained 309, 247, 178,and 111bp of IGF-I exon 1, respectively, at the 5'end and 148bp of IGF-I exon 3 at the 3'end; these four cDNA products corresponded to four class 1 IGF-I mRNA variants transcribed from four differet transcription start sites within exon 1. The fifth cDNA band contained 90bp of IGF-I exon 2 and the same 148bp exon 3 region; this cDNA band represented a class 2 IGF-I mRNA. The results studied by us indicated that the IGF-I gene in Songliao black pig is also expressed as class 1 and class 2 IGF-I mRNAs. Generation of multiple IGF-I transcripts differing in the leader exon appears to be a mechanism conserved in mammals, and the conservation of this mechanism across species suggests that it may be important for control of IGF-I function. Multiple dispersed transcription start sites in exon 1 and exon 2 have been reported for humans, rats, sheep, and bovine, which are probably a reflection of the absence of typical transcriptional regulatory signals, such as a TATA or CCAAT box, as well as regions of GC-rich, in the proximal promoter.By assembling the cloned 3'-end , class 1, class 2 Songliao black pig IGF-I cDNA sequences, and the published pig IGF-I cDNA sequence (GeneBank No. NM₂14256), the complete sequences of class 1 and class 2 IGF-I mRNA from Songliao black pig have been obtained and deposited in the GeneBank database libraries and given the accession No. DQ784687 and DQ784688.To determine the distribution and transcription levels of Songliao black pig class 1 and class 2 IGF-I mRNAs, TaqMan real-time RT-PCR was carried out. The result shows that both classes of IGF-I mRNAs were expressed in all tissues examined, including heart, liver, spleen, lung, kidney, stomach, small intestine, fat, skeletal muscle, brain, thalamencephalon, hypophysis, testis, ovary, and uterus. The ubiquitous presence of both classes of IGF-I mRNAs suggests that at least some of the factors controlling transcription from exon 1 and exon 2 in the Songliao black pig IGF-I gene are ubiquitous transcription factors. Both classes IGF-I mRNAs were expessed at relatively high levels in the liver, fat, uterus, test, spleen, and ovary. The differential expression of IGF-I mRNA between tissues suggests that IGF-I gene expression is also controlled by tissue-specific transcription factors. Differential tissue-specific expression of both classes IGF-I mRNAs could well be an essential element in the complex mechanism of regulation of IGF-I expression.Class 1 IGF-I mRNA appeared to be much more abundant than class 2 IGF-I mRNA in all tissues.The results of TaqMan real-time RT-PCR revealed that the expression levels of class 1 IGF-I mRNA or class 2 IGF-I mRNA in livers or muscles were not significantly different among the five breeds, including Songliao black pig, Sus scrofa, Duroc pig, Landrace pig, and Large White pig, which may reflects the conservation and importance of IGF-I gene function. In livers and muscles, the expression levels of class 1 IGF-I mRNA were significantly higher than class 2 IGF-I mRNA (P<0.05), this suggests that the expression of class 1 IGF-I mRNA may be essential to provide basal levels of IGF-I for normal growth and development and other IGF-I dependent processes. The expression levels of class 1 IGF-I mRNA in the livers were significantly higher than those in the muscles(P<0.05), indicating that the liver is likely to be the major source of circulating IGF-I.Genetic correlations analysis show that there was a positive genetic correlation between class 1 and class 2 IGF-I mRNAs expression levels in muscles, and daily gain (r=0.940 and r=0.897, respectively; P<0.05). Moreover, the positive correlation between class 1 IGF-I mRNA expression levels in muscles and hog percent was also found (r=0.887;P<0.05). There was no correlation (P>0.05) between the expression levels of class 1 and class 2 IGF-I mRNAs in livers and performance traits measured. It was suggested that the IGF-I gene plays a more important role during the growth and muscle differentiation of animals.The polymorphism of the IGF-I gene in Songliao black pig was analyzed by PCR-SSCP. The results showed that the polymorphisms were not detected by PCR-SSCP in the 5'UTR of the IGF-I gene. And two alleles, A, and B, three genotypes, AA, AB, and BB, were detected in the exon4 of the IGF-I gene. The polymorphism fragments were cloned and sequenced. The sequencing results indicated that there was one single nucleotide mutation:A→G in the exon4 of the the IGF-I gene. The Chi-square test for goodness of fit showed that the frequencies of the IGF-I genotypes in the population of Songliao black pig agreed with Hardy-Weinberg equilibrium.The genetic correlations were obtained between genotypes of the exon4 of IGF-I gene and growth and carcass traits by the least square analysis.The results indicated that different IGF-I genotypes significantly affected average daily gain after weaning.Average daily gain after weaning for AA genotype on IGF-I was significantly higher than for AB and BB. The pigs with AA genotype had a lower lean percentage than those with AB genotype and BB genotype(P<0.01), and had a higher average backfat thickness than those with AB genotype and BB genotype(P<0.01). Other traits were not significantly affected. Therefore, the genetic analysis of IGF-I gene revealed that IGF-I locus significantly affected average daily gain after weaning, lean percentage,and average backfat thickness.As above-mentioned, the IGF-I gene is expressed as class 1 and class 2 IGF-I mRNA variants in Songliao black pig, which use almost exclusively, exon 6 and code for IGF-IA. Both classes of IGF-I mRNA are expressed in a variety of tissues of Songliao black pig.The expression level of class 1 IGF-I mRNA is higher than that of class 2 IGF-I mRNA. Class 1 IGF-I mRNA was expressed in greater quantity in liver as compared with in muscles. There were no statidtically differences of class 1 or class 2 IGF-I mRNA in livers or muscles among five breeds. The two classes of IGF-I mRNAs expression levels in muscles were positively correlated with daily gain (DG), and the expression level of class 1 IGF-I mRNA in muscles was also positively correlated with hog percent. The Songliao black pigs with AA genotype had higher daily gain, lower lean percentage, and higher average backfat thickness. It could be preliminarily deduced that IGF-I gene was probably a major gene or a QTL linked gene which associated with growth and carcass traits in pig.