| Insulin-like growth factor genes(IGF1)are candidate genes for animal growth and development.It has been found that there are many base variations in the promoter region of pig IGF1 which are closely related to pig growth traits.Polymorphic(CA)n and poly-T microsatellites in the promoter region of porcine IGF1 have been detected,and(CA)n microsatellites may form potential Z-DNA structures and affect IGF1 transcription.In this study the(CA)n microsatellite variation in the promoter region of IGF1,(CA)n-Tn haplotypes and their relationship with growth traits,and the effection on the IGF promoter were focused.The dual luciferase recombinant vectors of different(CA)n-Tn haplotype and(CA)n microsatellite variant were constructed to elucidate the effect of microsatellite variation on IGF1 promoter activity.The gel retardation experiments were used to detected whether the(CA)n microsatellite could form a Z-DNA conformation.The results of this study are expected to further explore the functional analysis markers related to pig growth and development.The results of this study are as follows:(1)The haplotype(CA)17-T11,(CA)18-T11,(CA)19-T11 and(CA)20-T12 homozygous individuals accounted for 0.23%,22.81%,16.13% and 0.46% of the Landrace pig population,respectively,and(CA)18-T11 and(CA)19-T11 are dominant haplotypes.Correlation analysis showed that haplotype(CA)n-Tn was not significantly associated with birth weight,115 kg day age,115 kg average daily gain,and 115 kg backfat thickness,but was significantly associated with body length traits.The body length of(CA)17-T11 was significantly higher than those of(CA)19-T11 and(CA)20-T12.(2)The construction of haplotype(CA)17-T11,(CA)18-T11,(CA)19-T11 and(CA)20-T12 dual luciferase recombinant vectors were transfected into porcine fetal fibroblast,and the results showed that relative activity of haplotype(CA)17-T11 was highest,followed by(CA)18-T11 and(CA)19-T11,and the relative activity of(CA)20-T12 had the lowest.(3)With the(CA)n microsatellite deletion,the activity of T11 was higher than that of T12,and the activity of T11 was significantly lower than that of haplotype(CA)n-T11,while the T12 activity was higher than that of(CA)20-T12.After the poly-T deletion,the activity of(CA)17,(CA)18,(CA)19 and(CA)20 was higher than that of the corresponding(CA)n-Tn,and the relative activity of the IGF1 gene decreased with the increasing of the number of microsatellite repeats.When the(CA)n microsatellite and poly-T in the(CA)n-Tn haplotype fragment were simultaneously deleted,their relative activities were lower than the relative activities of(CA)n-Tn,(CA)n and Tn.(4)Though there are two Z? domains in the porcine ZBP1 protein,the human recombinant ZBP1 protein could not form a gel retardation with the p MD-18 T recombinant vector,indicating that the(CA)n microsatellite may not form the Z-DNA.In conclusion,it can be concluded that different(CA)n-Tn haplotypes affect the activity of IGF1 promoter and thus affect the growth traits of Landrace pigs.As the(CA)n microsatellite repeat number increases,the IGF1 transcriptional activity gradually decreases,and the(CA)n microsatellite may cooperate with poly-T to maintain the porcine IGF gene transcriptional activity. |
