Study On The Transcriptional Regulation Mechanism Of The Major Gene IGF2BP1 For Chicken Growth Traits

Growth traits are the most economic traits in the development of livestock.As a significant meat product in China,the analysis of the genetic mechanism of broiler growth and development is of great theoretical significance.It is in line with industrial needs and the development direction of the breeding industry.Insulin-like growth factor 2 m RNA binding protein 1(IGF2BP1),a member of the insulin-like growth factor 2 family,plays a crucial role in the development of the body and is a central effector gene affecting growth/body size traits.In our previous studies,two mutant alleles in the promoter region of chicken IGF2BP1 contributed positively to growth and body size traits.However,the transcriptional regulation mechanism of the chicken IGF2BP1 gene has been unclarified.This study uses polymorphism analysis of the structural variation of IGF2BP1 promoter in fast and large broiler breeds,dual-purpose local chicken breeds,high-yielding egg-laying breeds,and "Gushi × Anka" F2 resource group was analyzed using molecular biology techniques.Specify the transcriptionally active region of IGF2BP1 by a dual luciferase reporter gene.Construction of a chicken IGF2BP1 promoter mutant DF-1 cell line using CRISPR/Cas9 to explore the effect of mutation on cell proliferation.DF-1 cell(WW-type)and KO_L1 cell(L1L1-type)were used to study further mutation-induced changes in the signaling pathway.DF-1 and KO_L1 cell lines were demethylated separately according to the results of methylation site detection to determine the mechanism of action of promoter methylation on the transcriptional activity of allele W.These will allow us to probe into the differences in transcriptional activity of IGF2BP1 promoter fragment deletion and refine the genetic regulatory mechanism of structural variants affecting IGF2BP1 gene expression.The main experimental results obtained in this study are as follows:1.Analyze the relationship between promoter polymorphisms in the IGF2BP1 gene and growth traits in chickens.Different genotypes of fragment deletion variants in the promoter region of the IGF2BP1 gene in pheasant,commercial and local chickens by allele-specific PCR.The results showed six genotypes of IGF2BP1,WW,L1 W,L1L1,L2 W,L2L2,and L1L2.The W allele is dominant in the wild and local varieties.Phenotype correlation analysis with the Gushi x Anka F2 population showed that the L1L1 genotype exhibited better traits of size,body weight,and carcass weight(p<0.05).The results suggest that structural variation in the IGF2BP1 promoter can be a marker for molecular selection for growth traits in chickens.The Dual-Luciferase reporter gene results for the different alleles showed that p GL3-L1 transcriptional activity was significantly higher than p GL3-L2 and p GL3-W(p<0.01).2.Construction of chicken IGF2BP1 promoter mutant cells on CRISPR/Cas9Chicken fibroblast cells with genotypes L1L1 and L2L2 by gene editing techniques were constructed based on the sequence characteristics of alleles W and L1.The results showed that the expression of the IGF2BP1 gene,cell viability,and cell proliferation levels was significantly higher in L1L1 and L2L2 cells than in wild-type WW cells(p<0.01).Comparative analysis of differential m RNA expression after IGF2BP1 promoter fragment knockdown by transcriptome sequencing of KO_L1 and DF-1 cells.Differential genes enriched in the TGF-β signaling pathway,Wnt signaling pathway,Fox O signaling pathway,MAPK signaling pathway,and circadian rhythm pathway.3.Mechanism of action of DNA methylation on the allele W transcriptional activityMethylation prediction and TBS-seq have three CpG islands within the ~3.2 kb deletion region of the KO_L1 mutant,with one methylation region located in the ~1.5 kb deletion interval of the KO_L2 mutant.The results showed that there was no significant difference in the methylation level of the promoter of IGF2BP1 between DF-1 and KO methyltransferase treated with decitabine(p>0.05),the methylation of the W region of chicken IGF2BP1 allele was the main cause of the decrease of transcription activity and cell viability.The research starts with IGF2BP1 promoter variation to investigate the relationship between IGF2BP1 gene structure variation and growth traits.CRISPR/Cas9-acquired mutant cell exhibit elevated IGF2BP1 gene expression and accelerated cell proliferation.The signal pathways with potential research value during growth and development were screened by the transcriptome sequencing of mutant cells.The W region of the chicken IGF2BP1 promoter was the main reason for the decrease in its activity as demonstrated by in vitro methylation experiments.This study clarifies the regulatory mechanism of IGF2BP1 gene promoter variation on gene expression,providing a theoretical basis for the analysis of the genetic mechanism of chicken growth traits,and also serves as a foundation for broiler breeding as a growth genetic marker.