| Zinc finger protein(ZNF)refers to a class of protein that contains a short,stable,self-folding "finger" structure by binding to Zn2+.It is a transcription factor with a finger-like domain that plays a role in gene regulation.Important role.The zinc finger structure of zinc finger protein regulates gene expression at the transcription and translation level through specific binding to the target molecule DNA,RNA,DNA-RNA sequence,as well as to itself or other zinc finger proteins.Transposons are genetic elements that can"jump" to different positions in the genome.Retrotransposons play an important role in the composition of mammalian protein-coding genes.Molecular markers based on Retrotransposon-based Insertion Polymorphism(RIP)have become an important method for selection and breeding.In this paper,bioinformatics and comparative genomics methods were used to annotate the retrotransposon insertion sites on six ZNF(Zinc finger protein)genes in pigs,and PCR electrophoresis was performed to identify the polymorphic sites.At the same time,analyze the polymorphic distribution of these retrotransposons-mediated polymorphic sites in different populations,and analyze the association between the SINE insertion polymorphisms in ZNF2 and ZNF7 genes and the production and reproductive performance data of large white pigs.Finally,ZNF2 The core promoter region of the gene was excavated,and it was further verified whether the insertion of SINE in the first intron has an effect on the activity of the core promoter of the ZNF2 gene.The results are as follows:1.Annotate the transposons in the ZNFs gene family on pigs by RepeatMasker,and combine multiple sequence alignments to obtain 24 large structural variant sites(>50bp)that are suspected to be mediated by retrotransposons.PCR amplification was performed in the pool DNA of pig breeds,and finally 6 polymorphic loci were obtained,located in Intronl and Intron3 of ZNF2 gene,5’Flank and Intron2 of ZNF3 gene,3’UTR of ZNF7 gene,ZNF12 Gene Intron2.2.Select 2 valuable RIP sites(ZNF2-SINE-RIP and ZNF7-SINE-RIP)for PCR amplification and detection in 10 pig breeds,count and analyze population genetic information,the results show that these 2 RIP sites.The polymorphism in foreign breeds is better than that in domestic breeds.The frequency of SINE+ alleles at ZNF2-SINE-RIP marker locus is the highest in Duroc,indicating that this allele may come from foreign pigs Kind.The frequency of SINE+ alleles at the ZNF7-SINE-RIP marker locus is the highest in Sicilian pigs,indicating that this allele may also come from foreign pig breeds.PIC in foreign varieties is mostly moderately polymorphic,while PIC in domestic varieties is mostly low-polymorphic3.Perform PCR detection on ZNF2-SINE-RIP and ZNF7-SINE-RIP in large white populations(459 heads),and perform correlation analysis with large white pig production and reproductive performance data.The results showed that the corrected backfat thickness of ZNF2-SINE-RIP individuals with SINE insertion(SINE+/-)was significantly greater than that of individuals without insertion(SINE-/-);ZNF7-SINE-RIP was not significantly correlated with the production and reproductive performance data of large white pigs.4.The ZNF2 gene is highly conserved in multiple species,and it is predicted that there are multiple promoter regions near the SINE insertion site in intron 1 of the ZNF2 gene.In this study,the predicted promoter region was cloned into a dual luciferase reporter vector.After transfection of HeLa,PK15 and PEF cell lines,it was found that Frag.2 region exhibited extremely significant strong promoter activity(p<0.01).It may be the core promoter region of the ZNF2 gene to further verify the influence of SINE insertion on the core promoter.The results show that SINE insertion significantly inhibits(p<0.01)the activity of the core promoter,indicating that SINE tablets may be a repressor. |
