Study On The Structural Variation Of 264 Bp In The 3' Flanking Region Of CHD3 Gene In Xiang Pi

CHD3(chromodomain helicase DNA binding protein 3)belongs to the ATP-dependent chromatin regulatory domain helicase DNA binding protein(CHD)family,which includes nine members and three subfamilies.Among them,CHD3,CHD4 and CHD5 belong to the second subfamily(CHDⅡ).CHD3,also known as Mi-2α,was first identified as an autoantigen in patients with dermatomyositis.As dermatomyositis progresses,it often contributes to other complications,primarily increasing the risk of typical rashes with necrotizing myositis and malignant tumors.Pigs have very high similarity with humans in many aspects,such as physiological level,anatomical level,genetic level,etc.,so pigs are recognized as an ideal model animal for studying human diseases.Pig’s skin is strikingly similar to human skin in structure,thickness,hair follicle content,collagen,lipid composition and pigmentation.Xiang pigs belongs to small local pig species,which is characterized by short stature,early sexual maturity,fast reproduction and fast growth,and is more suitable for model animal research.There are a few individuals with wrinkled skin phenotype in the population of Xiang pigs,and some piglets with wrinkled skin phenotype still exist in the breeding piglets,indicating that the wrinkled skin phenotype can be passed on to the offspring.A 264 bp nucleotide insertion/deletion structural variation was found in the 3’-flanking region of CHD3 gene in Xiang pigs with systemic wrinkle.The purpose of this study was to explore the relationship between CHD3 gene structural variation and wrinkled skin in Xiang pigs,and to provide theoretical reference for studying the formation of wrinkled skin in Xiang pigs and aging of human skin.In this study,Large white pigs,ordinary Xiang pigs and Xiang pigs with wrinkle skin were used as test subjects.Skin histology study was conducted to observe the skin differences between ordinary Xiang pigs and Xiang pigs with wrinkle skin.Bioinformatics method was used to analyze the structural variation of CHD3 gene,and q PCR and Western blotting were used to detect the influence of structural variation on the expression level of CHD3 gene.The method of population polymorphism study was used to detect the differences in the distribution of CHD3 gene structural variation among pig populations.Finally,the relationship between CHD3 gene structural variation and the formation of wrinkled skin was preliminarily analyzed.The results of this study are as follows:1.The skin histological study was used to compare the difference between wrinkled skin and ordinary skin in Xiang pigs,the results showed that ordinary Xiang pigs skin smooth,skin collagen and elastic fibers in parallel arranged in tight mesh structure,to rule,wrinkled skin Xiang pigs skin is concave and convex changing significantly,however,the skin collagen fibers and elastic fiber fracture,mixed and disorderly and irregular.2.Based on the previous laboratory resequencing analysis results,a 264 bp nucleotide insertion/deletion structural variation existed in the 3’-flanking region of the CHD3 gene of Xiang pigs with systemic wrinkle skin.Specific primers were designed in the upstream and downstream of the structural variation,and the structural variation sites were amplified by PCR,and the existence of the structural variation was verified by sequencing.3.Repeat elements,miRNA binding sites and RNA binding protein(RBP)binding sites contained in the structural variation interval were predicted using UCSC,miRBase,miRanda and RBPsuite software.The results showed that there were 35miRNA binding sites and 10 RBP binding sites in the 264 bp structural variation of CHD3 gene,and a short-scatter element(SINE)was included in the variation range,belonging to the t RNA family.4.Rt-q PCR was used to detect the effect of the mutation locus on CHD3 mRNA expression.The results showed that the mRNA expression levels of DD(deletion)and ID(heterozygous)genotype of CHD3 were significantly higher than those of II(insertion)genotype(P<0.01).Western blotting was used to detect the effect of the structural variation on the protein expression of CHD3 gene.The results showed that the protein expression of DD and ID genotypes was significantly higher than that of II genotypes(P<0.01).5.Reverse transcription PCR(RT-PCR)was used to detect the presence of structural variation in mRNA.CHD3 was of type II,ID and DD in the genome,and still corresponded to type II,ID and DD in c DNA.6.PCR and SPSS chi-square test were used to detect population genotyping and polymorphism of structural variation loci,and to verify whether the distribution of structural variation in pigs was in accordance with Hardy-Weinberg Equilibrium.Genotyping results showed that II,ID and DD genotypes were detected in wrinkled skin Xiang pigs,ordinary Xiang pigs and Large white pigs,indicating that the structural variation of CHD3-SV264 in pigs presented rich polymorphism.The II genotypes were mainly found in ordinary Xiang pigs and Large White pigs.The frequency of D allele in Xiang pigs with wrinkle skin was significantly higher than that in ordinary Xiang pigs and Large white pigs(P<0.01).χ~2test showed that CHD3gene structure variation was in hardy-Weinberg equilibrium in Xiang pigs with wrinkle skin and ordinary Xiang pigs(P>0.05),and in hardy-Weinberg imbalance in Large white pigs(P<0.05).In conclusion,by analyzing and studying the structural variation of CHD3 and detecting the differences in the distribution of structural variation among pig populations,it is concluded that the 264 bp structural variation in the 3’flanking region of CHD3 gene may be one of the important factors for the formation of systemic skin folds in Xiang pigs.