Study on the Regulation of the Expression Mediated by a 282 bp Insertion/deletion Structural Variation of CD2AP Gene in Xiang Pigs (Sus scrofa)
WANG Dan1, RAN Xue-Qin2, WANG Wei1, NIU Xi1, LI Sheng1, HUANG Shi-Hui2, WANG Jia-Fu1,*
1 College of Life Sciences/Institute of Agro-bioengineering/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China; 2 College of Animal Sciences/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China
Abstract：Structural variation (SV) can affect gene phenotype by affecting the stability and expression regulation of related genes. To investigate the polymorphic distribution of the 282 bp SV in intron 1 of the CD2-associated protein (CD2AP) gene in the Xiang pig (Sus scrofa) population and its effect on the expression of the CD2AP gene, Xiang pig and Large White were taken as research subjects, and the SV locus was genotyped by PCR sequencing, and the distribution pattern and the association between this SV and pulmonary diseases in Xiang pig and Large White populations were studied. Bioinformatics methods were used to detect the repetitive elements and transcription factor binding sites in the SV region of intron 1 of the CD2AP gene. qPCR and Western blot were used to detect the effect of SV on CD2AP gene and protein expression. The effect of short interspersed nuclear element (SINE) on the transcriptional activity of CD2AP gene was examined by constructing pEGFP fluorescent expression vector. The results showed that the CD2AP-I1-sv282 structural variant fragment was 282 bp long and contained a 260 bp SINE element in the interval. It belonged to the tRNA family and was located in pig Chr7:42249739~42249998 bp, containing an RNA polymeraseⅢ promoter, 8 transcription factor binding sites, and 2 AluⅠsites. This SV exhibited polymorphism in both Xiang pig and Large White genomes, with II genotype (homozygous insertion), DD genotype (homozygous deletion), and ID genotype (heterozygous). χ2 test showed that the SV was in Hardy Weinberg equilibrium between the Xiang pig and Large White populations (P>0.05), and there was extremely significant difference in genotype frequencies between the Xiang pig and Large White populations (P<0.01). The frequency of the D allele was significantly higher than that of the I allele (P<0.001). The D allele was the dominant allele in the diseased lung samples, while the I allele was the dominant allele in the healthy lung samples. The Kendall's tau-b correlation analyses indicated that the presence of the pathological features was positively correlated with genotype, with the DD genotype group being more susceptible to structural mutations. As detected by using qPCR and Western blot assays, it was found that the mRNA and protein expression of the CD2AP gene in Xiang pig spleens was highly significantly higher in the DD genotype than in the ID genotype (P<0.01). The pEGFP fluorescent reporter vector carrying the SINE element showed a decrease in fluorescence intensity and EGFP protein in cells. In summary, the 282 bp structural variation in intron 1 of the CD2AP gene exhibited polymorphism in Xiang pig population, and was predominantly deletion type, leading to upregulation of gene expression. The results indicated that this SV affected gene transcriptional activity, and was associated with susceptibility to lung disease in Xiang pigs. The results are of great significance to analyze the molecular mechanism of disease susceptibility in Xiang pigs.
王丹, 冉雪琴, 王维, 牛熙, 李升, 黄世会, 王嘉福. 香猪CD2AP基因第1内含子282 bp插入/缺失结构变异介导的表达调控研究[J]. 农业生物技术学报, 2025, 33(2): 330-343.
WANG Dan, RAN Xue-Qin, WANG Wei, NIU Xi, LI Sheng, HUANG Shi-Hui, WANG Jia-Fu. Study on the Regulation of the Expression Mediated by a 282 bp Insertion/deletion Structural Variation of CD2AP Gene in Xiang Pigs (Sus scrofa). 农业生物技术学报, 2025, 33(2): 330-343.
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