【摘要】 结构变异(structural variation, SV)可以通过影响相关基因的稳定性和表达调控来影响基因表型。为探究CD2相关蛋白(CD2-associated protein, CD2AP)基因内含子1中的282 bp结构变异在香猪(Xiang pig)(Sus scrofa)群体中的多态分布以及对CD2AP基因表达的影响，本研究以香猪、大白猪(Large White)为研究对象，采用PCR测序方法对该SV位点进行鉴定和基因分型，研究群体分布规律以及CD2AP基因结构变异与疾病的关联；采用生物信息学方法检测CD2AP基因内含子1中SV区间所包含的重复元件和转录因子结合位点；用qPCR和Western blot检测SV对CD2AP基因和蛋白表达的影响；通过构建pEGFP荧光表达载体检验CD2AP基因SV中功能元件短散在核元件(short interspersed nuclear element, SINE)对基因转录活性的影响。结果显示，CD2AP-I1-sv282结构变异片段长度为282 bp，区间存在1个260 bp的SINE元件，属于tRNA家族，位于猪基因组Chr7:42249739～42249998 bp，含有8个转录因子结合位点；在SINE元件序列片段中具有RNA聚合酶Ⅲ启动子和2个AluⅠ限制性内切酶识别位点。PCR扩增和测序分析表明，该SV在香猪和大白猪基因组中均呈现多态性，有II (纯合插入型)、ID (杂合型)和DD (纯合缺失型) 3个基因型，χ2检验表明，该SV在香猪和大白猪群体中处于Hardy-Weinberg平衡状态(P>0.05)，香猪和大白猪群体之间的基因型频率差异极显著(P<0.01),D等位基因频率极显著高于I等位基因频率(P<0.001)。在肺部患病样本中，D基因型为优势等位基因；在肺部健康样本中，I基因型为优势等位基因；Kendall’s tau-b相关性分析表明，该结构变异与猪的肺部疾病具有相关性，患病群体与DD基因型显著正相关。qPCR和Western blot检测发现，DD基因型的香猪脾脏中CD2AP基因的mRNA和蛋白的表达量极显著高于ID基因型(P<0.01)。针对SINE元件所在序列构建了pEGFP荧光报告载体，转染细胞后检测其荧光强度及蛋白表达量，结果显示，含SINE序列的表达载体细胞与不含SINE序列相比，荧光强度和EGFP蛋白的表达量均降低。上述结果表明，CD2AP基因内含子1中282 bp结构变异在猪群中存在，且均以缺失型为主，可以导致基因的表达发生上调，结构变异区域存在的SINE元件影响基因转录活性，CD2AP基因的结构变异与香猪易感肺部疾病存在相关性。研究结果对解析香猪疾病易感分子机制具有重要意义。更多还原

【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 p EGFP fluorescent expression vector. The results showed that the CD2API1-sv282 structural variant fragment was 282 bp long and contained a 260 bp SINE element in the interval. It belonged to the t RNA family and was located in pig Chr7: 42249739～42249998 bp, containing an RNA polymerase Ⅲ promoter, 8 transcription factor binding sites, and 2 Alu Ⅰ sites. 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). χ2test 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 q PCR 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.更多还原