大白猪CDO 1基因克隆、生物信息学分析及组织表达定位

Cloning,Bioinformatics Analysis and Tissue Expression Localization of CDO 1 Gene in Large White Pigs

【目的】克隆大白猪半胱氨酸双加氧酶1(cysteine dioxygenase type 1,CDO1)基因并进行生物信息学分析,检测CDO 1基因在大白猪不同组织中的表达情况,并定位CDO1在乳腺中的位置,为进一步探究CDO 1基因在乳腺发育过程中的调控作用提供依据。【方法】通过PCR扩增大白猪CDO 1基因CDS全长序列,将CDO 1基因序列与pMD18-T载体连接后转化大肠杆菌DH5α感受态细胞,挑取单个阳性菌扩增培养,菌液通过PCR鉴定后测序,对测序结果进行不同物种间序列相似性比对及系统进化树构建;利用在线预测软件对CDO1蛋白进行生物信息学分析;利用实时荧光定量PCR检测CDO 1基因在大白猪不同组织中的表达水平;利用免疫组化方法检测CDO1蛋白在母猪乳腺中的定位。【结果】大白猪CDO 1基因CDS区序列全长603 bp,编码200个氨基酸,家猪与大白猪CDO 1基因核苷酸序列相似性最高。系统进化树结果显示,大白猪与家猪先聚为一类,且与猕猴亲缘关系较近。CDO1蛋白的分子质量为23.018 ku,等电点为5.98,不稳定系数为33.58(<40),为稳定亲水性蛋白,定位在胞浆内,存在糖基化和磷酸化位点,CDO1蛋白二级结构和三级结构模型预测结果一致。组织表达分析结果显示,CDO 1基因在大白猪肝脏中表达量最高,显著高于其他组织(P<0.05),在乳腺、背最长肌和心脏中表达次之,显著高于除肝脏外的其余组织(P<0.05),在十二指肠中表达量最低。免疫组化结果显示,CDO1定位在母猪乳腺终端芽(TEB)体细胞、上皮细胞和脂肪细胞中。【结论】大白猪CDO 1基因CDS区全长603 bp,编码200个氨基酸,在心脏、肝脏、乳腺中高表达;CDO1蛋白为稳定亲水性蛋白,在母猪乳腺TEB体细胞、上皮细胞和脂肪细胞中富集。研究结果为进一步探究CDO1蛋白调控母猪乳腺发育和影响母猪泌乳功能提供参考。

【Objective】This study was aimed to clone the cysteine dioxygenase type 1(CDO1)gene and conduct bioinformatics analysis,detect the expression of CDO 1 gene in different tissues of Large White pigs,and locate the site of CDO1 in mammary gland,so as to provide the basis for further exploring the regulatory role of CDO 1 gene in mammary gland development.【Method】The full-length sequence of CDO 1 gene CDS in Large White pigs was amplified by PCR.The CDO 1 gene sequence was ligated with pMD18-T vector to transformed into E.coli DH5αcompetent cells.After PCR identification and sequencing of the amplified culture solution of positive bacteria,the sequence similarity among different species were compared and the phylogenetic tree was constructed.The bioinformatics analysis of CDO1 protein was carried out using online prediction software.The expression of CDO 1 gene in different tissues of Large White pigs was detected by Real-time quantitative PCR,the localization of CDO1 protein in the mammary gland of Large White pigs was detected by immunohistochemistry.【Result】The full-length sequence of CDO 1 gene CDS in Large White pigs was 603 bp,encoding 200 amino acids.There was the highest similarity with the nucleotide sequence of CDO 1 gene between Large White pigs and Sus scrofa.The phylogenetic tree results showed that Large White pigs and Sus scrofa were first clustered into a group and were closely related to Macaca mulatta.The molecular mass of CDO1 protein was 23.018 ku,the isoelectric point was 5.98,and the instability coefficient was 33.58(<40),which was a stable hydrophilic protein and located in the cytoplasm.CDO1 protein had glycosylation and phosphorylation sites.The secondary structure and tertiary structure model of CDO1 protein were consistent.Tissue expression analysis results showed that the expression of CDO 1 gene in liver of Large White pigs was the highest,which was significantly higher than that in other tissues(P<0.05),it was next expressed in mammary gland,longissimus dorsi muscle and heart,which were significantly higher than that in other tissues except for liver(P<0.05),the expression of CDO 1 gene in duodenum was the lowest.Immunohistochemical results showed that CDO1 was localized in somatic cells,epithelial cells and adipocytes of terminal bud(TEB)of mammary gland in sows.【Conclusion】The total length of CDO 1 gene CDS in Large White pigs was 603 bp,encoding 200 amino acids,which was highly expressed in heart,liver and mammary gland.CDO1 protein was a stable hydrophilic protein,which was enriched in somatic cells,epithelial cells and adipocytes of TEB of mammary gland in sows.The results provided a reference for further exploring the effect of CDO1 protein on mammary gland development and lactation function of sows.