香苏杂交猪IGFBP2基因克隆、组织表达及生物信息学分析

IGFBP2 gene cloning, tissue expression and bioinformatics analysis of Xiangsu hybrid pig

  • 摘要: 【目的】探究胰岛素样生长因子结合蛋白2基因(IGFBP2)在香苏杂交猪不同生长时期及组织中的表达差异,为进一步分析IGFBP2基因对香苏杂交猪生长发育分子机制提供基础数据。【方法】采用PCR扩增得到香苏杂交猪IGFBP2基因的编码区(CDS)序列,利用在线软件对IGFBP2基因的CDS序列进行生物信息学分析,利用克隆的CDS构建系统发育进化树。使用实时荧光定量PCR检测IGFBP2基因在香苏杂交猪不同阶段各组织中表达情况。【结果】猪IGFBP2基因CDS全长951 bp,共编码316个氨基酸残基,蛋白二级和三级结构以无规卷曲为主,为亲水性稳定蛋白,且IGFBP2蛋白氨基酸序列中以甘氨酸(13.0%)和亮氨酸(12.3%)为主。同源性分析发现,香苏杂交猪IGFBP2基因与野猪、牛、绵羊和山羊的亲缘关系较近。实时荧光定量PCR结果显示,IGFBP2基因在3日龄、6月龄和12月龄香苏杂交猪的心脏中相对表达量存在极显著差异(P<0.01,下同),且3日龄的相对表达量较高;在肝脏中,12月龄的相对表达量显著高于3日龄和6月龄(P<0.05,下同),3日龄与6月龄无显著差异(P>0.05,下同);在脾脏中,3日龄的相对表达量极显著高于6月龄和12月龄,12月龄的相对表达量显著高于6月龄;在肺脏中,3日龄的相对表达量极显著高于6月龄和12月龄,而6月龄与12月龄的相对表达量无显著差异;在肾脏中,3日龄、6月龄和12月龄的相对表达量间均存在显著差异;在背最长肌中,6月龄与12月龄的相对表达量存在显著差异,且IGFBP2基因在3个年龄阶段的背最长肌中的相对表达量均较低。【结论】IGFBP2基因在香苏杂交猪不同生长时期各组织中均差异表达,且在与甘油三酯和脂肪酸合成代谢相关组织(肝脏)中高特异性表达,可能表明IGFBP2基因对脂肪的合成、代谢和沉积具有重要调控作用。

     

    Abstract: 【Objective】In order to explore the expression difference of insulin-like growth factor binding protein 2gene(IGFBP2) in different tissues of Xiangsu hybrid pigs at different growth stages, and to provide basic data for further analysis of the molecular mechanism of IGFBP2 gene on growth and development of Xiangsu hybrid pigs. 【Method】The coding region(CDS) sequence of IGFBP2 gene of Xiangsu hybrid pig was amplified by PCR. The CDS sequence of IGFBP2 gene was analyzed by online software and the phylogenetic tree was constructed by using cloned CDS. Real-time fluorescence quantitative PCR was used to detect the expression of IGFBP2 gene in various tissues of Xiangsu hybrid pigs at different stages. 【Result】The total length of the CDS of porcine IGFBP2 gene was 951 bp, encoding 316 amino acid residues. The secondary and tertiary structures of the protein was mainly random coil, and it was a hydrophilic stable protein, and the amino acid sequence of IGFBP2 protein was mainly glycine(13.0%) and leucine(12.3%). Homology analysis showed that IGFBP2 gene of Xiangsu hybrid pig was closely related to boar, cow, sheep and goat. The results of realtime fluorescence quantitative PCR showed that the relative expression of IGFBP2 gene in the heart of 3 days of age, 6 months of age and 12 months of age was extremely significantly different by comparing and analyzing the expression levels of different tissues at different stages(P<0.01,the same below). The relative expression level was higher at 3 days of age. The relative expression level in liver at 12 months of age was significantly higher than that at 3 days of age and 6 months of age(P<0.05,the same below), but there was no significant difference between 3 days of age and 6 months of age(P>0.05,the same below). The relative expression level in spleen at 3 days of age was extremely significantly higher than that at 6 and 12 months, and the relative expression level at 12 months of age was significantly higher than that at 6 months of age. The expression of 3 days of age was extremely significantly higher than 6 months of age and 12 months of age, but there was no significant difference between 6 months of age and 12 months of age. There were significant differences in the relative expression levels of kidney at 3 days, 6 months and 12 months of age. The relative expression level of IGFBP2 gene in longissimus dorsi at 6 months and 12 months of age was significantly different, and the relative expression level of IGFBP2 gene in longissimus dorsi at 3 age stages was lower. 【Conclusion】IGFBP2 gene is differentially expressed in various tissues of Xiangsu hybrid pigs at different growth stages, and is highly specifically expressed in tissues related to triglyceride and fatty acid synthesis metabolism(liver), which may indicate that IGFBP2 gene plays an important role in the regulation of fat synthesis, metabolism and deposition.

     

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