猪DNM2基因克隆、序列分析与组织表达谱研究

Cloning,Sequence Analysis and Tissue Expression of Porcine DNM2 Gene

本研究旨在对猪发动蛋白2(dynamin-2,DNM2)基因进行克隆和生物信息学分析,并探讨DNM2基因在猪不同组织中的表达情况。利用RT-PCR结合RACE方法克隆猪DNM2基因cDNA部分序列,与猪表达序列标签进行拼接,获得猪DNM2基因cDNA全长,并对其进行生物信息学分析,同时采用实时荧光定量PCR检测DNM2基因在猪不同组织中的表达情况。结果表明,猪DNM2基因的开放阅读框(open reading fram,ORF)为2 616bp,共编码871个氨基酸。DNM2相对分子质量为98 071.30,等电点(pI)为7.04;无信号肽和跨膜结构域,即该蛋白不属于分泌蛋白;DNM2蛋白的二级结构预测发现,构成α-螺旋、β转角、无规则卷曲、延展链的氨基酸数量分别为361、53、335和122个。多重分析结果显示,猪DNM2基因与牛、人、小鼠、大鼠的序列同源性分别为92.6%、91.8%、88.6%和89.3%;进化树分析表明,DNM2基因在物种间具有较高的保守性,不同物种间DNM2基因序列的差异符合物种间的进化性。实时荧光定量PCR结果显示,DNM2基因在脾脏中表达量较高,在乳腺、腿肌、输卵管、卵巢和子宫中表达量均较低。本研究结果为今后深入研究DNM2基因的生物学功能奠定了基础。

This study was aimed to clone porcine dynamin-2（DNM2）gene and analyze the gene structure using bioinformatics methods,the DNM2 gene mRNA level in different tissues was also investigated.We got the DNM2 gene cDNA sequence through stitching the expressed sequence tags（EST）and partial cloned sequence of DNM2 gene using RT-PCR and RACE methods.The mRNA level of porcine DNM2 gene in different tissues were detected with Real-time quantitative PCR.The results showed that DNM2 gene included an 2 616 bp whole length open reading frame（encoding 871 amino acids）.DNM2 had a relative molecular mass of 98 071.30 and an isoelectric point（pI）of 7.04,and there was no signal peptide and transmembrane domain,therefore,it did not belong to the secretory protein.The DNM2 second structure containedα-helix（361amino acids）,β-sheet（53amino acids）,random coil（335amino acids）and extended chain（122amino acids）.The sequence multi-aligned results showed that porcine DNM2 gene shared 92.6%,91.8%,88.6% and 89.3% of similar nucleotide sequence with that of cattle,human,mouse and rat,respectively.The phylogenetic analysis showed that DNM2 gene was highly conserved among species.Real-time quantitative PCR results indicated that the expression level of DNM2 gene was relatively higher in spleen while that was relatively lower in breast,leg muscle,fallopian tube,ovary and uterus.This research could provide the basis for the further study of the bio-logical function of DNM2 gene.