南极冰鱼和伯氏肩孔南极鱼EPO基因预测及生物信息学分析

CLONING AND BIOINFORMATICS ANALYSIS OF EPO GENE IN ANTARCTIC ICEFISH CHIONODRACO HAMATUS AND TREMATOMUS BERNACCHII

运用PCR、TA克隆及二代测序技术获得了独角雪冰鱼(Chionodraco hamatus)、伯氏肩孔南极鱼(Trematomus bernacchii)的EPO区域基因组DNA序列,并结合生物信息学方法进行了基因结构与保守功能域等生物学特性研究。结果表明:克隆获得的独角雪冰鱼、伯氏肩孔南极鱼EPO基因序列分别为2727 bp和2820 bp,包括5个外显子和4个内含子结构,编码区全长皆为558 bp,推定编码185个氨基酸。理化性质分析得到两者的EPO蛋白皆为相对稳定的酸性可溶性蛋白。其三级结构预测结果都显示出典型的4个反相平行的α螺旋结构特征,保持了其在生物体内发挥的原有作用。多重序列比对显示,独角雪冰鱼EPO与伯氏肩孔南极鱼的氨基酸同源性为95.2%,而与其它物种的同源性在53.9%—85.5%之间。系统发育树显示,独角雪冰鱼、伯氏肩孔南极鱼的EPO基因与鱼类的同源基因聚类,系统进化情况同亲缘关系一致,表明EPO基因在进化过程中具有较强保守性。本研究为揭示EPO基因在南极冰鱼内的遗传特性提供理论依据,并为进一步探讨EPO基因在极端低温高氧环境中的鱼类适应性进化研究奠定了基础。

We used PCR（polymerase chain reaction）, TA（based on thymine（T） and adenine（A）） cloning, and nextgeneration sequencing technology to isolate genomic DNA sequences of EPO（erythropoietin） gene from healthy Chionodraco hamatus and Trematomus bernacchii, and then we used bioinformatics methods to analyze their genetic characteristics, such as genetic structure and conserved domain. The EPO gene of C. hamatus and T. bernacchii are 2727 bp and 2820 bp, respectively. Both of them contain five exons and four intron structures, and the coding region is 558 bp in length, encoding 185 amino acids. In addition, EPO of both fish are a relatively soluble and acidic protein. The tertiary structure prediction shows a typical structure characteristic that they fold into a four-parallel-α-helix structure, which assue EPO keep original function in the organism. Amino acids alignment shows 95.2% identity in homology of the EPOs between C. hamatus and T. bernacchii. Compared with other species, the EPOs shared 53.9%—85.5% identities. The phylogenetic tree of the EPO nucleic acid sequences shows a close relationship among C. hamatus, C. hamatus and other fishes. The phyletic evolution agrees with their genetics, showing a conservative evolution progress of the EPO gene. This study may provide a theoretical basis for revealing the genetic characteristics of the Antarctic icefish EPO gene, and lay foundation to EPO study on long-term biological adaption under an extremely low temperature and high oxygen environment.