仿刺参EGFR基因的克隆与表达分析

Cloning and expression analysis of the EGFR gene in Apostichopus japonicus

表皮生长因子受体(EGFR)是多种细胞因子的受体,在细胞增殖、迁移及分化中具有重要的作用。应用RACE法首次从仿刺参体腔细胞中克隆出EGFR基因的全长cDNA序列。该cDNA全长3 826 bp,包括821 bp的5'-UTR,281 bp的3'-UTR,开放阅读框2 724 bp,编码907个氨基酸。推导的氨基酸序列55-184aa和365-487aa符合EGFR基因所具有的L1和L2受体结构域,在203-344aa和503-823aa含有EGFR家族特征区域CR1和CR2半胱氨酸富集区,并同为跨膜糖蛋白,在结构上具有一致性。经BlastP与GenBank已知物种氨基酸序列进行同源性比对,仿刺参EGFR基因氨基酸序列与果蝇EGFR相似性为49%,同源性为34%,与斑马鱼EGFR相似性为47%,同源性为34%,与淡水椎实螺EGFR相似性为49%,同源性为31%。据此推断,仿刺参EGFR基因属于EGFR家族成员。利用Real-time PCR技术检测了该基因在仿刺参各组织中的表达,结果显示在肠、呼吸树、表皮、体腔细胞、纵肌中EGFR均有表达,且体腔细胞和表皮表达量较高。结果表明,该基因可能在仿刺参组织发育和再生过程中起到重要的调控作用。

As a variety of cytokines receptors,epidermal growth factor receptor（EGFR）plays an important role in cell proliferation,migration and differentiation.It was not only involved in regulating growth and development in tissues,but also in the wound healing.In the present study,cDNA full-length sequence of the EGFR gene was cloned in coelomocytes of Apostichopus japonicus,by using 3′ RACE and 5′ RACE,and the sequence and structural analysis of the EGFR gene by using bioinformatics methods.The results showed that the cDNA length was 3 826 bp,including the 821 bp of the 5′-UTR and 281 bp of the 3′-UTR.The open reading frame was 2 724 bp,which could encode 907 amino acids.The deduced amino acid sequence of 55-184aa and 365-487aa was L1 and L2 receptor domain of the EGFR gene.The CR1 and CR2 cysteine-rich regions,which were transmembrane glycoprotein,were located in the 203-344aa and 503-823aa regions.Identity rates of deduced amino acid sequence of EGFR with those of known species were subjected to BlastP searching in NCBI.Results showed that the structure of EGFR gene in the study was similar to that of other species EGFR gene,and the amino acid sequence of EGFR gene of A.japonicus was 49% in similarity and 34% in homology with EGFR of Drosophila melanogaster and 47% in similarity and 34% in homology with EGFR of Danio rerio and 49% in similarity and 31% in homology with EGFR of Lymnaea stagnalis.All results indicate that the EGFR gene of A.japonicus detected in this study is one of the EGFR family members.Based on the results above,expression of EGFR in different tissues by relative quantitative RT-PCR,the internal standard gene was Cytb.The result showed EGFR expresses in every tissue of A.japonicus,especially higher in coelomocyte and epidermis,which was 143.87 and 51.67 times of the respiratory tree,and the difference was significant to other tissues（P0.05）.The expression of intestine and longitudinal muscle was low,only 10.82 times and 4.47 times of respiratory tree,and the difference was not significant（P0.05）.These results indicate that the EGFR plays an important role in cell growth and tissue regeneration of A.japonicus.