鲫鱼γ-氨基丁酸受体β_3亚基基因克隆及其分析

Cloning and analysis of γ-aminobutyric acid receptor β_3 submit of Carassius auratus

γ-氨基丁酸受体(GABAR)主要存在于脊椎动物和非脊椎动物的中枢神经系统(CNS),是氟虫腈、阿维菌素、硫丹和林丹等杀虫剂的作用靶标。为了阐明GABAR拮抗剂类农药影响鱼类安全性的分子作用机理,并研究其与农药分子的亲和作用,运用生物信息学方法,在NCBI基因组数据库中找到已经公布的同源的GABAARβ3亚基基因序列,通过多序列同源比对寻找高度保守区,再根据高度保守区设计特异性引物,通过RT PCR和RACE PCR技术,成功地克隆了鲫鱼GABAR A型β3亚基基因的全长cDNA序列(GenBank登录号:KC964110),该基因长2 767 bp,开放阅读框(ORF)为1 506 bp,编码502个氨基酸,与已知的其他物种β3亚基具有高度的保守性。应用qRT-PCR扩增,检测到该基因在鲫鱼不同组织器官的差异性表达。

γ-aminobutyric acid receptor （ GABAR ） mainly exist in the central nervous system of vertebrates and invertebrates （ CNS ） , it is the important targets of pesticides such as fipronil, abamectin, endosulfan and lindane. In order to clarify the molecular mechanism of GABA antagonists pesticides effects on the fishes, and the affinity with pesticide molecules, bioinformatic methods were used to search GABAA R β3 gene sequences that had released in the NCBI genome databases and did homologous alignment. Degenerate primers were designed in the highly conservative district. The full length cDNA sequence of GABAAR β3 of Carassius auratus was cloned successfully by RT-PCR and RACE PCR technology. The gene is 2 767 bp （ GenBank ID： KC964110 ） , with an open reading＆nbsp;frame （ ORF） of 1 506 bp, encoding 502 amino acids. Multiple sequence alignment indicated that it was highly conserved with other known β3 subunits. QRT-PCR amplification was used to detect the activity of transcription of the gene in different tissues and organs of C. crassius. The results indicated that it was differentially expressed in organs of Carassius auratus.