采用RT-PCR方法,从鲢、鲤、鲫3种鱼类肝脏总RNA中克隆出了谷胱甘肽转移酶Pi型(GSTPi)cDNA序列,推导了其编码的氨基酸序列。3种鱼类GSTPi的ORF全长627bp,编码208个氨基酸。翻译起始密码均为ATG,终止密码子均为TGA。鱼类与哺乳动物、两栖类爪蟾以及节肢动物丝虫之间GSTPi氨基酸序列相似度平均值分别为50%、33%、15%左右。5种鱼类之间的氨基酸序列相似度较大,其中鲤科鱼类之间平均为85%左右。我们以GSTPi为分子标记,用最大简约数法(MP)构建了13个物种的系统进化树,识别出两个大的单系类群:哺乳类组成类群一(bootstrap 100);鱼类组成类群-(bootstrap 93)。通过比较鱼类与哺乳类GSTPiN末端和c末端功能域的氨基酸组成差异,探讨了淡水鱼类GSTs承担较强的微囊藻毒素去毒能力的可能分子机制。
Using RT-PCR method, the glutathione transferase Pi cDNAs were cloned from Cyprinus carpio, Hypophthalmichthys molitrix, and Carassius auratus. The open reading frames (ORFs) from the 3 fishes were 627 bp long (encoding for 208 amino acids) with the initial code ATG and the terminal code TGA. The sequence similarity was 50% between fish and mammals, 33% between fish and amphibian, and 15% between fish and arthropoda, respectively. The sequence similarity was big among fishes, and the average value of the 4 cyprinids was about 85%. Phylogenetic tree was constructed for 13 species based on GST Pi amino acid sequences using MP (Maximum Parsimony) method. Two major clusters were recognized: cluster one consisted of Mammals (bootstrap 100) and cluster two consisted of fishes (bootstrap 93). Based on the sequences analyses of N/C domain of GST Pi, we proposed the detoxification mechanism of freshwater fishes that were thought to have stronger tolerance to microcystins.