多鳞铲颌鱼RPL34基因3’端序列的克隆与表达

采用同源克隆和cDNA末端快速扩增技术,从多鳞铲颌鱼雄生殖腺中首次克隆得到核糖体蛋白L34 3’末端cDNA序列。该3’末端cDNA序列长297bp,预测开放阅读框为162bp,编码53个氨基酸的蛋白质,经BLAST比对,该cDNA序列与斑马鱼、斑点叉尾鱼回、非洲爪蟾、大西洋鲑同源率达到82%～85%。利用实时定量RT-PCR检测核糖体蛋白L34mRNA在多鳞铲颌鱼组织中的表达,以及注射激素后在生殖腺中的表达。研究结果表明,核糖体蛋白L34基因在多鳞铲颌鱼雄生殖腺中特异性表达;核糖体蛋白L34在雌性生殖腺、心、脑、鳃、肠和肌肉中表达量较少,其中在雌性生殖腺表达量最低,雄性生殖腺表达量高于雌性生殖腺、肝、心、脑、鳃、肠、肌肉、脾,差异极显著(P0.01)。在雄性生殖腺注射甲基睾丸酮后,核糖体蛋白L34基因的表达量对照组高于注射组,差异显著(P0.05),在雌性生殖腺注射雌二醇后,核糖体蛋白L34基因表达量对照组高于注射组,差异极显著(P0.01)。

Cloning and Expression of RPL34 Gene 3' Terminal Sequence in Onychostoma macrolepis

In this study,the ribosomal protein L34 gene 3’terminal cDNA was cloned from testis of Onychostoma macrolepis by homology cloning and rapid amplification of cDNA ends(RACE).The 3’end of the cDNA 297 bp,predicted open reading frame(ORF),was found to be 162 bp,encoding 53 amino acids of the protein.By the BLAST alignment,the homologous rate was shown to be of 82% to 85% of the cDNA sequences of zebrafish Danio rerio,channel catfish Ictalurus punctatus,Xenopus laevis and Atlantic salmon Salmo salar.Real-time quantitative RT-PCR was used to test the expression of ribosomal protein L34 mRNA in tissue as well as the expression of gonad in Onychostoma macrolepis injected by hormone.The results showed that the RPL34 gene was specifically expressed in the testis,lower expression in ovary,heart,brain,gill,intestine and muscle,and the minimal expression in the female gonads.There was significantly higher expression level in the testis than that in the ovary,liver,heart,brain,gills,intestine,muscle,and spleen(P<0.01).There was significantly higher RPL34 gene expression level in the testis in the control group fish by injection of methyltestosterone than that injected group(P<0.05).There was very significantly higher RPL34 gene expression level in the ovary in the control group fish by injection of estradiol than that in the injected group(P<0.01).The results were evaluated by using RPL34 as the feasibility of housekeeping genes,providing scientific basis.