金钱鱼Sox3基因cDNA克隆及组织表达

cDNA Cloning and m RNA Expression Analysis of Sox3 in Scatophagus argus

金钱鱼是中国重要经济型鱼类,但其人工繁殖难以取得成功。本研究利用cDNA末端快速扩增法(rapid-amplification of cDNA ends,RACE)克隆了金钱鱼(Scatophagus argus)Sox3基因cDNA全长序列,分析该基因序列在不同物种间的差异,并用RT-PCR半定量方法分析其在雌、雄组织中的表达差异。结果表明:金钱鱼Sox3 c DNA全长1270 bp,包括5’非翻译区(5’-UTR)217 bp、3’非翻译区(3’-UTR)150 bp和开放阅读框(open reading frame,ORF)903 bp,编码300个氨基酸,该氨基酸序列34104位为HMG保守盒。金钱鱼Sox3氨基酸序列与大黄鱼(Larimichthys crocea)的同源性最高,为99.3%,其次是金头鲷(Sparus aurata),为98.7%。Sox3前体蛋白的系统进化树显示,金钱鱼与大黄鱼亲缘关系最近。金钱鱼Sox3基因的组织表达有明显的组织特异性及性别差异,在性腺中表达量最高,雄性个体脑和垂体次之,肌肉、肾脏、心脏和肠有低量的表达,肝脏、脾脏、胃和鳃中未检测到表达;雌性个体在卵巢中观察到表达水平最高,脑、垂体和鳃次之,脾脏、肌肉、肾脏、心脏和肠中有低量的表达,肝脏和胃中未检测到表达。本研究为研究Sox3基因在金钱鱼人工繁殖与养殖产业方面提供理论参考。

S catophagus argus is a kind of important economic fish in China,but its artificial breeding rarely develops.The full length c DNA of Sox3 was cloned from gonads of S.argus using RT-PCR and rapid amplification of c DNA ends(RACE).Clustal alignment was used to analyze the sequence divergence of Sox3 between S.argus and other species by studying alignment and phylogenetic trees.And the tissue expression of Sox3 was performed using semi-quantitative RT-PCR.The results showed that full length of S.argus Sox3 cDNA of 1270 bp contained a 217 bp 5’-untranslated region(5’-UTR),a 150 bp 3’-untranslated region(3’-UTR)and a 903 bp open reading frame(ORF)encoding 300 amino acids.A HMG(high mobility group)box sited in 34~104 th amino acids of S.argus Sox3 amino acids sequence.The amino acids sequence had a high similarity to Sox3 of other species,and the percent identity compared with Larimichthys crocea and Sparus aurata was 99.3%and 98.7%.Based on the results from phylogenetic tree analysis,the S.argus Sox3 was most closely related to the Sox3 of L.crocea.In addition,S.argus Sox3 displayed tissue and sex specific expression patterns.In the male fish,strong expression of Sox3 was observed in the testis but only moderate expression was found in the brain and pituitary.Low expression was observed in the muscle,kidney,heart and intestine.No expression signal was detected in the liver,spleen,stomach and gill.In the females,high expression level was observed in the ovary and only moderate m RNA level was found in the brain,pituitary and gill.No expression was detected in the liver and stomach.Low expression of Sox3 was detected in the spleen,muscle,kidney,heart and intestine.This research provides the referenced theory of Sox3 using in artificial breeding and culture industry of S.argus.