中华鳖GHITM cDNA基因克隆及表达分析

Molecular Cloning and Expression Analysis of HGITM cDNA in Trionyx sinensis

为研究GHITM基因在中华鳖(Trionyx sinensis)胚胎发育及生长中的作用,本研究通过RT-PCR和RACE方法获得了中华鳖GHITM全长c DNA序列;采用实时荧光定量PCR对GHITM m RNA组织表达及不同温度孵化胚胎发育过程中的表达特性进行分析。结果显示,中华鳖GHITM c DNA序列长度为2650 bp,开放阅读框为1050 bp,5’非编码区为123 bp,3’非编码区为1477 bp,编码349个氨基酸,编码蛋白的等电点为10.01,分子量为37.12kDa。GHITM编码氨基酸序列由胞外区、跨膜区和胞内区组成,7个跨膜域组成跨膜区。GHITM编码氨基酸序列的同源性分析显示,中华鳖与锦龟(Chrysemys picta bellii)和绿海龟(Chelonia mydas)同属一个分支,3种鳄鱼构成一个分支,7种鸟类形成一个分支。实时荧光定量检测显示,GHITM基因在肝脏、肌肉和脑垂体中的表达水平较高,显著高于心脏、性腺、肠、肾脏和脾脏组织(P<0.05);50 g左右和500 g左右雄性个体肝脏中的GHITM基因表达量显著高于雌性个体(P<0.05);低温胁迫孵化能显著抑制胚胎肝脏中GHITM基因的表达(P<0.05)。上述研究结果表明,GHITM基因与中华鳖生长和胚胎发育密切相关,其在中华鳖胚胎中的表达受孵化温度调节。本研究为探讨中华鳖胚胎发育和生长提供理论依据。

In this study, we obtained the full-length cDNA of the GHITM gene from Trionyx sinensis for the first time using the RACE(rapid-amplification of cDNA ends) method. The full-length cDNA sequence was 2650 bp, including a 123 bp 5’-UTR, 1477 bp 3’-UTR, and 1050 bp open reading frame(ORF) that encoded 349 amino acid residues. The isoelectric point(pI) of this peptide was 10.01, and the molecular mass was 37.12 kDa. The amino acid sequence was composed of the extracellular region, the transmembrane region, and the intracellular region. The transmembrane region was composed of 7 transmembrane domains. The phylogenetic analysis of the amino acid sequences showed that T. sinensis, Chrysemys picta bellii, and Chelonia mydas belonged to the same branch, the three species of crocodiles formed a branch, and birds formed another branch. The expression of GHITM in different tissues was also analyzed with quantitative real-time PCR. The results showed that GHITM was expressed in all the tested tissues, including the liver, pituitary, muscle, spleen, kidney, heart, intestine, and gonad, with the high expression observed in the liver, muscle, and pituitary. At the specifications of 50 g and 500 g, the expression of GHITM was significantly higher in the liver of males than females(P<0.05). Low temperature incubation can inhibit the expression of GHITM in fetal embryos(P<0.05). The results indicated that GHITM was related to the growth and embryo development of T. sinensis.