黄鳍棘鲷IGF-2基因克隆及IGF-1/2在胚胎发育过程中的表达特性分析

Gene cloning of IGF-2 gene and differential expression of IGF-1/2 during embryonic development in Acanthopagrus latus

类胰岛素样生长因子(Insulin-like Growth Factors,IGFs)是生物生长轴下游的关键调控因子,其对促进细胞分化与机体生长具有重要的作用。为深入研究IGF-1和IGF-2基因在黄鳍棘鲷(Acanthopagrus latus)胚胎发育过程中表达机制及可能发挥的作用,本文利用分子克隆技术分离鉴定了黄鳍棘鲷IGF-2基因cDNA序列,并对其进行了生物信息学分析。同时,在对其胚胎发育过程连续观察的基础上,利用实时荧光定量PCR的方法,测定并分析了黄鳍棘鲷胚胎发育不同时期IGF-1和IGF-2基因mRNA的表达情况。实验结果表明,IGF-2基因cDNA序列全长为1736 bp,其中开放阅读框648 bp,共编码215个氨基酸。多重序列比对分析发现,黄鳍棘鲷IGF-2氨基酸序列与同属鲷科鱼类的大西洋鲷(Sparus aurata)相似性为99.95%,与三长棘赤鲷(Pagrus auriga)相似性为98.14%,与其他硬骨鱼类的IGF-2也有较高的相似性,显示出IGF-2在硬骨鱼类进化关系上的保守性。在水温为(26.5±0.5)℃,pH为8.0和盐度为28的条件下,黄鳍棘鲷由受精卵至孵化出膜历时25.5 h。实时荧光定量PCR结果显示:IGF-1和IGF-2基因mRNA在黄鳍棘鲷胚胎发育不同时期均有表达。IGF-1基因表达量呈现先升高后降低的趋势,在肌肉效应期达到最高表达量;IGF-2基因表达量呈现先升高后降低再升高的趋势,在原肠期、肌肉效应期与出膜后3 d表现高表达量,这表明IGF-1和IGF-2基因在胚胎发育时期可能发挥重要作用。

Insulin-like growth factors(IGFs)are key regulators downstream of the growth axis and play an important role in promoting cell differentiation and growth.In order to further study the expression mechanism and possible roles of IGF-1 and IGF-2 genes in the embryonic development of Acanthopagrus latus.Our research cloned and identified the cDNA sequence of IGF-2 gene from A.latus and proceeded a biological information analysis.At the same time,based on the foundation of continual observation of the embryonic development of A.latus.RT-qPCR was carried out to explore the expression levels of IGF-1 and IGF-2 genes.The results suggested that the full length and open reading frame of IGF-2 gene are 1736 bp and 648 bp respectively,encoding a predicted peptide of 215 amino acids.Multiple alignment showed that A.latus IGF-2 was 99.95%similar to Sparus aurata and 98.14%similarity with Pagrus auriga,as well as relatively high homology with other teleosts,indicating that conservation of IGF-2 in teleost evolutionary relationships.Through continuous observation of the embryonic development of A.latus,it was found that under the conditions of water temperature of(26.5±0.5)℃,pH of 8.0 and salinity of 28,the time from fertilization of egg to hatching of membrane of A.latus was 25.5 h.Expression analysis showed that IGF-1 and IGF-2 genes constitutively expresses in various tested stages of embryo of A.latus.The expression of IGF-1 gene showed a trend of first increasing and then decreasing,reaching a relatively high expression in the muscle ef-fect period,and the expression of IGF-2 gene showed a trend of first increasing and then decreasing and then in-creasing,IGF-2 gene was expressed in the gastrula stage,the muscle effect stage and 3 days after membrane emer-gence.Summarizing,all the data of our study above showed that IGF-1 and IGF-2 genes may play a crucial role in embryonic development in A.latus.