Gene cloning of IGF-2 gene and differential expression of IGF-1/2 during embryonic development in Acanthopagrus latus
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摘要: 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1 736 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。Abstract: 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 1 736 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 effect period, and the expression of IGF-2 gene showed a trend of first increasing and then decreasing and then increasing, IGF-2 gene was expressed in the gastrula stage, the muscle effect stage and 3 days after membrane emergence. 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.
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图 1 黄鳍棘鲷IGF-2基因全长序列及其编码的氨基酸序列
*代表氨基酸终止密码子；左右两列数字分别代表最左边与最右边碱基或者氨基酸在整个基因或者氨基酸序列中的位置，
代表poly(A)加尾信号 Fig. 1 Complete sequence of IGF-2 gene cDNA and deduced amino acid sequence from Acanthopagrus latus
* Represents stop codon TGA; the left and right columns represent the positions of the leftmost and rightmost bases or amino acids in the entire gene or amino acid sequence;
represents poly(A) signal 
图 2 黄鳍棘鲷IGF-2基因氨基酸序列多重序列比对
不同颜色的字母代表不同的氨基酸；左右两边数字分别代表最左边与最右边的氨基酸在整个氨基酸序列中的位置
Fig. 2 Amino acid sequence multiple alignment of IGF-2 gene from Acanthopagrus latus
Different colored letters represent different amino acids; the left and right numbers represent the positions of the leftmost and rightmost amino acids in the amino acid sequence
图 3 黄鳍棘鲷和其他物种IGF-2基因氨基酸序列的系统进化树分析
Fig. 3 Phylogenetic analysis of IGF-2 gene amino acid sequence from Acanthopagrus latus and other species
图 4 黄鳍棘鲷的胚胎发育
a. 2细胞期；b. 4细胞期；c. 8细胞期；d. 16细胞期；e. 32细胞期；f. 64细胞期；g. 多细胞期；h. 桑葚胚期；i. 高囊胚期；j. 低囊胚期；k. 原肠前期；l. 原肠中期；m. 原肠晚期；n. 胚孔封闭期；o. 视囊形成期；p. 肌节出现期；q. 晶体形成期；r. 尾芽期；s. 尾芽游离期；t. 肌肉效应期；u. 心跳期；v. 将孵期；w. 破膜期
Fig. 4 Embryonic development of Acanthopagrus latus
a. 2-cell stage; b. 4-cell stage; c. 8-cell stage; d. 16-cell stage; e. 32-cell stage; f. 64-cell stage; g. multicellular stage; h. morula stage; i. high blastula stage; j. low blastula stage; k. early-gastrula stage; l. mid-gastrula stage; m. late-gastrula stage; n. closure of blastopore stage; o. eye vesicle formation stage; p. muscle burl stage; q. crystal formation stage; r. tail-bud stage; s. tail-bud free stage; t. muscular effect stage; u. heart-beating stage; v. pre-hatching stage; w. hatching stage
图 5 黄鳍棘鲷胚胎发育过程中IGF-1/IGF-2基因mRNA的相对表达量
标有不同字母的组间平均值差异显著（p<0.05）
Fig. 5 The relative expression of IGF-1/IGF-2 genes mRNA in different embryo development stages of Acanthopagrus latus
The mean values marked with different letters between groups are significantly striking（p<0.05）
表 1 本实验所用引物
Tab. 1 The primers used in the experiments
引物名称 序列（5′-3′） 用途 IGF-2-F TCATCTCAGCCGCACCAACT CDS序列克隆 IGF-2-R AAAAGGTGCTGGAACAGGAATC CDS序列克隆 5′-GSP1 GGCATCACGGGTAAGACCTGTA 5'末端序列克隆 5′-GSP2 GTGGCAAAGTGAGTGGCGTC 5'末端序列克隆 3′-GSP1 TCTGAACTCTTTCGCTCCCTCT 3'末端序列克隆 3′-GSP2 ATTAGATTCCTGTTCCAGCACCTT 3'末端序列克隆 M13F-47 CGCCACCCTTTTCCCAGTCACGAC 菌液检测 M13R-48 AGCGGATAACAATTTCACACAGGA 菌液检测 IGF-1-F1 TAGCCACACCCTCTCACTACTG 荧光定量 IGF-1-R1 AAGCCTCTCTCTCCACACACAA 荧光定量 IGF-2-F1 CCGTAGCTGTGACCTCAACC 荧光定量 IGF-2-R1 TCCTCTGCCACACCTCGTAT 荧光定量 β-actin-F ACCCAGATCATGTTCGAGACC 内参基因 β-actin-R ATGAGGTAGTCTGTGAGGTCG 内参基因 
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表 2 海水鱼类胚胎发育特征的比较
Tab. 2 Comparison of embryonic developmental characteristics of marine fish
物种 受精卵直径/
mm油球直径/
mm孵化水温/
°C胚胎发育
时间黄鳍棘鲷
（Acanthopagrus latus）0.900±0.050 0.250±0.020 26.5±0.5 25 h 30 min 条石鲷
（Oplegnathus fasciatus）[28]0.910±0.038 0.210±0.028 23.9±0.4 30 h 32 min 黑棘鲷
（Acanthopagrus schlegelii）[29]0.895±0.055 − 21.0±0.5 32 h 大西洋鲷
（Sparus aurata）[30]0.870±0.050 0.205±0.025 19±0.5 44 h 48 min 军曹鱼
（Rachycentron canadum）[31]1.245±0.065 0.325±0.027 27.0±0.5 26 h 30 min 鞍带石斑鱼
（Epinephelus lanceolatus）[32]0.820±0.030 0.230±0.030 29.0±0.5 18 h30 min 多纹钱蝶鱼
（Selenotoca multifasciata）[21]0.605±0.005 0.237±0. 013 27.0±1.0 18 h 30 min 黄姑鱼
（Nibea albiflora）[33]0.830±0.018 0.240±0.012 23.2±0.15 22 h 59 min
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