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| 黄条鰤igfbp-1和igfbp-2基因克隆表达及其生长调控作用 |
| Molecular cloning, expression profiles and regulation effect on growth of insulin-like growth factor-binding protein-1 (igfbp-1) and igfbp-2 in yellowtail kingfish (Seriola lalandi) |
| 投稿时间：2022-12-13 修订日期：2023-02-13 |
| DOI： |
| 中文关键词: cDNA 克隆 igfbp-1 igfbp-2 黄条鰤 组织表达 生长调控 |
| 英文关键词: cDNA cloning Seriola lalandi igfbp-1 igfbp-2 tissue expression growth regulation |
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| 中文摘要: |
| 胰岛素样生长因子结合蛋白(insulin-like growth factor binding proteins, igfbps)在调节胰岛素生长因子(insulin-like growth factor, igf)的生物学活性和生长调控中起着至关重要的生理作用。本研究克隆了黄条鰤(Seriola lalandi) igfbp-1和igfbp-2a、igfbp-2b等3个基因cDNA编码序列(ORF),分析了其组织分布特征,并检测了工厂化不同养殖密度下黄条鰤肝脏中5个基因igfbp-1、igfbp-2a、igfbp-2b和igf-1、igf-2对生长的调控作用。结果表明, igfbp-1的 ORF长为741 bp, 共编码 246 个氨基酸; igfbp-2a的 ORF长为 882 bp , 共编码 293个氨基酸; igfbp-2b的 ORF长为 810 bp , 共编码 269个氨基酸。它们均具有广泛的组织表达特性,其中在肝脏中显著高表达。工厂化养殖条件下低密度组实验鱼生长速度最快且igfbp-1、igfbp-2a、igfbp-2b和igf-1、igf-2表达量均最高,与中、高密度组有显著性差异(P＜0.05);而中高密度组实验鱼的生长及igfbp-1、igfbp-2a、igfbp-2b、igf-1、igf-2表达量均无显著差异。表明igfbp-1、igfbp-2a、igfbp-2b参与了不同密度下黄条鰤生长的调控过程,且与igf-1、igf-2对生长的表达调控存在正向协同效应。研究结果为阐释黄条鰤生长的分子机制以及工厂化条件下适宜养殖密度的调控提供了理论依据。 |
| 英文摘要: |
| Insulin-like growth factor binding proteins (igfbps) play crucial roles in the regulation of biological activities of insulin-like growth factors (igfs) and growth performance in vertebrates. igfbp is a six-member protein family (igfbp1-6) with a high affinity for igf. igfbp affects the distribution, stability and biological activity of igf by regulating the interaction between igf ligands and receptors. Recently, igfbp-1 has been identified as a regulator of growth, reproduction, and development in bony fish, such as juvenile Atlantic salmon, where igfbp-1 interacts with cortisol to regulate growth. In orange-spotted grouper (Epinephelus coioides), igfbp-1 regulates cell metabolism and growth through interaction with insulin in primary hepatocytes incubated in vitro. In bony fish, igfbp-2 has a wide range of tissue expression characteristics, and the regulation of igf may be through autocrine or paracrine pathways. For example, in zebrafish, long-term fasting induced increased igfbp-2 mRNA expression in the liver. In addition, igfbp-2 can inhibit the activity of igf ligand through its high affinity with igf, thus playing an inhibitory and regulatory role in the growth process of zebrafish. The cloning, molecular characterization and expression analysis of igfbp-2 in goldfish (Carassius auratus) showed that igfbp-2 mRNA expression in goldfish liver was significantly up-regulated after fasting, and quickly recovered to normal level after feeding. The results indicated that the expression of igfbp-2 mRNA may be related to the anabolism and catabolism of goldfish, and is regulated by metabolic factors.
Yellowtail kingfish(Seriola lalandi) belongs to the Carangidae family. It is a warm and temperate oceanic fish with long distance migration characteristics and is distributed in the global middle and upper oceans with high economic value and nutrition. This species has a large size and fast growth rate, and is suitable for used in land-based industrial circulating water, deep-water cages, enclosures and breeding boats. Our laboratory has carried out studies on the regulatory mechanism of the rapid growth of yellowtail kingfish and have cloned gh, igf-1, igf-2, ghr and other growth-related functional genes and revealed their molecular regulation in the early growth and development. As an important growth regulator, igfbp regulates the growth, development and nutrient metabolism of fish through the interaction of growth axis. The mechanism of igfbp in the regulation of yellowtail kingfish growth has not been reported. In this paper, we will further study the growth of yellowtail kingfish by analyzing the key growth axis factors.
In this study, RNAiso Plus reagent (TaKaRa) was used to extract total RNA from tissues, the integrity of RNA was detected by agarose gel electrophoresis, and the quality of RNA was determined by NanoDrop2000C spectrophotometer (Thermo, USA). First cDNA strand was synthesized with PrimeScript? RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription Kit (TaKaRa). Secondly, according to the predicted sequence of yellowtail kingfish igfbp gene searched by NCBI GenBank database, primers were designed and the product amplification, gel recovery, linking, transformation, positive clones were selected and tested. qRT-PCR was used to analyze the distribution of yellowtail kingfish tissues and the expression patterns of liver tissues under different density of industrial culture.
The results showed that the length of ORF domains of igfbp-1, igfbp-2a, igfbp-2b were 741 bp, 882 bp, 801 bp, and encoding 246 amino acids, 293 amino acids, 269 amino acids, respectively. The conserved domain of insulin growth factor-binding protein homologues (IB) was present in the N-terminal of the three igfbps, and the conserved domain of thyroglobulin type-1 repeat (Ty-1) was present in the C-terminal. The analysis of gene expression characteristics showed that they had a wide range of tissue expression characteristics, and were highly expressed in liver. The expression level of igfbp-1 was the highest in the liver of both male and female fish (P < 0.05), and was also highly expressed in the gonads, and the expression level of igfbp-1in male fish was higher than that in female fish (P < 0.05), but the expression level of igfbp-1 in other tissues was very low and had no significant difference. The expression level of igfbp-2a in the liver of both male and female fish was the highest (P < 0.05), followed by the gonads (P < 0.05). The expression level of igfbp-2a in other tissues was relatively low, and the expression level of igfbp-2a in the liver of female fish was higher than that of male fish (P < 0.05). The expression level of igfbp-2b was the highest in the liver of both male and female fish (P < 0.05), but was lower in other tissues with no significant difference. The expression level of igfbp-2b in male fish liver was higher than that in female fish (P < 0.05).Under industrial culture conditions, the fish in the low-density group showed the greatest growth rate and the highest expression levels of igfbp-1, igfbp-2a, igfbp-2b and igf-1, igf-2, which were significantly different from those from the medium and high-density groups (P<0.05). However, there were no significant differences in the growth and liver genes expression levels were observed between the middle and high -density groups.
It indicated that igfbp-1, igfbp-2a and igfbp-2b participated in the growth regulation of yellowtail kingfish, and a positive synergistic effect with the expression regulation of igf-1 and igf-2 was found in growth regulation of yellowtail kingfish. Article of this study, ORF regions of igfbp-1, igfbp-2a and igfbp-2b genes of yellowtail kingfish were cloned, and the structural characteristics, tissue expression characteristics and their relationships with growth performance under different culture densities were analyzed. The results provide a theoretical basis for interpreting the molecular mechanism of growth of yellowtail kingfish and the regulation of suitable culture densities under industrial conditions. |
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