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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 |
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张文静1,2, 徐永江2, 崔爱君3, 王滨4, 姜燕5, 王开杰6, 周鹤庭7
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1.浙江海洋大学国家海洋设施养殖工程技术研究中心 浙江 舟山 316022;2.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266071;3.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266072;4.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266073;5.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266074;6.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266075;7.中国水产科学研究院黄海水产研究所
青岛海洋科学与技术试点国家实验室深蓝渔业工程联合实验室 山东 青岛 266076
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| 摘要: |
| 胰岛素样生长因子结合蛋白(insulin-like growth factor binding proteins, IGFBPs)在调节胰岛素样生长因子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 aureovittata) igfbp-1、igfbp-2a和igfbp-2b等3个基因cDNA编码的开放阅读框(ORF)序列，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、igf-1和igf-2对生长的调控作用。结果显示，igfbp-1的ORF长为741 bp，共编码246个氨基酸；igfbp-2a的ORF长度为882 bp，共编码293个氨基酸；igfbp-2b的ORF长度为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，且具有性别二态性表达特性。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、igf-1和igf-2表达量均最高，与中、高密度组有显著性差异(P<0.05)，且与血清IGF-1、GH表达趋势相同，与皮质醇含量及葡萄糖浓度的趋势相反；而中、高密度组实验鱼的生长及igfbp-1、igfbp-2a、igfbp-2b、igf-1和igf-2表达量均无显著差异。本研究表明，igfbp-1、igfbp-2a和igfbp-2b参与了不同养殖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。 |
| 关键词: cDNA克隆 igfbp-1 igfbp-2 黄条鰤 组织表达 生长调控 |
| DOI：10.19663/j.issn2095-9869.20221213001 |
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| Molecular cloning, expression profiles of insulin-like growth factor-binding protein-1 (igfbp-1) and igfbp-2 and their regulation effects on growth of yellowtail kingfish (Seriola aureovittata) |
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ZHANG Wenjing1,2, XU Yongjiang2, CUI Aijun3, WANG Bin4, JIANG Yan5, WANG Kaijie6, ZHOU Heting7
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1.National Engineering Research Center for Marine Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071, China;3.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266072, China;4.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266073, China;5.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266074, China;6.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266075, China;7.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Joint Laboratory for Deep Blue Fishery Engineering, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266076, China
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| Abstract: |
| Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating 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. It affects the distribution, stability, and biological activity of IGF by regulating the interaction between IGF ligands and receptors. Recently, IGFBP-1 was 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. IGFBP-1 regulates cell metabolism and growth through interaction with insulin in primary hepatocytes incubated in vitro in orange-spotted grouper (Epinephelus coioides). IGFBP-2 has a wide range of tissue expression characteristics in bony fish, and IGF regulation may occur through autocrine or paracrine pathways. For example, long-term fasting induced increased igfbp-2 mRNA expression in the liver of zebrafish. In addition, IGFBP-2 can inhibit the activity of IGF ligand through its high affinity with IGF to play an inhibitory and regulatory role in zebrafish growth. igfbp-2 mRNA expression was significantly up-regulated in Carassius auratus (goldfish) liver after fasting, and quickly recovered to normal levels after feeding. This indicated that igfbp-2 mRNA expression may be related to the anabolism and catabolism of goldfish, and is regulated by metabolic factors.
Yellowtail kingfish (Seriola aureovittata) of the Carangidae family has high economic value and nutrition value. It is a warm and temperate oceanic fish with long distance migration characteristics that is distributed in the middle and upper oceans globally. This species is large with a fast growth rate, and is a promising candidate for aquaculture in land-based industrial circulating water system, deep-water cages, net pen systems, and aquaculture craft, etc. Our laboratory studied the regulatory mechanism of the rapid growth of yellowtail kingfish and cloned gh, igf-1, igf-2, ghr, and other growth-related functional genes to reveal their molecular regulation in early growth and development. igfbp genes regulate the growth, development, and nutrient metabolism of fish through their interaction with growth axis. The mechanism of IGFBP and how it influences the regulation of yellowtail kingfish growth is unreported. This paper further studied the growth of yellowtail kingfish by analyzing the key growth axis factors.
RNAiso Plus reagent (TaKaRa) was used to extract total RNA from tissues. RNA integrity was detected by agarose gel electrophoresis, and RNA quality was determined by a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, USA). First, cDNA strand was synthesized using a PrimeScript™ RT reagent kit with a gDNA Eraser (Perfect Real-Time) reverse transcription kit (TaKaRa). Primers were designed to clone the predicted sequence of yellowtail kingfish igfbp gene according to the NCBI GenBank database. The product was amplified by polymerase chain reaction, purified from agarose gel electrophoresis, linked with T4 Ligases, transformed into Trans1-T1 Phage Resistant Chemically Competent Cell, and positive clones were selected and tested. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyze the distribution of yellowtail kingfish tissues and the expression patterns of liver tissues under different densities of industrial culture.
The lengths of igfbp-1, igfbp-2a, igfbp-2b open reading frame (ORF) domains were 741 bp, 882 bp, and 801 bp, and encoded 246 amino acids, 293 amino acids, and 269 amino acids, respectively. The conserved domain of insulin growth factor-binding protein homologues (IB) was present in the N-terminus of the three igfbps, and the conserved domain of thyroglobulin type-1 repeat (Ty-1) was present in the C-terminus. They had a wide range of tissue expression characteristics and were highly expressed in the liver. There were differences between the expression of the same gene in the same tissues of male and female fish. For example, the expression of igfbp-1 and igfbp-2b genes was significantly higher in the liver of male fish compared with female fish, while igfbp-2a expression was significantly higher in the liver of female fish compared with male fish. The tissue differential expression of genes between sexes indicates that igfbp may have sex dimorphism when it plays a physiological role. However, the specific characteristics of this difference between males and females and the possible signaling pathway are unclear. The fish in the low-density group showed the greatest growth rate and the highest expression levels of igfbp-1, igfbp-2a, igfbp-2b, igf-1, and igf-2 under industrial culture conditions. These expression levels were significantly different from those of the medium and high-density groups (P<0.05). However, there were no significant differences in the expression levels of growth and liver genes between the middle and high-density groups.
igfbp-1, igfbp-2a, and igfbp-2b participated in the growth regulation of yellowtail kingfish. The expression regulation of igf-1 and igf-2 had a positive synergistic effect with growth regulation of yellowtail kingfish. The ORF regions of igfbp-1, igfbp-2a, and igfbp-2b genes of yellowtail kingfish were cloned, and the structural characteristics, tissue expression characteristics, and relationships with growth performance were analyzed under different culture densities. The content of serum IGF-1, GH, cortisol, and glucose concentration was detected in the early stage. The expression trend of igfbp-1, igfbp-2, igf-1, and igf-2 was the same as that of serum IGF-1 and GH in the liver of yellowtail kingfish rapidly growing at the low-density group, but contrary to the expression trend of cortisol content and glucose concentration. This indicated that the key factors of growth axis, cortisol, and glucose participated in the growth regulation of yellowtail kingfish at different densities. However, the specific regulatory mechanism requires further study. The results provide a theoretical basis for interpreting the molecular mechanism of the growth of yellowtail kingfish and the regulation of suitable culture densities under industrial conditions. |
| Key words: cDNA cloning Seriola aureovittata igfbp-1 igfbp-2 Tissue expression Growth regulation |
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