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)

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.

Keywords

• cdna cloning
• seriola aureovittata
• igfbp-1
• igfbp-2
• tissue expression
• growth regulation