Molecular characterization, expression pattern and genetic variant of insulin-like growth factor 2 mRNA-binding protein 3 gene in goose (Anser cygnoides)

ABSTRACT As a member of the insulin-like growth factor mRNA binding proteins (IGFBPs) family, IGF2BP3 plays important role in skeletal muscle development by regulating IGF2, which has been considered a key gene related to growth and reproductive traits in many agricultural animal species. However, study on the goose IGF2BP3 gene is still scarce. In this study, the full-length cDNA sequence of goose IGF2BP3 gene was cloned and characterized. The goose IGF2BP3 cDNA is 1825 bp in length, containing an open reading frame (ORF) of 1755 bp that encodes a protein of 584 amino acids, which consists of two RNA-recognition motifs (RRMs) and four K-homologous (KH) domains. RT-qPCR analysis indicates that the IGF2BP3 mRNA expression level is the highest in ovary tissue, followed in hypothalamus, spleen, leg muscle and liver, and low level in breast muscle and muscular stomach of the adult Zhedong White goose. Meanwhile, 25 single nucleotide polymorphisms (SNPs) and 5 insertions/deletions (InDels) variants were identified in about 10,764 bp of goose IGF2BP3 genomic DNA sequence. These results serve as a foundation for further investigations of the function of IGF2BP3 gene in geese.


Introduction
The insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) belong to a conserved family of RNA binding, which contain three members including IGF2BP1, IGF2BP2 and IGF2BP3, and act in various important aspects of cell functions, such as cell polarization, migration, morphology, metabolism, proliferation and differentiation (Bell et al. 2013).
As a member of the IGF2BPs, IGF2BP3 is also known as IMP3/ VICKZ3 and plays crucial role during cell developmental processes (Nielsen et al. 1999;Nassiry et al. 2005;Yisraeli 2005;Bell et al. 2013). Studies indicated that IGF2BP3 is a secreted protein that can bind to insulin-like growth factor 2 (IGF2), regulate IGF2 localization and play important roles in cell proliferation and migration (Jones and Clemmons 1995;Nielsen et al. 2002;Bell et al. 2013). Additionally, IGF2 is an important growth factor that can promote cell development and growth. It can bind to the two types of cell surface receptors known as IGF1 receptor and IGF2 receptor, stimulate cell proliferation and inhibit apoptosis (Bergman et al. 2013). Meanwhile, IGF2 gene is well known to be located within the linkage regions and has been considered as a positional candidate gene of growth traits in many animal species (Sewalem et al. 2002;McElroy et al. 2006;Wang et al. 2005). Therefore, IGF2BP3-mediated regulation of IGF2 function may play key role in animal muscle development (Lin et al. 2017).
In recent years, a series of studies have focused on the function of IGF2BP3 gene in different species. In humans, a study reported that IGF2BP3 as an RNA N6-methyladenosine reader can regulate cell cycle and angiogenesis in colon cancer, and it also had been associated the most with distinct cancer types (Hammer et al. 2005;Lederer et al. 2014;Yang et al. 2020;Zhang et al. 2022). A study on chicken performed by Ye et al. (2014) showed that IGF2BP3 gene participated in adipocytokine and insulin signalling pathways. Subsequently, Lin et al. (2017) demonstrated that IGF2BP3 was a direct target gene of let-7b, which can regulate myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. Furthermore, a study performed by Yang et al. (2021) indicated that DNA methylation regulated the expression and function of IGF2BP3 by modulating SP1 binding in the skeletal muscle development of pigs.
The goose is a popular poultry species, and in the past two decades, the goose industry has become highly profitable across the globe. As an important meat poultry, its growth and development have always been focused, and increased muscle growth in geese is economically beneficial for the poultry industry. IGF2BP3 plays an important role in skeletal muscle development, but the information on goose IGF2BP3 gene is limited. In this study, we isolated and analysed the full-length coding sequence of the goose IGF2BP3 gene, investigated its expression pattern in various tissues and screened the genetic variants located in this gene.

Animals and samples collection
Six healthy female adult (70 days) Zhedong White geese were provided by Wenjie goose breeding of Xiangshan Co. LTD, China. All geese had access to open ground with a pool for swimming and reared under commercial management conditions. Several tissue samples, including heart, liver, spleen, lung, kidney, breast muscle, leg muscle, brain, skin, muscular stomach, hypothalamus, pituitary and ovary tissues were collected immediately, frozen in liquid nitrogen, and then stored at −80°C prior to RNA isolation. Tissues pooled from leg muscle and brain were used for cloning the full-length cDNA sequence of goose IGF2BP3 gene.
Blood samples were collected under the goose wing vein to the disposable blood collection tubes those including EDTA anticoagulant, stored at −20°C before genomic DNA isolation. Blood samples from the three Chinese native goose breeds were used to screen the genetic variants located in the genomic DNA sequence of the IGF2BP3 gene.

RNA extraction, DNA isolation and cDNA synthesis
Total RNA was extracted with Trizol Reagent (Invitrogen, USA) and treated with RNase-free DNaseI (TaKaRa, Dalian) to remove any contaminating genomic DNA. First-strand cDNA was synthesized using ReverTra Ace Kit (TaKaRa, Dalian) and the concentration of each sample was adjusted to 200-300 ng/µL, stored at −20°C for quantitative real-time PCR (qPCR) analysis.
Genomic DNA was isolated from the blood samples using AxyPrep TM blood Genomic DNA Miniprep Kit (AxyGEN, USA). DNA concentration and quality were measured with the spectrophotometer ND-1000 (Nano-Drop, USA), and the concentration of each sample was adjusted to 50-300 ng/µL and stored at −20°C.

Molecular cloning and sequence analysis of goose IGF2BP3 gene
Based on the conserved region of IGF2BP3 gene in chicken (ID: NM_001006359) and duck (ID: XM_038174133), two pairs of primers (BP3-F1/R1 and BP3-F2/R2) were designed to amplify the whole CDS sequence of goose IGF2BP3 gene (Table 1).
The PCR program included denaturation for 5 min at 94°C, followed by 36 cycles of 30 s at 94°C, 45 s at annealing temperature, 90 s at 72°C, and an extension step of 5 min at 72°C. The purified PCR products were cloned into the PEASY-T1 vector (TransGen, China) and sequenced commercially (Sangon, China).
The obtained cDNA sequences were matched using DNAMAN software. The open reading frame (ORF) and the amino acid sequences were deduced using SeqMan (DNAstar, USA). Sequence similarity searches in GenBank were performed by using the BLAST2.1 search tool (http://www.ncbi.nlm.nih. gov/blast).
A phylogenetic tree was constructed based on the above amino acid sequence alignment by the neighbour-joining algorithm of the MEGA 5.0 software (Tamura et al. 2011). The reliability of the branching was tested by bootstrap re-sampling (1000 replicates). The evolutionary distances were computed using the Jones-Thornton-Taylor matrix-based method (Jones et al. 1992).

Tissue expression pattern analysis of goose IGF2BP3 mRNA
To detect the IGF2BP3 mRNA levels in different tissues of the female adult (70 days) Zhedong White geese, qPCR was performed with a 384-well C1000 Touch TM Thermal (Bio-Rad, USA) using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Bio-Rad, USA). IGF2BP3-QF/QR (ID: ON908307) and β-actin-F/R (ID: M26111.1) primers were designed and used for qPCR analysis ( Table 1). The PCR program included a denaturation step of 2 min at 95°C, followed by 40 cycles of 5 s at 95°C, 30 s at 60°C. The specificity of qPCR primers for each gene was confirmed by melting curve analysis. Melting curve analysis parameters were 1 cycle at 95°C for 10 s, 1 cycle at 65°C for 5 s, and then the temperature was increased 0.5°C/cycle to 95°C while continuously monitoring fluorescence. The relative quantification of gene expression was replicated three times for each sample. The data were analysed with one-way ANOVA, followed by Tamhane's T2 post hoc test. The results were expressed as the mean ± SEM. p < 0.05 was considered statistically significant, and p < 0.01 was considered very significant.

Genetic variants identification of goose IGF2BP3 gene
The three goose populations were applied to construct the DNA pooling. According to the genomic DNA sequence of Peking duck IGF2BP3 (NC_040047), 14 pairs of primers (BP3-SF1/SR1⍰BP3-SF14/SR14, Table 1) were synthesized to identify the genetic variants in the regions of the whole CDS, several introns and partial 3'-untranslated region (UTR) of goose IGF2BP3 gene. PCR products were amplified from the DNA pools and sequenced directly by the SangonBiotech (Shanghai, China). The obtained sequences were aligned by SeqMan of DNASTAR software.

Multiple sequence alignments and evolutionary relationships of IGF2BP3
Multiple sequence alignment analysis revealed that the avian IGF2BP3 proteins were a little longer than those of the mammalian counterparts. Meanwhile, the IGF2BP3 proteins were highly conserved between the avian and mammalian species, and the IGF2BP3 proteins were completely conserved in Anser cygnoides, Anas platyrhynchos, Taeniopygia guttata, Falco peregrinus, Calypte anna and Falco cherrug (Figure 2).
Based on the multiple sequence alignments, a phylogenetic tree was constructed using MEGA 5.0 software (Figure 3). The dendrogram was clustered into two subgroups, the avian species including Anas platyrhynchos, Gallus gallus, Taeniopygia guttata, Coturnix japonica, Columba livia, Falco peregrinus, Calypte anna and Falco cherrug belonging to one group, and the mammal species including Sus scrofa, Bos Taurus and Ovis aries belonging to the other. The phylogenetic tree also displayed that the deduced goose IGF2BP3 protein was more closely related to the avian IGF2BP3, especially to that of Anas platyrhynchos, Taeniopygia guttata, Falco peregrinus, Calypte anna and Falco cherrug (Figure 3). Tissue expression pattern of goose IGF2BP3 gene As shown in Figure 4, qPCR analysis showed that the goose IGF2BP3 mRNA was differentially expressed in the detected tissues of the female adult (70 days) Zhedong White geese. The highest level of IGF2BP3 mRNA expression was observed in ovary tissue, and the levels in the hypothalamus, spleen, leg muscle and liver tissues were higher than that in heart, lung, kidney, brain, pituitary and skin, little expressed in breast muscle and muscular stomach tissues (Figure 4).

Discussion
The insulin-like growth factor-2 mRNA-binding proteins (IGF2BP1, IGF2BP2 and IGF2BP3) belong to a highly conserved family of RNA-binding, which carry six typical canonical RNAbinding domains, including two RRMs in their N-terminal part and four KH domains in the C-terminal region (Nielsen et al. 2001;Yaniv and Yisraeli 2002;Yisraeli 2005). In this study, we first cloned and characterized the goose IGF2BP3 cDNA sequence, which consists of an open reading frame of 1755 nucleotides with the capacity to encode a protein of 584 amino acids. Multiple sequence alignment implied that the number and sequences of IGF2BP3 amino acids were strongly conserved among the avain species. Meanwhile, the completely conserved IGF2BP3 amino acids sequences highlight it may act similar role on the Anser cygnoides, Anas platyrhynchos, Taeniopygia guttata, Falco peregrinus, Calypte anna and Falco cherrug. The sequence information from this study will facilitate further research on the function of the IGF2BP3 gene in geese.
The expression of eukaryotic genes is temporarily and multidimensional controlled (Shahsavari et al. 2021). Only a relatively small set of the entire genome is expressed in each type of tissue, and the expression of genes depends on the stage of development . Meanwhile, the amount of gene products that are made in the same tissue as well as in other tissues that make up that product, regulates the expression of that gene (Direkvandi et al. 2020). One of the basic activities in domestic animals is the study of genes and proteins at the cellular or chromosomal level which are to economic traits (Mohamadipoor-Saadatabadi et al. 2021). Compared to the high expression level in the embryo, IGF2BP3 mRNA was reported to be expressed at negligible levels in adult mouse and human organs, but except reproductive tissues including ovaries, testis and brain tissues (Hammer et al. 2005). In Zebrafish, a high expression level also was detected in ovary tissue (Ren et al. 2020). In the present study, the highest IGF2BP3 mRNA expression level also was detected in the ovary tissue of adult Zhedong White goose, which implies that the goose IGF2BP3 may act important roles in ovarian follicle development, and be a strong performance target for goose reproductive traits. Therefore, further work will be necessary to confirm its influence on reproductive traits.
The determination of gene polymorphism is important for farm animals breeding to define genotypes of animals and their associations with productive, reproductive and economic traits (Yaripour et al. 2017). IGF2BP3 is a translational activator of IGF2, which was reported as a key gene located in the linkage  The relative expression level of IGF2BP3 mRNA was analysed with the Comparative Ct method, employing goose β-actin as the reference gene in each sample (n = 3); Values are displayed as the mean ± SEM. "*" shows significant differences (p < 0.05), "**" and "***" represent extremely significant differences (p < 0.01). regions affecting growth, carcass and reproductive traits of many agricultural animal species, such as pig, cattle, chicken, Muscovy duck and goose (Van Laere et al. 2003;Wang et al. 2005;Tang et al. 2010;Huang et al. 2014;Wang et al. 2021). Meanwhile, the whole-genome analysis revealed the association between IGF2BP3 gene and birth weight of humans (Warrington et al. 2019). Recently, Yang et al. (2021) reported that IGF2BP3 might be considered as a candidate gene in breeding for meat production traits in the pig industry. In this study, 19 variants located in the intronic regions, only one synonymous mutation (A264G, Pro180pro) located in the exonic region, which reveals that the IGF2BP3 amino acid sequence is highly conserved in geese. Although these variants do not directly change the amino acid structure of the protein, they may affect the gene function by affecting translation with codon bias, changing the stability of the mRNA or controlling transcription of the gene (Zhang et al. 2005;Van Laere et al. 2003;Kimchi-Sarfaty et al. 2007;Hou et al. 2010). The potential effects of the large number of variations on goose economic traits will be investigated in our following work.
In this study, we first cloned and characterized the whole CDS sequence of the goose IGF2BP3 gene, and found the IGF2BP3 mRNA was highly expressed in the ovary tissues of the female adult Zhedong White geese, and identified 25 SNPs and 5 InDels variants in three native goose populations. All the present findings will provide important resources for research on the functions of the goose IGF2BP3 gene.