Gene combination related to larimichthys crocea figure and application of gene combination in breeding

Abstract

The invention discloses a gene combination related to a larimichthys crocea figure and application of the gene combination in breeding, and belongs to the technical field of fry and fingerling breeding. The invention relates to a gene combination related to a large yellow croaker figure. The gene combination comprises an INHBB gene, a Nodal gene, a GDF3 gene, an ACVR2A gene, an ALK4 gene and a Smad2 gene. The invention is applied to the aspect of larimichthys crocea body shape screening, and provides a molecular marker for larimichthys crocea breeding screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fry and seed fish breeding, and particularly relates to a gene combination related to the body shape of Larimichthys crocea and application thereof in breeding. BACKGROUND

[0002] The depletion of global fishery resources has prompted people to shift from traditional capture fisheries to aquaculture. Marine aquaculture has now become a key method for sustainable production of marine products. China, as the world's largest aquaculture producer, has taken the lead in this transition, and aquaculture will become the main source of aquatic dietary protein. Larimichthys crocea plays an important role in aquaculture, and therefore, growth-related traits are crucial for many aquaculture species, directly affecting yield. However, challenges such as climate change, disease outbreaks, and loss of genetic diversity due to inbreeding hinder the growth and body shape of Larimichthys crocea. In most selective breeding programs, growth-related traits are considered to be quantitative traits controlled by multiple genes distributed throughout the genome. The body shape of Larimichthys crocea is crucial to the aquaculture industry, as it affects swimming, feeding, and consumer preferences. At the same time, as most consumers prefer slender Larimichthys crocea, body shape has become an important economic feature and greatly affects commercial value. Given its importance, it is crucial to identify genes that regulate the growth and body shape of Larimichthys crocea.

[0003] The transforming growth factor-beta (TGFβ) family plays a crucial role in coordinating a variety of biological processes, including cell growth, proliferation, and differentiation. The TGFβ signaling pathway involves ligand binding to membrane receptors, activation of type I receptors through phosphorylation of type II receptors, phosphorylation of Smad proteins, and initiation of signal transduction. The TGFβ superfamily includes a wide variety of ligands, such as TGFβs (transforming growth factor beta, TGFB1-5), BMPs (bone morphogenetic proteins, BMP2-16), GDFs (growth and differentiation factors, GDF1-15), Nodal, activins (INHBA and INHBB), and inhibins. The receptors for these ligands are mainly divided into two types: type I and type II. Specifically, type I receptors include ALK (activin-like kinase) 1 to 7, while type II receptors include TGFBR2, ACVR2, ACVR2B, AMHR2, and BMPR2. Smad proteins, which play a key role in downstream signaling pathways of these receptors, can be further divided into three different categories: receptor-activated or pathway-restricted Smads (R-Smads), common pathway Smads (Co-Smads), and inhibitory Smads (I-Smads). This classification system provides a theoretical framework for exploring the complex mechanisms of interaction within the TGFβ superfamily and its signaling pathways. Essentially, embryonic cells at various developmental stages and various cells in adult bodies have the ability to perceive TGFβ signals. Currently, the number and types of TGFβ family members have been evaluated in model organisms, including worms, flies, and mammals. Most, if not all, cell types respond to at least a subset of TGFβ family ligands, thereby regulating numerous cellular activities.

[0004] The TGFβ signaling pathway not only plays a key role in the mediation of biological growth and development, but also is deeply involved in the regulation of body shape-related traits in Pseudosciaena crocea, as revealed by recent whole-genome association studies. However, as of now, there is a lack of research on which specific members of the signaling pathway are involved and how they are precisely regulated. This lack of research is mainly due to the completion of whole-genome sequencing and the construction of a fine genetic map of P. crocea in 2014. In P. crocea, the TGFβ family members include 24 ligands, 19 receptors, and 3 Smads proteins. In addition, some members have more detailed subtype classifications, such as BMPR1BX1 (XM_010732488.3), BMPR1BX2 (XM_027275036.1), and BMPR1BX3 (XM_027275041.1). However, there are few studies on the systematic classification of these gene entries, and even fewer studies on the specific mechanisms of each member in the regulation of growth and body shape in P. crocea. SUMMARY

[0005] In order to solve the problems in the prior art, the application provides a gene combination related to the body shape of large yellow croaker and application thereof in breeding, which can be used for screening research of large yellow croaker with beautiful body shape and provides a molecular marker for screening of large yellow croaker breeding.

[0006] To solve the technical problem, the application adopts the technical scheme of:

[0007] The application provides a gene combination related to the body shape of large yellow croaker, which contains INHBB gene, Nodal gene, GDF3 gene, ACVR2A gene, ALK4 gene and Smad2 gene.

[0008] In a preferred embodiment, the gene combination is INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2 and GDF3-ACVR2A-ALK4-Smad2.

[0009] The transforming growth factor beta (TGFβ) signaling axis plays a key role in coordinating a series of biological functions such as cell growth, proliferation and differentiation. The present application determines the TGFβ signaling pathway members and their expression patterns in different culture modes of Pseudosciaena crocea. The TGFβ signaling pathway and its expression patterns in Pseudosciaena crocea cultured in two different modes: group N (2,400 fish in a 120 cubic meter net cage) and group V (168,000 fish in a 5,600 cubic meter ship cabin). After 120 days, it is found that the growth rate of fish in group V is faster and the body shape is slimmer compared with group N. The present application uses bioinformatics technology to systematically classify the 48 TGFβ sequence information included in the National Center for Biotechnology Information (NCBI) database. Through rigorous data analysis and comparison, 21 kinds of TGFβ ligands, 10 kinds of TGFβ receptors and 3 kinds of Smads proteins in Pseudosciaena crocea are accurately identified. In order to further explore the mechanism of action of these signaling molecules, the present application further extracts RNA from Pseudosciaena crocea tissue samples containing the above identified components, and uses advanced molecular biology techniques to detect the mRNA expression level. The experimental results show that these signaling molecules mainly regulate the growth rate and body shape of Pseudosciaena crocea through the following three specific ligand-receptor-R-Smad signaling pathways: INHBB-ACVR2A-ALK4-Smad2 signaling pathway; Nodal-ACVR2A-ALK4-Smad2 signaling pathway; GDF3-ACVR2A-ALK4-Smad2 signaling pathway. Based on the above innovative research results, the present application successfully determines a set of gene combinations closely related to the body shape regulation of Pseudosciaena crocea, which provides an important theoretical basis and technical support for Pseudosciaena crocea breeding and related industries.

[0010] In a preferred embodiment, the INHBB gene encodes a protein with an amino acid sequence of SEQ ID NO: 1; the Nodal gene encodes a protein with an amino acid sequence of SEQ ID NO: 2; the GDF3 gene encodes a protein with an amino acid sequence of SEQ ID NO: 3; the ACVR2A gene encodes a protein with an amino acid sequence of SEQ ID NO: 4; the ALK4 gene encodes a protein with an amino acid sequence of SEQ ID NO: 5; and the Smad2 gene encodes a protein with an amino acid sequence of SEQ ID NO: 6.

[0011] In a preferred embodiment, the INHBB gene has a nucleic acid sequence of SEQ ID NO: 7 encoding CDS; the Nodal gene has a nucleic acid sequence of SEQ ID NO: 8 encoding CDS; the GDF3 gene has a nucleic acid sequence of SEQ ID NO: 9 encoding CDS; the ACVR2A gene has a nucleic acid sequence of SEQ ID NO: 10 encoding CDS; the ALK4 gene has a nucleic acid sequence of SEQ ID NO: 11 encoding CDS; and the Smad2 gene has a nucleic acid sequence of SEQ ID NO: 12 encoding CDS.

[0012] In another aspect, the present application provides application of the gene combination related to the body shape of Pseudosciaena crocea in screening Pseudosciaena crocea with different body shapes.

[0013] The present application further provides a preparation for detecting the expression amount of each gene in the gene combination related to the body shape of Pseudosciaena crocea, which is a PCR amplification primer.

[0014] The present application further provides a product for screening Pseudosciaena crocea with different body shapes, which is a fluorescent quantitative PCR amplification detection kit, and the product screens Pseudosciaena crocea with different body shapes by detecting the expression amount of each gene in the gene combination related to the body shape of Pseudosciaena crocea.

[0015] The present application further provides a method for screening Pseudosciaena crocea with different body shapes, which screens Pseudosciaena crocea individuals by detecting the expression amount of each gene in the gene combination.

[0016] In a preferred embodiment, the method screens individuals with low expression amount of INHBB gene, Nodal gene, GDF3 gene, ACVR2A gene, ALK4 gene, and Smad2 gene.

[0017] The TGF beta signal pathway is activated by the combination of ligand and membrane receptor, the phosphorylation of type II receptor activates type I receptor, and then phosphorylates Smad protein to start signal transduction. The screening of the body shape related gene combination of large yellow croaker is first based on the completeness of the signal pathway in large yellow croaker. According to the integrity of the nucleotide and amino acid sequence of large yellow croaker in the NCBI database, we first screened 15 signal pathways. According to the expression amount of ligand, receptor and R-Smads in the experimental group, we further clarified the promotion and inhibition of each gene. Further, the feasible way of regulating body shape is combed out, and there are 5 pathways in the two subfamilies. According to the expression amount of each gene involved in the above 5 pathways in the muscle of V group, it is found that INHBB, Nodal, GDF3, ACVR2A, ALK4, Smad2 and N group are inhibited, and three signal pathways INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2 and GDF3-ACVR2A-ALK4-Smad2 are further locked.

[0018] In a preferred embodiment, the method is detected by a fluorescent quantitative PCR amplification method; wherein a primer pair for detecting the INHBB gene, the sequences of the upstream and downstream primers are SEQ ID NO: 13 and SEQ ID NO: 14; a primer pair for detecting the Nodal gene, the sequences of the upstream and downstream primers are SEQ ID NO: 15 and SEQ ID NO: 16; a primer pair for detecting the GDF3 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 17 and SEQ ID NO: 18; a primer pair for detecting the ACVR2A gene, the sequences of the upstream and downstream primers are SEQ ID NO: 19 and SEQ ID NO: 20; a primer pair for detecting the ALK4 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 21 and SEQ ID NO: 22; a primer pair for detecting the Smad2 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 23 and SEQ ID NO: 24.

[0019] Compared with the prior art, the beneficial effects of the present application are that:

[0020] The present application analyzes the TGF beta signal pathway and its expression mode in large yellow croaker fed in different breeding modes, finds that the INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2 and GDF3-ACVR2A-ALK4-Smad2 axis plays a leading role in regulating the growth and body shape of large yellow croaker, and the population with low gene expression amount grows fast and has slender body shape, which can be used as a molecular marker for screening large yellow croaker with fast growth potential and slender body shape. Attached Figure Description

[0021] Figure 1 The growth and body shape of the large yellow croaker provided in Embodiment 2 of the present invention, wherein A is the fish in the early stage of aquaculture, B is the fish cultured in net cages for 120 days, and C is the fish cultured in aquaculture boats for 120 days.

[0022] Figure 2 A phylogenetic tree was constructed based on the nucleotide sequences of ligands belonging to the TGFβ / activin / Nodal subfamily of large yellow croaker provided in Example 2 of this invention;

[0023] Figure 3 This is the phylogenetic tree constructed based on the nucleotide sequences of the BMP / GDF / MIS subfamily ligands of large yellow croaker provided in Example 2 of the present invention;

[0024] Figure 4 This is the phylogenetic tree constructed based on the receptor nucleotide sequence of large yellow croaker provided in Embodiment 2 of the present invention;

[0025] Figure 5 This is the phylogenetic tree constructed based on the R-Smads nucleotide sequence of large yellow croaker provided in Embodiment 2 of the present invention;

[0026] Figure 6 The expression of the ligand provided in Example 2 of this invention in the liver of large yellow croaker;

[0027] Figure 7 The expression of the ligand provided in Example 2 of this invention in the muscle of large yellow croaker;

[0028] Figure 8 The relative expression level of receptors in the liver of large yellow croaker provided in Example 2 of the present invention;

[0029] Figure 9 The relative expression of receptors in the muscle of large yellow croaker provided in Embodiment 2 of the present invention;

[0030] Figure 10 The relative expression of Smads in the liver provided in Embodiment 2 of the present invention;

[0031] Figure 11 This is the relative expression of Smads in muscle provided in Embodiment 2 of the present invention. Detailed Implementation

[0032] The average total length (32.41 ± 1.16) cm and average weight (350.4 ± 34.24) g of P. major (group C) were selected and divided into two experimental groups. Among them, group N consisted of 2,400 fish, which were raised in a 120 cubic meter net cage; group V consisted of 168,000 fish, which were raised in a 5,600 cubic meter ship cabin of a farming work ship. After 120 days of feeding, 15 fish were randomly selected from each group for further detailed experimental analysis. The parameters of body weight, total length, body length, body height, chest height, caudal peduncle length, caudal peduncle width, caudal peduncle length to caudal peduncle width ratio (CPL / CPW), body length to body height ratio (BL / BD), caudal peduncle length to body length ratio (CPL / BL), and condition factor were used to describe growth and body shape.

[0033] The present application will be described in detail below in conjunction with specific examples and accompanying drawings.

[0034] Example 1: Sequence analysis of P. major growth and body shape related genes

[0035] First, the protein sequences of all TGFβ ligands, receptors and Smads of P. major were downloaded from the National Center for Biotechnology Information (NCBI) database, and BLAST software was used to compare and analyze them with corresponding sequences of other species; then, ClustalW multiple sequence alignment tool (http: / / www.ebi.ac.uk / clustalw / ) was used to carry out multiple sequence alignment, and the amino acid sequences of ligands, receptors and Smads with close genetic relationship were screened and downloaded according to the alignment results; then, the neighbor-joining (NJ) method of MEGA7.0 software was used to construct the individual phylogenetic tree, and the robustness of the phylogenetic tree was evaluated by 1000 times of guided repetition to ensure its reliability.

[0036] Table 1 Oligonucleotide primers for amplifying TGFβ signaling pathway genes of P. major

[0037]

[0038]

[0039]

[0040]

[0041] All data were statistically analyzed using GraphPad Prism 5.0 software, and evaluated using one-way ANOVA. Tukey's Honestly Significant Difference (HSD) test was used for post-hoc multiple comparisons to evaluate the differences between the mean values. The results were expressed as mean ± SD, and P value less than 0.05 was considered statistically significant.

[0042] Example 2: Genes differentially expressed in Group N and Group V

[0043] (1) Growth and body shape of P. major in two culture modes

[0044] After 120 days of feeding, significant differences were observed in body weight, total length, body length, caudal peduncle length, caudal peduncle length / width, body length / body height, caudal peduncle length / body length, and condition factor between Group V and Group N, with Group V growing faster and having a more elongated body shape than Group N (Table 2). To more intuitively represent the differences in these data, one fish was selected from each of the three groups as a representative ( Figure 1 ).

[0045] Table 2 Effects of cage and factory ship culture on growth and body shape of P. major (n = 15)

[0046] Indicator Early stage of culture (Group C) Group N Group V Body weight (g) 350.4 ± 34.24 c ]] 508 ± 29.02 b ]] 535.33 ± 22.24 a ]] Total length (cm) 32.41 ± 1.16 c ]] 35.31 ± 1.22 b ]] 37.28 ± 0.92 a ]] Body length (cm) 28.29 ± 1.15 c ]] 31.35 ± 0.93 b ]] 33.78 ± 1.24 a ]] Body height (cm) 7.9 ± 0.49 b ]] 9.04 ± 0.35 a ]] 9.06 ± 0.44 a ]] Chest height (cm) 7.27 ± 0.6 b ]] 8.31 ± 0.22 a ]] 8.06 ± 0.29 a ]] Caudal peduncle length (cm) 7.49 ± 0.55 c ]] 8.07 ± 0.69 b ]] 9.34 ± 0.42 a ]] Caudal peduncle width (cm) 2.03 ± 0.13 c ]] 2.19 ± 0.15 b ]] 2.3 ± 0.08 a ]] Caudal peduncle length / Caudal peduncle width (CPL / CPW) 3.7 ± 0.28 b ]] 3.7 ± 0.32 b ]] 4.07 ± 0.24 a ]] Body length / Body height (BL / BD) 3.59 ± 0.21 b ]] 3.47 ± 0.18 b ]] 3.73 ± 0.17 a ]] Caudal peduncle length / Body length (CPL / BL) 0.26 ± 0.01 b ]] 0.26 ± 0.02 b ]] 0.28 ± 0.01 a ]] Fatness (g / cm 3 )]]> 1.55 ± 0.15 a ]] 1.66 ± 0.17 a ]] 1.4 ± 0.15 b ]]

[0047] (2) Phylogenetic analysis of ligand sequences

[0048] The types and numbers of ligands are shown in Figure 2 and Figure 3 . NCBI contains 24 ligand entries, which can be classified into 21 types. It is worth noting that GDF15 in P. major (XM_010733382.3) does not cluster with other GDF types in the phylogenetic tree, but is more closely related to GDF8 ( Figure 3 ). Except for AMH / MIS, which has four subtypes, each of the remaining 20 types of ligands has only one subtype. In P. major, the identified ligand types include GDF8, GDF6 / BMP13, GDF5 / BMP14, BMP3, GDF9, AMH, BMP10, BMP4 / BMP2B, BMP2 / BMP2A, GDF3, BMP8 / BMP8B, BMP5, BMP7, BMP6, NDR2, INHBB / ActivinB, INHBA / Activin A, TGFB1, TGFB2, and TGFB3.

[0049] (3) Phylogenetic analysis of receptors

[0050] By studying Figure 4The receptor phylogenetic tree in the database reveals that the 19 receptor nucleotide sequence entries in the gene pool can be divided into 10 different groups. These include five type I receptors: ALk3 / BMPR1A, ALK6 / BMPR1B, ALK2 / ACVR1, ALK4 / ACVR1B, and ALK7 / ACVR1C. There are also five type II receptors: ACVR2A / ACTRⅡA, ACVR2B / ACTRⅡB, TGFBR2 / TBRⅡ, BMPR2, and AMHR2 / MISR2. Some of these receptors exhibit different subtypes. For example, AMHR2 has two subtypes, AMHR2X1 and AMHR2X2; ACVR2A has five subtypes, ACVR2AX1, ACVR2AX2, ACVR2AX3, ACVR2AX4 and ACVR2AX5; BMPR1A has two subtypes, BMPR1AX1 and BMPR1AX2; and BMPR1B has four subtypes, BMPR1BX1, BMPR1BX2, BMPR1BX3 and BMPR1BX4.

[0051] (4) Phylogenetic analysis of R-Smads

[0052] exist Figure 5 In the phylogenetic tree shown, the nucleotide sequences of each type of R-Smad are grouped together. The five Smad genes found in large yellow croaker are classified into three classes: Smad1, Smad2, and Smad3. Among them, Smad2 includes three different subtypes: Smad2X1, Smad2X2, and Smad2X3. In large yellow croaker, although Smad5 and Smad8 belong to the R-Smads family and primarily respond to BMP signaling, they were not identified.

[0053] (5) ligand mRNA level

[0054] In this invention study, the expression of the ligand in the liver tissue of large yellow croaker is shown in [reference needed]. Figure 6 Among them, TGFB2, BMP6, and GDF9 showed significantly upregulated expression levels in both groups N and V; TGFB1, BMP3, BMP4, BMP10, and GDF5 showed decreased expression levels in group N and increased expression levels in group V; BMP5, BMP7, and GDF6 showed no significant difference in expression levels between groups N and V; in muscle tissue ( Figure 7 The expression patterns of ligands in muscle tissue are distinctly different from those in the liver, except for GDF9. Furthermore, the expression levels of BMP4, BMP5, and AMH were significantly increased in both the N and V groups. These findings suggest that ligand expression in muscle tissue may be influenced by regulatory mechanisms different from those in the liver.

[0055] (6) Receptor mRNA level

[0056] In the research of this invention, Figure 8 The expression levels of ligands in the liver tissue of large yellow croaker were shown. ALK2 and AMHR2 expression levels showed no significant difference between groups N and V, while ALK3 and ALK7 expression levels decreased in group N and increased in group V. Notably, except for ALK4, the mRNA expression trends of ALK6, TGFBR2, and ACVR2B in muscle tissue differed from those in liver tissue. Figure 8 Furthermore, in muscle tissue, there was no significant difference in ALK6 expression levels between groups N and V, while the expression of ALK7, TGFBR2, and ACVR2B was significantly increased in both groups N and V. Figure 9 ).

[0057] (7) mRNA level of R-Smads

[0058] In the study of this invention, Figure 10 and Figure 11 The expression patterns of R-Smads in the liver and muscle tissues of large yellow croaker were presented separately. Specifically, in both liver and muscle tissues, there were no significant changes in Smad3 expression levels between the experimental and control groups, and no significant difference in Smad3 expression between the two experimental groups. Regarding Smad1 and Smad2, their expression patterns in the liver and muscle tissues of group N showed a differentiated trend. In liver tissue, the expression of these two genes differed from the control group, while no similar differences were found in muscle tissue; conversely, genes that showed significant differences in muscle tissue compared to the control group did not show differences in liver tissue. This result indicates that the expression of Smad1 and Smad2 in group N is affected by tissue-specific regulatory mechanisms. However, in group 5, the expression of Smad1 and Smad2 did not appear to be affected by the tissue type used in the experiment; that is, the expression patterns of the two genes remained consistent in both tissues, and no inter-tissue differences were observed. In summary, under different treatment methods and tissue types, the expression of Smad3 in R-Smads of large yellow croaker is relatively stable, while the expression of Smad1 and Smad2 shows tissue-specific variation patterns under different treatment conditions.

[0059] Analysis results

[0060] In contrast to the elongated body shape of wild P. major, the farmed varieties of P. major tend to have a more rounded appearance. Slender fish exhibit superior swimming, foraging, and predator avoidance abilities. Consumer preference for slender P. major greatly influences its market value. In a 120-day farming experiment, P. major reared on a factory ship grew faster and had a more slender body shape than P. major reared in a net cage. The observed faster growth rate can be attributed to the uninterrupted forced water exchange between the rearing tank and the external natural seawater achieved by the advanced water exchange system of the factory ship. In addition, the factory ship also utilizes a deep water intake device to obtain seawater of the optimal temperature and salinity required for rearing, ensuring that P. major remains in an optimal growth state. It is thus hypothesized that the swimming behavior and schooling tendency of P. major reared in a drifting mode factory ship rearing system are very similar to those of wild P. major, which is the main reason for its fast growth rate and slender body shape.

[0061] Based on extensive research on the specificity of TGFβ superfamily ligand-receptor-R-Smad interactions, experience in teasing out the TGFβ signaling pathways of A. japonicus and Crassostrea gigas, and detection of related nucleotide sequences in the P. major genome ( Figure 2-5 ) and mRNA expression levels ( Figure 6-11 ), the corresponding relationships between ligand-receptor-R-Smad in P. major were summarized in Table 4. In the TGFβ / activin / Nodal subfamily, the detected components included ligands (INHBA, INHBB, and Nodal), receptor I (ALK4 and ALK7), receptor II (ACVR2A and ACVR2B), and R-Smad (Smad2, 3). Conversely, in the BMP / GDF / AMH subfamily, a series of ligands (BMP2, BMP4, BMP5, BMP6, BMP7, BMP8A, GDF3, GDF5, GDF6, GDF8, GDF9, and AMH), receptor I (ALK2, ALK3, ALK4, ALK6, and ALK7), receptor II (ACVR2A, ACVR2B, BMPR2, and AMHR2), and R-Smads (Smad1, 2, 3) were detected. In Table 4, family members exhibiting complete signaling pathways are listed. Certain members, including TGFB1-3, have nucleotide and amino acid sequences in the P. major genome, and their mRNA expression levels under different culture conditions were evaluated using RT-PCR technology. Nonetheless, after reviewing the receptor types of TGFB1-3 reported in the current literature, we did not find these genes in the genome of P. major. Therefore, due to their incomplete signaling pathways, these members were not included in Table 4.

[0062] Table 4 Specificity of TGFβ superfamily ligand-receptor-R-Smad

[0063]

[0064]

[0065] Based on the promoting and inhibiting effects observed in the experimental group by ligands, receptors, and R-Smads ( Figure 6-11 Table 5 lists feasible pathways regulating liver growth and body shape, including 11 pathways across two subfamilies. Table 6 records feasible pathways regulating muscle growth and body shape, with 5 pathways across two subfamilies. Fewer feasible pathways exist in muscle compared to the liver, likely because different culture methods do not activate Smad1. Notably, Smad1 is the only recognizable R-Smad among various BMPs in large yellow croaker. This protein can bind to Co-Smad (Smad4) to form a complex, which then enters the nucleus to regulate gene expression, as mentioned in related studies. The five pathways regulating growth and body shape are also present in the liver of large yellow croaker muscle tissue. However, whether these pathways maintain consistent tissue stability in different organs (such as the heart, spleen, kidneys, intestines, gonads, and brain) remains unanswered and requires further experimental verification in these other tissues. In group V muscle, the expression levels of each member in the three signaling pathways differed: the expression levels of each member in the INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2, and GDF3-ACVR2A-ALK4-Smad2 signaling pathways were inhibited. Figure 7 , 9 (11) INHBB, Nodal, and GDF3 act as ligands that bind to specific receptors to transmit signals; reduced expression of these ligands may lead to weakened signaling capabilities. ACVR2A and ALK4 can bind to these three ligands, thereby activating downstream signal transduction of Smad2. Reduced expression of these ligands may further inhibit TGFβ signal transduction. As a downstream signaling molecule, reduced Smad2 expression indicates decreased activity of the TGFβ signaling pathway. Although ACVR2B and ALK7 can also bind to the aforementioned three ligands, their expression levels are increased. They may bind to other ligands to transmit different signals or compensate for reduced function of ALK4 and ACVR2A under specific conditions.

[0066] Table 5. TGFβ superfamily ligand-receptor-R-Smad specificity in the liver.

[0067]

[0068] Table 6. TGFβ superfamily ligand-receptor-R-Smad specificity in muscle

[0069]

[0070] In summary, the present application discloses that there are 34 members belonging to two TGFβ subfamilies in Larimichthys crocea. These signaling molecules can affect growth rate and body shape through five different ligand-receptor-R-Smad signaling pathways. Among them, INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2 and GDF3-ACVR2A-ALK4-Smad2 signaling axes play a leading role in regulating the body shape of Larimichthys crocea.

Claims

1. A gene combination related to the body shape of Larimichthys crocea, characterized in that, The gene combination comprises INHBB gene, Nodal gene, GDF3 gene, ACVR2A gene, ALK4 gene and Smad2 gene.

2. The gene combination related to the body shape of Pseudosciaena crocea according to claim 1, characterized in that, The gene combination is INHBB-ACVR2A-ALK4-Smad2, Nodal-ACVR2A-ALK4-Smad2 and GDF3-ACVR2A-ALK4-Smad2.

3. The gene combination related to the body shape of Pseudosciaena crocea according to claim 1, characterized in that, The INHBB gene encodes a protein with an amino acid sequence of SEQ ID NO: 1; the Nodal gene encodes a protein with an amino acid sequence of SEQ ID NO: 2; the GDF3 gene encodes a protein with an amino acid sequence of SEQ ID NO: 3; the ACVR2A gene encodes a protein with an amino acid sequence of SEQ ID NO: 4; the ALK4 gene encodes a protein with an amino acid sequence of SEQ ID NO: 5; and the Smad2 gene encodes a protein with an amino acid sequence of SEQ ID NO:

6.

4. The gene combination related to the body shape of Pseudosciaena crocea according to claim 1, characterized in that, The INHBB gene encodes a CDS with a nucleic acid sequence of SEQ ID NO: 7; the Nodal gene encodes a CDS with a nucleic acid sequence of SEQ ID NO: 8; the GDF3 gene encodes a CDS with a nucleic acid sequence of SEQ ID NO: 9; the ACVR2A gene encodes a CDS with a nucleic acid sequence of SEQ ID NO: 10; the ALK4 gene encodes a CDS with a nucleic acid sequence of SEQ ID NO: 11; and the Smad2 gene encodes a CDS with a nucleic acid sequence of SEQ ID NO:

12.

5. Use of the gene combination related to body shape of Pseudosciaena crocea in claim 1-4 as a molecular marker for screening Pseudosciaena crocea with different body shapes.

6. A reagent for detecting the expression amount of each gene in the combination of genes related to the body shape of Pseudosciaena crocea according to any one of claims 1 to 4, characterized by, The preparation is a PCR amplification primer.

7. A product for screening different body shapes of large yellow croaker, characterized in that, The product is a fluorescent quantitative PCR amplification detection kit, and the product screens Pseudosciaena crocea with different body shapes by detecting the expression amount of each gene in the gene combination related to body shape of Pseudosciaena crocea in claim 1-4.

8. A method for screening different body shapes of large yellow croaker, characterized in that, The method screens Pseudosciaena crocea individuals by detecting the expression amount of genes in the gene combination in claim 1-4.

9. The method for screening different body shapes of large yellow croaker according to claim 8, characterized in that, Screen individuals with low expression amount of INHBB gene, Nodal gene, GDF3 gene, ACVR2A gene, ALK4 gene and Smad2 gene.

10. The method for screening different body shapes of Pseudosciaena crocea according to claim 8, wherein, The method is detected by a fluorescent quantitative PCR amplification method; wherein, a primer pair for detecting the INHBB gene, the sequences of the upstream and downstream primers are SEQ ID NO: 13 and SEQ ID NO: 14; a primer pair for detecting the Nodal gene, the sequences of the upstream and downstream primers are SEQ ID NO: 15 and SEQ ID NO: 16; a primer pair for detecting the GDF3 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 17 and SEQ ID NO: 18; a primer pair for detecting the ACVR2A gene, the sequences of the upstream and downstream primers are SEQ ID NO: 19 and SEQ ID NO: 20; a primer pair for detecting the ALK4 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 21 and SEQ ID NO: 22; a primer pair for detecting the Smad2 gene, the sequences of the upstream and downstream primers are SEQ ID NO: 23 and SEQ ID NO: 24.

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