Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1–28 aa), a transforming growth factor-β (TGF-β) propeptide (38–272 aa), and a mature TGF-β peptide (314–416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.


Introduction
The transforming growth factor β (TGF-β) family is a multifunctional group of secretory signal transducers that are involved in cell proliferation, differentiation, apoptosis, embryonic development, growth, bone formation and reconstruction, and other important biological processes [1][2][3][4][5]. The TGF-β superfamily includes TGF-βs, growth differentiation factors (GDFs), bone morphogenetic proteins (BMPs), Inhibins/Activins, and Nodal. All members of the TGF-β superfamily release mature polypeptides by division of precursors at specific sites, and the activity relies on the formation of dimers from the same or different subunits [6,7]. Although the TGF-β family contains many diverse factors, they all signal via conserved signaling pathways. The TGF-β family ligands bind to receptors and then mediate the phosphorylation of Smad proteins. The interaction between Smads and other signaling proteins mediates regulatory signals, controlling the expression of target genes, the translation of mRNA translation, and the regulation of cytoplasmic protein [8][9][10][11]. In recent years, the TGF-β superfamily has been found to play a critical role in the growth and development of shellfish [7,[12][13][14].
BMPs, as the largest secreted cytokines in the TGF-β superfamily, are originally named for their ability in bone formation [15]. BMPs are widely expressed during the development of mammals, with many biological activities, such as development, proliferation,

Sample Collection
All abalones (H. discus hannai and Lvpan abalone) used in this study were obtained from Fuda Aquaculture and were consistent with the previous study [43]. The adductor muscles were collected in different stages of development of H. discus hannai, that is, at 1, 4, 7, 10, 12, 14, 16, and 18 months. All experimental samples used to analyze the expression pattern in tissues were consistent with the previous study [43]. The adductor muscle tissue samples of the larger H. discus hannai ("L-DD-group"), the smaller H. discus hannai ("S-DD-group"), the larger Lvpan abalone ("L-DF-group"), and the smaller Lvpan abalone ("S-DF-group") were also consistent with the previous study [43]. All the samples were stored at −80 • C.

Cloning and Sequence Analysis
The isolation of total RNA and the synthesis of cDNA followed the methods in the previous study by Huang et al. (2023) [43]. The primers (hdh-BMP7-F and hdh-BMP7-R), used to amplify the hdh-BMP7 open reading frame (ORF), were designed using the software of Primer3Plus (Table 1). The ORF sequences of hdh-BMP7 were uploaded to GenBank (accession numbers: OP856631). Lasergene software tools were used to determine the amino acid sequence of hdh-BMP7. SingalP 5.0 Server was used for signal peptide prediction; Prop 1.0 server was used for proteolytic processing site analysis; SOPMA was used for secondary structure analysis; CDD was used for protein domains; the ClustalW2 program was used for aligning the sequence of BMP7, and the MEGA program with the neighbor-joining algorithm was used for constructing the phylogenetic tree.

Single Nucleotide Polymorphism Analysis
To explore the SNPs of hdh-BMP7, the abalones, the foot muscle samples, the method of DNA extraction, the growth-related traits, and the method of hdh-BMP7 ORF sequence genotype were consistent with the previous study [43]. The POPGENE 1.32 software was used to evaluate the PIC of the SNPs according to the manufacturer's instructions.

RNA Interference of hdh-BMP7
A pair of specific primers, dsBMP7-F and dsBMP7-R, were designed to amplify a 303 bp fragment of the hdh-BMP7. EGFP acted as a control gene, amplifying a 497 bp fragment. The information of specific primers is shown in Table 1. The dsRNA synthesis and purity were performed as previously described [47]. For the hdh-BMP7 RNAi assays, approximately 80 H. discus hannai (~4-5 g) were randomly divided into two groups: the hdh-BMP7 RNAi group and the EGFP control group. The abalones were intramuscularly injected. The RNAi experimental methods were consistent with the previous study [43].

Real-Time Quantitative Reverse Transcription PCR
The qRT-PCR was used to detect the gene expression levels of hdh-BMP7 (gene ID: evm.model.scaffold76053. 25 The data processing used the 2 −∆∆CT method. The qRT-PCR runs and the cycling parameters used were performed using similar conditions as described previously [48]. The PCR amplification was performed in 20 µL reaction mixtures containing the following components: 10 µL FastStart Universal SYBR Green Master (ROX), 1 µL forward and reverse primers (10 µM each), 5 µL of cDNA (100-times diluted), and 4 µL distilled water [29].

Statistical Analysis
All experimental data in this study were analyzed using one-way ANOVAs (ANOVA) in SPSS 19.0 (IBM, Tulsa, OK, USA). The significance level for these analyses was specified at p < 0.05.
All experimental data in this study were analyzed using one-way ANOVAs (ANOVA) in SPSS 19.0 (IBM, Tulsa, OK, USA). The significance level for these analyses was specified at p < 0.05.

Expression Detection of Hdh-BMP7
The expression levels of hdh-BMP7 in tissue and developmental stage-specific abalones were determined by qRT-PCR. The highest expression levels of hdh-BMP7 were found

Expression Detection of Hdh-BMP7
The expression levels of hdh-BMP7 in tissue and developmental stage-specific abalones were determined by qRT-PCR. The highest expression levels of hdh-BMP7 were found

Expression Detection of Hdh-BMP7
The expression levels of hdh-BMP7 in tissue and developmental stage-specific abalones were determined by qRT-PCR. The highest expression levels of hdh-BMP7 were found in the gills and cerebral ganglion (p < 0.05, Figure 4a). Hdh-BMP7 mRNA was widely expressed at different developmental stages of H. discus hannai (Figure 4b). The expression of hdh-BMP7 mRNA was lower in the S-DD-group than in the L-DD-group (p < 0.01, Figure 4c). in the gills and cerebral ganglion (p < 0.05, Figure 4a). Hdh-BMP7 mRNA was widely expressed at different developmental stages of H. discus hannai (Figure 4b). The expression of hdh-BMP7 mRNA was lower in the S-DD-group than in the L-DD-group (p < 0.01, Figure 4c).

SNPs in Hdh-BMP7
Sequence comparisons detected eight single nucleotide polymorphisms (SNPs) in hdh-BMP7 ( Table 2). The average Polymorphic information content (PIC) was 0.147, which is considered a low polymorphism level (PIC < 0.25). Four SNPs (744 A > G, 819 A > G, 834 T > A, and 852 C > G) were related to growth traits in H. discus hannai. The MW/TW of AA was higher than that of abalones with the genotype of GG at the 744 A > G locus (p < 0.05). The MW/TW of AA was higher than that of abalones with the GG and GA genotype at the 819 A > G locus (p < 0.05). The MW/TW of TT was lower than that of abalones with the AT genotype at the 834 T > A locus (p < 0.05). The MW/TW of CC was higher than that of abalones with the genotype of GG at the 852 C > G locus (p < 0.05).

SNPs in Hdh-BMP7
Sequence comparisons detected eight single nucleotide polymorphisms (SNPs) in hdh-BMP7 ( Table 2). The average Polymorphic information content (PIC) was 0.147, which is considered a low polymorphism level (PIC < 0.25). Four SNPs (744 A > G, 819 A > G, 834 T > A, and 852 C > G) were related to growth traits in H. discus hannai. The MW/TW of AA was higher than that of abalones with the genotype of GG at the 744 A > G locus (p < 0.05). The MW/TW of AA was higher than that of abalones with the GG and GA genotype at the 819 A > G locus (p < 0.05). The MW/TW of TT was lower than that of abalones with the AT genotype at the 834 T > A locus (p < 0.05). The MW/TW of CC was higher than that of abalones with the genotype of GG at the 852 C > G locus (p < 0.05).

Effects of RNA Interference
The expression levels of hdh-BMP7 in the adductor muscle decreased about 40-50% after RNAi experiment at 1 day, 15 days, and 30 days (p < 0.01, Figure 5a) compared to the EGFP control group. After RNAi experiment for 1 day, hdh-BMPR I and myosin heavy chain (hdh-MHC) mRNA levels were significantly lower in the hdh-BMP7 RNAi group than in the EGFP control group (Figure 5b,e). After RNAi experiment for 15 and 30 days, hdh-BMPR I, hdh-BMPR II, hdh-Smad1, and hdh-MHC mRNA levels were lower in the hdh-BMP7 RNAi group compared with the EGFP control group (p < 0.01, Figure 5). After RNAi experiment for 30 days, the decrease in shell length, shell width, and total weight in the hdh-BMP7 RNAi group was significantly higher than in the EGFP control group (p < 0.05, Figure 6a-c). After RNAi experiment for 30 days, the decrease in shell length, shell width, and total weight in the hdh-BMP7 RNAi group was significantly higher than in the EGFP control group (p < 0.05, Figure 6a-c).

Verification of the Lvpan Abalone
In order to further verify the function of BMP7 in abalone, we compared the expression level of BMP7 mRNA between H. discus hannai (DD) and Lvpan abalone (DF). The expression of df-BMP7 mRNA in Lvpan abalone was lower in the S-DF-group than in the L-DF-group (p < 0.05, Figure 7). In addition, the expression of BMP7 mRNA in the H. discus hannai (DD-group) was lower than in the Lvpan abalone (DF-group) (p < 0.05).

Discussion
BMP7 is one of the members of the TGF-β superfamily. In this paper, we cloned the ORF frame of the BMP7 from H. discus hannai. We found that the hdh-BMP7 protein contained 416 amino acid residues encoded by 1251 nucleotides. Our results also demonstrate that hdh-BMP7 has a TGF-β propeptide. The hdh-BMP7 protein has the same TGF-β func-

Verification of the Lvpan Abalone
In order to further verify the function of BMP7 in abalone, we compared the expression level of BMP7 mRNA between H. discus hannai (DD) and Lvpan abalone (DF). The expression of df-BMP7 mRNA in Lvpan abalone was lower in the S-DF-group than in the L-DF-group (p < 0.05, Figure 7). In addition, the expression of BMP7 mRNA in the H. discus hannai (DD-group) was lower than in the Lvpan abalone (DF-group) (p < 0.05).

Verification of the Lvpan Abalone
In order to further verify the function of BMP7 in abalone, we compared the expression level of BMP7 mRNA between H. discus hannai (DD) and Lvpan abalone (DF). The expression of df-BMP7 mRNA in Lvpan abalone was lower in the S-DF-group than in the L-DF-group (p < 0.05, Figure 7). In addition, the expression of BMP7 mRNA in the H. discus hannai (DD-group) was lower than in the Lvpan abalone (DF-group) (p < 0.05).

Discussion
BMP7 is one of the members of the TGF-β superfamily. In this paper, we cloned the ORF frame of the BMP7 from H. discus hannai. We found that the hdh-BMP7 protein contained 416 amino acid residues encoded by 1251 nucleotides. Our results also demonstrate that hdh-BMP7 has a TGF-β propeptide. The hdh-BMP7 protein has the same TGF-β func-

Discussion
BMP7 is one of the members of the TGF-β superfamily. In this paper, we cloned the ORF frame of the BMP7 from H. discus hannai. We found that the hdh-BMP7 pro-tein contained 416 amino acid residues encoded by 1251 nucleotides. Our results also demonstrate that hdh-BMP7 has a TGF-β propeptide. The hdh-BMP7 protein has the same TGF-β functional domain as other BMP members, which is required for the BMP ligand to be functional [49]. Hdh-BMP7 has seven conserved cysteine residues, which is consistent with the BMP7 in Meretrix meretrix [50]. However, not all mollusks have seven highly conservative cysteine residues, such as Sinonovacula constricta [7] and T. granosa [37]. Highly conserved cysteine residues are the structural characteristics that are typical of the TGF-β superfamily. Therefore, the data indicate that hdh-BMP7 is a member of the BMP family [51]. The result of the phylogenetic tree suggests that H. discus hannai is most closely related to these shellfish of T. granosa, M. coruscus, P. imbricata, and C. gigas.
BMPs are widely expressed during the development of mammals, with many biological activities [7,16]. BMP7 is a member of the BMPs. To explore the potential roles of BMP7 in H. discus hannai, we analyzed the expression of hdh-BMP7 mRNA in various tissues of individuals in different development stages. The qRT-PCR results revealed that hdh-BMP7 was widely expressed in the cerebral ganglion, gills, mantle, and other tissues, which is consistent with results previously found in zebrafish [52], M. meretrix [50] and T. granosa [37]. Previous studies have suggested that the BMP system plays a role in pituitary function in vertebrates [53], and BMP7 is mainly involved in shell growth and nacre formation [7,37,50]. Thus, our results of tissues distribution suggest that hdh-BMP7 may be related to shell growth and shell formation and play an important role in the nervous system. In addition, the hdh-BMP7 mRNA was widely expressed in different developmental stages of abalone, and its expression level was higher in the L-DD-group than in the S-DD-group. Thus, we speculate that hdh-BMP7 may be related to the growth and development of H. discus hannai.
SNP is an important molecular marker, widely divided in the genome of organisms. The SNPs in candidate genes were analyzed to find the loci related to the target trait, which helps to effectively screen individuals. For example, in Haliotis diversicolor supertexta, a total of nine SNP sites were identified in the MSTN gene. The association analysis of SNP sites and growth traits showed that the SNP site g909C > T in the coding region of the MSTN gene in H. diversicolor supertexta was significantly correlated with the shell length, shell height, and body weight [54]. In the myostatin gene of H. discus hannai, nine SNPs were significantly associated with growth traits [43]. In previous studies, BMP7 has been identified as a potential growth-related gene in SNP studies in bovines [33] and chickens [34] but not in abalone. In order to explore the SNPs and growth correlation of the BMP7 gene of H. discus hannai, hdh-BMP7 was experimentally measured by direct sequencing of PCR products, and the association between hdh-BMP7 SNPs and five growthrelated traits (shell length, shell width, total weight, muscle weight, and muscle weight/wet weight ratio) was analyzed. These data identified eight SNPs in the CDS region of hdh-BMP7, four of which were significantly associated with growth traits, which suggests that hdh-BMP7 is closely correlated with growth traits of abalone. Previous research has shown that BMP signaling is the dominant pathway controlling muscle mass and the inhibition of BMP signaling causes muscle atrophy [35]. Overexpression of BMP7 promotes muscle growth and prevents muscle atrophy [36]. In our study, the higher ratio of MW/TW means that the adductor muscle contributes to a large part of the total weight, which suggests that hdh-BMP7 also may be related to the abalone muscle growth. There is a strong correlation between BMP7 and the growth of shellfish such as S. constricta [7], M. meretrix [50], and T. granosa [37]. Therefore, we speculate hdh-BMP7 may play a crucial role in the process of growth regulation of H. discus hannai. In this study, only four SNP sites related to growth traits were found in the CDS region. In order to provide more useful molecular markers for breeding, we plan to screen SNP in the intron and promoter regions next. Meanwhile, we will further expand the abalone samples to verify the accuracy of SNP.
Although there are some reports on functional genes related to abalone growth, most of these studies use high-throughput sequencing technology to mine relevant genes and pathways and lack further functional verification, or the functional verification only stays at the level of cloning and mRNA expression, and the in-depth study on the mechanism of abalone functional gene is still very lacking. The technology of RNAi refers to the phenomenon of the gene silencing by foreign dsRNAs at the mRNA level. At present, researchers often use RNAi to reveal the genetic function of aquatic animals, such as H. discus hannai [43], P. martensii [38], etc. To further investigate the interaction between the BMP system and growth in abalone, we successfully inhibited the expression of hdh-BMP7 by RNAi, which resulted in a significant decrease in shell length growth gain, shell width growth gain, and total weight gain compared with the EGFP control group. This further demonstrates that hdh-BMP7 can promote abalone growth. In vertebrates, BMPs can act as regulators of myoblasts and facilitate the development and regeneration of myoblasts after muscle injury [55,56]. Overexpression of BMP7 or ALK3 promotes muscle growth and prevents muscle atrophy [35,36]. A previous study [57] suggested that BMP signaling is a positive regulator of muscle mass in animals. In this study, the ratio of MW/TW was significantly different in several SNP loci. Muscle is the main edible part of abalone, accounting for about half of the body weight. The growth of muscle directly affects the overall growth. Some studies have investigated the mechanism of BMP7 in growth regulation on shellfish [7,37,50], although no reports have been made about abalone. In a future study, we will conduct more long-term RNAi experiments and detect the morphological change of abalone muscle fibers, so as to further reveal the regulatory function of BMP7 on regulating abalone muscle growth.
As we all know, BMPs initially bind to two BMP receptors, BMPR I and BMPR II, which activate Smads and target genes [58]. The interaction between Smads and other proteins mediates signals, controlling the expression of target genes, the translation of mRNA translation, and the regulation of cytoplasmic protein [8][9][10][11]. Previous studies have reported that TGF-β signaling was transduced from the extra-cellular space to the cell nucleus via Smad proteins to activate downstream pathways and regulate general life processes [59,60]. In Scylla paramamosain, BMP7 also obeys this signaling mechanism [15]. In this study, when hdh-BMP7 expression was inhibited, the expression levels of hdh-BMPR I and hdh-BMPR II changed in response, which indicates that hdh-BMPR I and hdh-BMPR II are the receptors of hdh-BMP7 in abalone. In addition, the expression levels of hdh-smad1 and hdh-MHC were significantly decreased (p < 0.01) compared to the EGFP control group at 15 days and 30 days, suggesting that hdh-BMP7 may promote muscle growth by hdh-Smad1 signaling in H. discus hannai.
In the end, we verified the role of BMP7 in Lvpan abalone. Lvpan abalone, a hybrid with fast growth, has become a good material for proving the mechanism related to abalone growth. In this research, we compared the expression level of BMP7 mRNA between H. discus hannai (DD) and Lvpan abalone (DF). We found that the expression in L-group DD and DF at the same age was higher than that in S-group equivalents. The BMP7 expression level in the DF was higher than that in DD at the same age, which further indicates that BMP7 plays a positive role in regulating abalone growth. Ultimately, our data indicate that hdh-BMP7 potentially impacts the growth of H. discus hannai.

Conclusions
In the study, we cloned the ORF sequence and described the structure of hdh-BMP7 from H. discus hannai. The expression level of hdh-BMP7 mRNA was higher in the fastgrowing group (p < 0.05) than in the slow-growing group, suggesting that it may be correlated with the growth of abalone. Further results from RNAi indicated that the hdh-BMP7 gene plays a vital role in promoting growth of H. discus hannai. Association analysis identified four SNPs that were significantly associated with growth traits, suggesting that hdh-BMP7 is closely involved in abalone growth. The results of this paper would lay a foundation for further study on the function of hdh-BMP7 in abalone growth and contribute to molecular marker assisted breeding in H. discus hannai.