Journal of Oceanology and Limnology   2019, Vol. 37 issue(4): 1333-1341     PDF
Institute of Oceanology, Chinese Academy of Sciences

Article Information

WANG Qian, CUI Zhongkai, GUO Hua, ZHANG Nianwei, XU Wenteng, YANG Yingming, CHEN Songlin
Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis
Journal of Oceanology and Limnology, 37(4): 1333-1341

Article History

Received Jul. 1, 2018
accepted in principle Aug. 14, 2018
accepted for publication Sep. 6, 2018
Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis
WANG Qian1,2, CUI Zhongkai1,2, GUO Hua1,2, ZHANG Nianwei1,2, XU Wenteng1,2, YANG Yingming1,2, CHEN Songlin1,2     
1 Key Lab of Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266273, China
Abstract: The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sex-determining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1; functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1-Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.
Keywords: Cynoglossus semilaevis    dmrt1    promoter    transcription regulation    luciferase assay    gonad    

dmrt1 (doublesex and mab-3 related transcription factor 1), first isolated from the human testis cDNA library, is homologous to the Drosophila sex regulatory gene doublesex and the nematode worm sex regulator mab-3, both of which contain a zinc finger-like DNA-binding motif (DM domain) (Raymond et al., 1998). With expression mainly in the developing gonads of male embryos, dmrt1 is evolutionarily conserved among metazoan species including mammals, birds, reptiles and fishes, and plays an essential role in testis differentiation and development (Raymond et al., 1999, 2000; Jørgensen et al., 2008; Smith et al., 2009; Yoshimoto and Ito, 2011; Zhao et al., 2015).

In most mammals that employ an XX/XY sex-determining system, the Sry gene on the Y chromosomes triggers the male sex determination, while dmrt1 is dispensable in the sex determination process but maintains Sertoli cell in postnatal testis instead (Gubbay et al., 1990; Sinclair et al., 1990; Zhao et al., 2015). Nevertheless, in several non-mammalian vertebrates, such as chicken and frog with a ZZ/ZW system, dmrt1 is mapped to the Z chromosome and considered to be a male sex-determining gene (Koopman, 2009; Yoshimoto and Ito, 2011; Lambeth et al., 2014). Intriguingly, in a teleost fish medaka (Oryzias latipes) that uses an XX/ XY system, the duplicated copy of dmrt1 on the Y chromosome named DMY/Dmrt1Y is served as a male determinant (Matsuda et al., 2002; Nanda et al., 2002). DMY/Dmrt1Y is exclusively expressed in the somatic cells surrounding the germ cells in XY embryos during early gonadal formation and persists in a Sertoli cell in mature testis (Kobayashi et al., 2004). Over-expression of this particular gene can induce female-to-male sex-reversal (Matsuda et al., 2007), and knocking-down/out can result in fertile male-to-female sex-reversal (Paul-Prasanth et al., 2006; Luo et al., 2015; Chakraborty et al., 2016). In addition, a sexually dimorphic expression pattern of dmrt1 has been detected mainly during testicular differentiation in male individuals in many other fish species such as rainbow trout, zebrafish, orange-spotted grouper and tilapia (Guan et al., 2000; Marchand et al., 2000; Xia et al., 2007; Jørgensen et al., 2008). However, functional evidence of this gene in fish is still rare so far.

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine flatfish that is widely distributed in Chinese coastal waters, and the body length and weight of adult females are two to four times that of males (Chen et al., 2009; Ji et al., 2011). Thus, studies of sex determination in C. semilaevis will have both theoretical and economic significance. Our previous studies in C. semilaevis have suggested that it has ZZ/ZW sex chromosome system and dmrt1 is located on the Z chromosome with a corrupted pseudogenized copy on the W chromosome (Zhou et al., 2005; Chen et al., 2009, 2014). The C. semilaevis dmrt1 is highly expressed in the germ cells and pre-somatic cells of the undifferentiated gonad at the "sex-determining stage" (approximately 60-dph) and persists at high levels during testis development in the male individual. In the sex-reversed ZW males (genotype ZW) induced by high temperature, this gene exhibited similar expression level to normal male (ZZ) (Chen et al., 2014; Shao et al., 2014). Furthermore, knocking-out of dmrt1 in ZZ male using the TALENs technique causes an abnormal testis and disrupted spermatogenesis, and the dmrt1-deficient males show accelerate growth characteristics compared to wild-type males (Cui et al., 2017). All of these observations support the view that dmrt1 is a male determining gene in C. semilaevis, which might function in a dosage-dependent manner. Therefore, investigation of the promoter of this gene could provide further information for understanding its sex-related regulation mechanism. Besides, identifying of this promoter could supply a suitable regulatory element to drive interesting gene expression through a transgenic approach in the C. semilaevis.

In this paper, we cloned the promoter sequence of C. semilaevis dmrt1, which included 5′-upstream sequence and partial exon1. To investigate the potential role of regulatory elements in gene expression, we have constructed truncated promoters and analyzed their promoting activity. The results showed that there were two positive regulatory regions in the promoter. Furthermore, we combined these positive regions to construct a new promoter, in vitro analysis showed its high driving activity (3.25 times versus that of the full length promoter), and in vivo analysis confirmed its improved promoting capability for exogenous gene expression in tongue sole gonads.

2 MATERIAL AND METHOD 2.1 Material 2.1.1 Ethics statement

The collection and handling of the C. semilaevis used in this study were approved by the Animal Care and Use Committee at the Chinese Academy of Fishery Sciences, and all the experimental procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals at the Chinese Academy of Fishery Sciences.

2.1.2 Experimental fish maintenance

The C. semilaevis were kept in the Haiyang High-Tech Experimental Base (Haiyang, China). The fish were fed twice a day with commercial pellets and were reared in filtered seawater at 22–23 ℃.

2.2 Method 2.2.1 Acquisition and analysis of the C. semilaevis dmrt1 promoter sequence

Cynoglossus semilaevis genomic DNA was extracted from caudal fins using a TIANamp Marine Animals DNA Kit (TIANGEN, China). The dmrt1 promoter was isolated using specific primers (dmrt-F/R, Table 1) designed according to the archived dmrt1 mRNA sequence (GenBank: EU070761.1) and the assembled C. semilaevis Z chromosome sequence (GenBank: NC_024328.1). The PCR cycling conditions were: 5 min initial denaturation at 94 ℃; 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 1.5 min; and a final 10 min extension at 72 ℃. The 2 935 kb PCR product was sub-cloned into the pEASY-T1 cloning vector (Transgene, China), transformed into Trans1-T1 cells (Transgene, China) and sequenced.

Table 1 Sequences of primers

The putative transcriptional binding sites in the promoter sequences of dmrt1 were predicted using the TFBIND online software ( (Tsunoda and Takagi, 1999). To analyze the similarity of dmrt1 promoter among species, the corresponding sequence of zebrafish, medaka, frog, rat, and human were downloaded from the NCBI database and the multiple sequence alignment was conducted by the MAFFT online tool (

2.2.2 Construction of the luciferase transfection vectors

To evaluate the promoting activity of regulatory elements of dmrt1, six promoter-luciferase plasmids were constructed by serial-deletion, according to the distribution of predicted positive/negative transcription factors (Fig. 1). The primers used were listed in Table 1, with a Kpn Ⅰ and Xho Ⅰ recognition site added to the 5′ end of each forward and reverse primer, respectively. Purified PCR products were digested and cloned into the Kpn Ⅰ/Xho Ⅰ site of the pGL3-Basic vector (Promega, CA, USA) which contains a firefly luciferase gene. In addition, pGL-dmrt1-1 was digested to construct pGL-dmrt1-4 and pGL-dmrt1-6 with Kpn Ⅰ/Spe Ⅰ and Kpn Ⅰ/Pst Ⅰ, respectively, followed by end-blunting using T4 polymerase (TaKaRa, Dalian, China) and self-ligation with T4 ligase (Promega, WI, USA) (sketched in Fig. 2).

Fig.1 Nucleotide sequence of C. semilaevis dmrt1 promoter region and the potential transcription factor binding sites a. schematic map of the C. semilaevis dmrt1 promoter. The box represented the exon1. The numbers above the line indicated the positions (the first base of ATG was set as +1); b. nucleotide sequence of the C. semilaevis dmrt1 promoter and its potential functional components. Underlined boldface letters showed the CAAT box and TATA box. Putative positive factors were underlined in red, negative factors in green, neutral factors in purple, the ATG was in bold. (-) represented the location on the complementary chain.
Fig.2 Luciferase assays of the C. semilaevis dmrt1 truncated vectors The left panel showed the schematic map of the dmrt1 truncated promoter-luciferase plasmids and the effects of each region. The triangle, inverted triangle or circle represented the up-regulated, down-regulated or neutral effect of the corresponding region. The right panel exhibited the luciferase activities of corresponding truncated promoters. Statistical differences were indicated as different letters.

For pGL-dmrt1-Δ3 construction, dmrt-F and dmrt-Δ3-R (Table 1) were used to amplify the -2 932/-1 928 dmrt1 promoter fragment and Kpn Ⅰ and Spe Ⅰ were used to delete the -2 932 to -1 326 sequence of pGL-dmrt1-1. Thus, the -2 932/-1 928 PCR product was ligated to the deleted pGL-dmrt1-1 to form pGL-dmrt1-Δ3. For pGL-dmrt1-Δ4 and pGL-dmrt1-Δ3+Δ4 constructions, dmrt-F/dmrt-Δ4-R and dmrt-F/dmrt-Δ3 +Δ4-R primers (Table 1) were designed to amplify the -2 932/-1 326 and -2 932/-1 928 dmrt1 promoter fragments, respectively, similarly to the pGL-dmrt1-Δ3 formation. The two PCR products were digested by Kpn Ⅰ and then ligated separately to the pGL-dmrt1-5 vector (-867/+3) (sketched in Fig. 3). All plasmids were prepared using an EndoFree Plasmid Mini Kit (CWBIO, Beijing, China).

Fig.3 Luciferase assays of the new-constructed C. semilaevis dmrt1 promoters The left panel showed the schematic map of the region-deleted promoter-luciferase plasmids. The numbers above indicated the positions. The right panel exhibited the luciferase activities of corresponding promoters. Statistical differences were indicated as different letters.
2.2.3 Cell culture

Human embryonal kidney (HEK) 293T cells were routinely cultured in DME/F-12 medium (HyClone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Australia) and recombinant human FGF (Invitrogen, MD, USA) and maintained in 5% CO2 at 37 ℃.

2.2.4 Transfection and luciferase assay

Transfection experiments were performed in 24-well culture plates. For the dual-luciferase assay, the cells were co-transfected with 800 ng of promoter-luciferase constructs and 40 ng of pRL-TK plasmid (Renilla luciferase gene driven by HSV thymidine kinase promoter) using Lipofectamine 2000 Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. The pGL-3 Basic vector was used as negative control. At 48 hours post-transfection, cells were lysed and the activities of Firefly and Renilla luciferase were measured using dual-luciferase reporter gene assay kit (Beyotime, Shanghai, China) with a Varioskan Flash spectral scanning multimode reader (Thermo, Vantaa, Finland). Each experiment was performed in triplicate.

2.2.5 In vivo analysis of promoters

To test whether candidate dmrt1 promoters could drive the expression of reporter gene luciferase in C. semilaevis in vivo, plasmids pGL-dmrt1-4, pGL-dmrt1-Δ3, and pGL-dmrt1-Δ3+Δ4 were injected into C. semilaevis separately. Test C. semilaevis were divided randomly into 7 groups (n=3 per group) according to plasmids injected: PBS as blank control; pGL-3 Basic vector as a negative control; pGL-3 Control vector containing an SV40 promoter as positive control; pGL-dmrt1-1; pGL-dmrt1-4; pGL-dmrt1-Δ3 and pGL-dmrt1-Δ3+Δ4. Each group was kept in a separate 80 L tank for 2 days for acclimation. Then the injection was performed precisely into the gonad with a dose of 20 μg plasmid in a total volume of 40 μL with PBS (according to the preliminary experiments). At 48 h post injection, gonads of each C. semilaevis were sampled and stored in liquid nitrogen until RNA extraction.

2.2.6 qRT-PCR analyses of luciferase level

Total RNA of each C. semilaevis was extracted from gonads using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. After examined by agarose gel electrophoresis and quantified using NanoDrop 2000 Spectro-photometer (Thermo, USA), 2 μg RNA with DNA contamination removed was used to synthesis cDNA using FastQuant RT Kit (with gDNase) (TIANGEN, China). The amount of luciferase mRNA was determined by qRT-PCR using the 7500 Real-Time PCR System (ABI, USA). The 20 μL reactions contained 2× SYBR Premix Ex Taq, 2 pmol each primer (Luc-qF/R), 2 μL of 10× diluted cDNA templates and 0.4 μL of ROX Reference Dye. Each cDNA sample was amplified in triplicate with the cycling conditions: 95 ℃ for 30 s, and 40 cycles of 95 ℃ for 5 s and 60 ℃ for 30 s, followed by melt curve analysis. β-actin (β-actin-qF/R) was used as the internal control. Relative abundance of transcripts was calculated as R=2-ΔΔCt.

2.2.7 Statistical analysis

The results of the luciferase assay experiment were expressed as mean±SD. The values were compared by one-way ANOVA followed by Bonferroni's multiple comparison test using GraphPad Prism 6.0 (GraphPad, USA). Statistical significant differences were defined as P < 0.05.

3 RESULT 3.1 Acquisition and analysis of the C. semilaevis dmrt1 promoter sequence

The dmrt1 promoter sequence of 2 935 bp (-2 932/+3) was cloned from the C. semilaevis genomic DNA with specific primers dmrt-F/R (Table 1) and verified by sequencing. This sequence contained a 2 887-bp of a promoter sequence and a 48-bp of partial exon1. Intriguingly, although the coding region of dmrt1 gene had high identities among species, the promoter sequence showed low similarity to the same regions of zebrafish, medaka, frog, rat and human (data not shown). Prediction of putative transcriptional binding sites revealed that the promoter sequence harbored potential binding sites for multiple transcription factors including the TATA box and the inverted CAAT box, as well as positive transcription factors Sp1, Egr1, and HSF2; negative transcription factors CEBP, Sox5; and neutral factors Cdxa and Nkx2.5 (Fig. 1).

3.2 Luciferase assays of C. semilaevis dmrt1 regulatory regions

To explore the promoter activity of the isolated C. semilaevis dmrt1 sequence, we generated a set of truncated promoter-luc vectors according to the predicted transcription factor binding sites. Compared to the full-length promoter, four truncated fragments exhibited changes of promoter activities, either increased or decreased (P < 0.05), while pGL-dmrt1-6-luc showed similar activity (Figs. 1a, 2). The highest promoter activity was observed in pGL-dmrt1-4-luc (1.75 times of that of pGL-dmrt1-1-luc), while the lowest was in pGL-dmrt1-3-luc (39.14% of that of pGL-dmrt1-1-luc). Based on the results, we have made a pairwise comparison and classified the six regions into three categories by their regulatory behavior. Four of them showed positive effect including -2 932 to -2 485, -2 485 to -1 928, -867 to -285 and -285 to +3. One negative region was observed from -1 928 to -1 326, while region from -1 326 to 867 was rather neutral (Fig. 2).

3.3 Construction of new C. semilaevis dmrt1 promoter and in vitro activity analysis

To further analyze the regulatory role of the six regions mentioned above, precise deletion studies were performed to construct pGL-dmrt1-Δ3-luc, pGL-dmrt1-Δ4-luc and pGL-dmrt1-Δ3+Δ4-luc corresponding to a deletion of -1 928 to -1 326, -1 326 to -867, -1 928 to -867, respectively. Then their activities were analyzed in 293T cells (Fig. 3). Deletion of the negative region (-1 928 to -1 326) resulted in the up-regulated activity of pGL-dmrt1-Δ3-luc (P < 0.05), which confirmed the negative regulatory effect of this region. The specific deletion of the neutral region (-1 326 to -867) caused a slight but not significant increase, also consistent with the previous conclusion. The simultaneous deletion of the negative and neutral region (-1 928 to -867) significantly increased the activity, and the activity of the resulted of pGL-dmrt1-Δ3+Δ4-luc was ~3.25 times as that of pGL-dmrt1-1-luc (P < 0.05).

3.4 In vivo activity of newly constructed C. semilaevis promoter

The in vivo activities of pGL-dmrt1-1-luc, pGL-dmrt1-4-luc, pGL-dmrt1-Δ3-luc, and pGL-dmrt1-Δ3+Δ4-luc were tested by injecting them precisely into the C. semilaevis gonads. The qRT-PCR was performed to detect the expression of the luciferase gene. The result confirmed the successful in vivo driving activities of each newly constructed promoter, and the activity of dmrt1-Δ3+Δ4 was the highest among the tested constructs, which was 2.13 times of that of dmrt1-1 (P < 0.05) and 1.37 times of that of dmrt1-4 (P>0.05). Although dmrt1-4 and dmrt1-Δ3 performed higher activities than dmrt1-1, there was no statistical significant difference, which was different from the in vitro test (Fig. 4).

Fig.4 Expression of the luciferase gene in vivo after gonadal injection Data are shown as mean±SD of three independent samples. Statistical differences are indicated as different letters. pGL-3 Basic vector was used as negative control and pGL-3 Control vector was used as the positive control. Note: the error bar of the positive control was clipped due to the y-axis limit.

dmrt1 is a conserved transcription factor required for testis formation and somatic-cell masculinization in various vertebrate species (Yoshimoto and Ito, 2011; Luo et al., 2015; Zhao et al., 2015). Expression profiles of dmrt1 showed a strict specificity in germ cells and somatic cells of embryonic gonads and postnatal testis (Smith et al., 1999; Raymond et al., 2000; Shetty et al., 2002; Xia et al., 2007). In Chinese tongue sole, since an efficient protocol for microinjection of flatfish embryos have been developed recently (Cui et al., 2017), the research of sex determining the mechanism through a transgenic approach becomes possible. Now an essential step in developing transgenic C. semilaevis is to identify a suitable regulatory element to drive interesting gene expression. Here we focus on the promoter of dmrt1 mainly because (1) an endogenous promoter may be a better choice since its activity may be stronger in the native environment compared to an exogenous promoter (Friedenreich and Schartl, 1990; Sunilkumar et al., 2002), and (2) the expression profile of dmrt1 may provide a precise spatial and temporal control for target genes referred to sex-determining and growth.

The promoter region (-2 935 to +3) of C. semilaevis dmrt1 has been isolated by PCR. This region shows low similarity with other species, which is consistent with a recent research regarding the molecular evolution of dmrt1 promoters in a wide range of vertebrates including agnatha, bony fish, amphibian, reptile, bird, and mammal. There is no ncEx1 (non-coding first exon) in dmrt1 gene of C. semilaevis, as in the chicken and mice. It has been reported that species without an ncEx1 have evolved an intronic derived promoter to regulate both the somatic- and germ-cell expression of dmrt1 (Mawaribuchi et al., 2017). In addition, the C. semilaevis dmrt1 promoter has a TATA box located ~200 bp upstream of the transcriptional start site (TSS). A CAAT box, which is essential for the promoter activity, was also found at the location of 257 bp upstream of the TSS (Quitschke et al., 1989).

In the C. semilaevis dmrt1 promoter, we have predicted binding sites for multiple transcription factors. Among them, Sp1 and Egr1 have been reported as positive elements that are important for the dmrt1 promoter function. Sp1 zinc finger transcription factors expressed in a variety of cells and tissues play a critical role in the maintenance of differentiated cells (Marin et al., 1997; Cook et al., 1999). The Egr proteins are thought to be involved in cellular growth and differentiation in a variety of tissues and a previous research in mice showed that Egr1 had a sexually dimorphic function in male and female fertility (Topilko et al., 1998; Tourtellotte et al., 1999). HSF2 is most abundantly expressed in certain cell types of mice testis and the expression level is elevated during spermatogenesis. The increased HSF2 will induce the formation of heterotrimers with HSF1 and then provide a switch that integrates the transcriptional activation of their common target genes during the maturation of male germ cells (Åkerfelt et al., 2010). On the other hand, CEBP and Sox5 are thought to be negative regulators of the dmrt1 promoter. CEBP-α is a protein expressed in Sertoli cells and known to act as a transcriptional repressor (Slomiany et al., 2000; Lei and Heckert, 2002). Sox5 in zebrafish could bind the dmrt1 promoter and inhibit dmrt1 expression (Gao et al., 2005). These binding sites are useful for predicting the regulatory region in the C. semilaevis dmrt1 promoter.

To characterize the regulatory elements of the C. semilaevis dmrt1 promoter, we constructed a series of truncated promoter-luc vectors. The luciferase assay indicated that there were two positive regulatory regions, a distal regulatory region located at -2 932/-1 928 and a proximal region at -867/+3 (Fig. 2). The similar situation has been found in the rat dmrt1 promoter. It possesses a distal regulatory region -3.2 kb to -2.8 kb that is involved in Sertoli cell-specific expression of dmrt1 and proximal sequences between -1.3 kb to 400 bp that is for germ cell-specific expression of dmrt1 (Lei et al., 2009). However, we have demonstrated that there is no homology in the dmrt1 promoter sequence between the tongue sole and rat, thus the transcription elements of dmrt1 promoter should be investigated in future work.

Besides the two positive regulatory regions, there is a negative regulatory region (-1 928 to -1 326) and a region has no significant effect (-1 326 to -867). We have made separate and simultaneous deletion. The deletion of the negative regulation region (dmrt1-4 and dmrt1-Δ3) showed significantly higher promoter activity than the full-length promoter (dmrt1-1) in the in vivo test. For in vitro test, higher activities were also observed for dmrt1-4 and dmrt1-Δ3 compared to dmrt1-1, but the differences were not statistically significant. The reason may be the complex situation of in vivo experiment. The absorption and utilization of exogenous plasmids may be different among individuals and thus could affect the significance of the differences. Intriguingly, both in vivo and in vitro experiments illustrated that simultaneous deletion of the two regions (dmrt1-Δ3+Δ4) performed a significantly higher promoter activity (Figs. 3–4). The results suggest there may be an interaction between the two regions and together they have a higher negative effect. Through bioinformatics analysis, Sox5 and CEBP are most enriched elements located in the two regions. As the Sox5 can interact with CEBPs and cooperatively regulate the target genes involved in differentiation and development (Kfoury and Kapatos, 2009), we postulate that Sox5 and CEBP may be important elements for the negative regulatory effect. However, the hypothesis requires further investigation.


In summary, this study cloned the promoter sequence of C. semilaevis dmrt1 and functional analysis showed that there were two positive regulatory regions, one negative regulatory region, and one neutral regulatory region. Furthermore, we combined the positive regions and thus constructed several new promoters. After in vitro and in vivo analysis, one constructed promoter was confirmed with high driving activity and could drive reporter gene expression in the gonad. As the first report of the C. semilaevis dmrt1 promoter, our results will not only contribute to understanding the regulatory mechanism of the dmrt1 gene during C. semilaevis sex determination but also provide an endogenous promoter to facilitate the transgenic research for analyzing sex- and growth-related genes.


Based on previous studies, the dmrt1 promoter sequence data that support the findings of this study have been deposited in GenBank with the accession code NC_024328.1 (

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