Hanhua Cheng

Wuhan University, Wu-han-shih, Hubei, China

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Publications (49)154.61 Total impact

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    ABSTRACT: The swamp eel is a teleost fish with a characteristic of natural sex reversal and an ideal model for vertebrate sexual development. However, underlying molecular mechanisms are poorly understood. We report the identification of five DM (doublesex and mab-3) domain genes in the swamp eel that include Dmrt2, Dmrt2b, Dmrt3, Dmrt4 and Dmrt5, which encode putative proteins of 527, 373, 471, 420 and 448 amino acids, respectively. Phylogenetic tree showed that these genes are clustered into corresponding branches of the DM genes in vertebrates. Southern blot analysis indicated that the Dmrt1-Dmrt3-Dmrt2 genes are tightly linked in a conserved gene cluster. Notably, these Dmrt genes are up-regulated during gonad transformation. Furthermore, mRNA in situ hybridisation showed that Dmrt2, Dmrt3, Dmrt4 and Dmrt5 are expressed in developing germ cells. These results are evidence that the DM genes are involved in sexual differentiation in the swamp eel.
    Molecular Biology Reports 01/2014; · 2.51 Impact Factor
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    ABSTRACT: Loss and/or inactivation of VHL (the von Hippel Lindau tumour suppressor) cause various tumours. Using a yeast two-hybrid system, we have identified an AR co-activator, UXT, as a VHL-interacting protein. GST pull-down and co-immunoprecipitation assays show that UXT interacts with VHL. In addition, UXT recruits VHL to the nucleus. VHL associates with the DBD and hinge domains of the AR and induces AR ubiquitination. Moreover, VHL interaction with the AR activates AR transactivation upon DHT treatment. VHL knockdown inhibits AR ubiquitination and decreases transcriptional activation of the AR. Our data suggest that the VHL-UXT interaction and VHL-induced ubiquitination of AR regulate transcriptional activation of the AR.
    Biochemical Journal 08/2013; · 4.65 Impact Factor
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    ABSTRACT: Transfer RNAs (tRNAs) play an important role linking mRNA and amino acids during protein biogenesis. Four types of tRNA genes have been identified in living organisms. However, the evolutionary origin of tRNAs remains largely unknown. In this paper, we conduct a deep sequence analysis of diverse genomes that cover all three domains of life to unveil the evolutionary history of tRNA genes from tRNA halves. tRNA half homologs were detected in diverse organisms, and some of them were expressed in mouse tissues. Continuous tRNA genes have a conserved pattern similar to indels, which is, more closely flanking regions have higher single nucleotide substitution rates, while tRNA half homologs do not have this pattern. In addition, tRNAs tend to break into tRNA halves when tissues are incubated in vitro, the tendency of tRNA to break into tRNA halves may be a "side-effect" of tRNA genes evolving from tRNA halves. These results suggest that modern tRNAs originated from tRNA halves through a repeat element-mediated mechanism. These findings provide insight into the evolutionary origin of tRNA genes.
    Molecular Biology and Evolution 06/2013; · 10.35 Impact Factor
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    ABSTRACT: BACKGROUND INFORMATION: The translationally controlled tumor protein (TCTP) plays an important role in maintaining cell proliferation and its high expression is associated with many tumors. The tumor suppressor von Hippel-Lindau protein (VHL) has been shown to function as an E3 ubiquitin ligase. Although great progress has been made, biological roles of these factors and relevant molecular mechanisms remain largely unknown. RESULTS: In this study, we have shown that TCTP specifically binds to VHL through its β domain and competes with hypoxia-inducible factor-1α (HIF1α). TCTP overexpression decreased the protein level of VHL and the inhibition of TCTP expression by miRNA resulted in an increase of the VHL protein level. Moreover, TCTP overexpression promoted the K48-linked ubiquitination of VHL, thus degradation through the ubiquitin-proteasome pathway. In addition, we showed that TCTP increased the protein level of HIF1α, which promoted both VEGF (vascular endothelial growth factor)-HRE (hypoxic response element)-promoter-driven luciferase reporter and endogenous VEGF expression. CONCLUSIONS: These data have demonstrated that TCTP binds to the β domain of VHL through competition with HIF1α, which promotes VHL degradation by the ubiquitin proteasome system and HIF1α stability.
    Biology of the Cell 02/2013; · 3.49 Impact Factor
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    ABSTRACT: With an increasing incidence of male idiopathic infertility, identification of novel genes involved in spermatogenesis is an important aspect for the understanding of human testicular failure. In the present study, we have identified a novel gene Spata33, also called as 4732415M23Rik or C16orf55, which is conserved in mammalian species. Spata33 was predominantly expressed in the postpartum and adult mouse testes at mRNA and protein levels. Its expression was increased during the first wave of the spermatogenesis, indicating that Spata33 may be associated with the meiotic process. Further immunohistochemistry analysis revealed that Spata33 was mainly expressed in the spermatocytes, spermatogonia and round spermatids. Its expression was uniformly distributed in the nucleus and cytosol in these germ cells, which was further confirmed by Spata33-tagged with GFP staining in the GC-1 and TM4 cells. These results indicated that Spata33 was predominantly expressed in the mouse testis and associated with spermatogenesis. Identification and characterization of the novel testis-enriched gene Spata33 may provide a new route for understanding of spermatogenesis failure.
    PLoS ONE 01/2013; 8(7):e67882. · 3.73 Impact Factor
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    ABSTRACT: Gonad differentiation is one of the most important developmental events in vertebrates. Some heat shock proteins are associated with gonad development. Heat shock protein 70 (Hsp70) in the teleost fish and its roles in sex differentiation are poorly understood. We have identified a testis-enriched heat shock protein Hspa8b2 in the swamp eel using Western blot analysis and Mass Spectrometry (MS). Fourteen Hsp70 family genes were further identified in this species based on transcriptome information. The phylogenetic tree of Hsp70 family was constructed using the Maximum Likelihood method and their expression patterns in the swamp eel gonads were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). There are fourteen gene members in the Hsp70 family in the swamp eel genome. Hsp70 family, particularly Hspa8, has expanded in the species. One of the family members Hspa8b2 is predominantly expressed in testis of the swamp eel.
    PLoS ONE 01/2013; 8(6):e65269. · 3.73 Impact Factor
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    ABSTRACT: As an essential component of eukaryotic cells, the nuclear envelope (NE) plays a crucial role in many physiological processes. At present, a few membrane proteins from NE have been functional characterized. To determine whether the inner nuclear membrane (INM) protein Nurim is expressed in cancer cells with evidence of apoptosis, we identified three isoforms of this protein that are specific for human testicular seminoma and are generated by alternative splicing. We observed that Nurim is expressed in a broad range of cancer types and that its expression level is correlated with a higher tumor grade. Biochemical analysis showed that Nurim b, like a, is tightly bound to the nuclear envelope. Furthermore, knockdown using miR-Nurim resulted in an abnormal shape change of the nuclear envelope. Notably, Nurim knockdown obviously increased apoptosis induced by ultraviolet in HeLa cells. Together, these findings implicate that the INM protein Nurim plays an important role in the suppression of apoptosis.
    Current Molecular Medicine 10/2012; · 4.20 Impact Factor
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    ABSTRACT: Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type-specific expression of Miwi. CpG methylation at the USF-binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter-driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis.
    PLoS Genetics 05/2012; 8(5):e1002716. · 8.52 Impact Factor
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    ABSTRACT: Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry, however, a few genes have been identified in this species. We report here construction of a pituitary cDNA library from a 24 years old female Chinese sturgeon just after its spawning, and obtained 2,025 ESTs from the library. 885 unique sequences were identified, which were categorized into 12 functional groups. More than half of the unique sequences (57%) do not match with annotated sequences in the public databases. Three of these novel genes were further identified. Notably, a full-length of cDNA (1,143 bp) encoding somatolactin of 232 amino acids was identified. Phylogenetic analysis showed 97% amino acid identity with White sturgeon somatolactin. RT-PCR analysis indicated that the somatolactin mRNA was only detected in pituitary. Pituitary-specific expression of the somatolactin suggested that the protein may play important physiological functions in pituitary-endocrine system of the Chinese sturgeon.
    Molecular Biology Reports 04/2012; 39(4):4647-53. · 2.51 Impact Factor
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    ABSTRACT: Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ∼300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment.
    PLoS ONE 01/2011; 6(2):e17017. · 3.73 Impact Factor
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    ABSTRACT: Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3'end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2'-O-methylated at their 3' ends.
    Genetics 12/2010; 186(4):1175-85. · 4.39 Impact Factor
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    ABSTRACT: Androgen receptor (AR) plays a critical role in prostate cancer and male sexual differentiation. We have identified AR from a primitive vertebrate with a sex reversal characteristic, the rice field eel. AR of this species (eAR) is distinct from human AR, especially in the ligand binding domain (LBD), and its expression in gonads shows an increasing tendency during gonadal transformation from ovary via ovotestis to testis. eAR has a restricted androgen-dependent transactivation function after a nuclear translocation upon dihydrotestosterone exposure. A functional nuclear localization signal was further identified in the DNA binding domain and hinge region. Although nuclear export is CRM1-independent, eAR has a novel nuclear export signal, which is negatively charged, indicating that a nuclear export pathway may be mediated by electrostatic interaction. Further, our studies have identified critical sequences for ligand binding in the C terminus. A structure of three α-helices in the LBD has been conserved from eels to humans during vertebrate evolution, despite a distinct amino acid sequence. Mutation analysis confirmed that the LBD is essential for dihydrotestosterone-induced nuclear import of eAR and following transactivation function in the nucleus. In addition, eAR interacts with both Sox9a1 and Sox9a2, and their interaction regulates transactivation of eAR. Our data suggest that the primitive species conserves and especially acquires key novel domains, the nuclear export signal and LBD, for the eAR function in spite of a rapid sequence evolution.
    Journal of Biological Chemistry 11/2010; 285(47):37030-40. · 4.65 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: Androgen receptor (AR) plays a critical role in prostate cancer and male sexual differentiation. We have identified AR from a primitive vertebrate with a sex reversal characteristic, the rice field eel. AR of this species (eAR) is distinct from human AR, especially in the ligand binding domain (LBD), and its expression in gonads shows an increasing tendency during gonadal transformation from ovary via ovotestis to testis. eAR has a restricted androgen-dependent transactivation function after a nuclear translocation upon dihydrotestosterone exposure. A functional nuclear localization signal was further identified in the DNA binding domain and hinge region. Although nuclear export is CRM1-independent, eAR has a novel nuclear export signal, which is negatively charged, indicating that a nuclear export pathway may be mediated by electrostatic interaction. Further, our studies have identified critical sequences for ligand binding in the C terminus. A structure of three α-helices in the LBD has been conserved from eels to humans during vertebrate evolution, despite a distinct amino acid sequence. Mutation analysis confirmed that the LBD is essential for dihydrotestosterone-induced nuclear import of eAR and following transactivation function in the nucleus. In addition, eAR interacts with both Sox9a1 and Sox9a2, and their interaction regulates transactivation of eAR. Our data suggest that the primitive species conserves and especially acquires key novel domains, the nuclear export signal and LBD, for the eAR function in spite of a rapid sequence evolution.
    Journal of Biological Chemistry 11/2010; 285(47):37030-37040. · 4.65 Impact Factor
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    ABSTRACT: Dmrt1 (Dsx- and Mab3-related transcription factor-1), a conserved transcription factor in different phyla, is a key regulator in sex determination. Here, we report the novel ncRNA gene Dmr (Dmrt1-related gene), from mouse chromosome 5 that trans-splices with Dmrt1 from chromosome 19 to generate a Dmrt1 protein that lacks the C-terminus. Dmr is mouse and rat specific, and the surrounding genes are also conserved in both species. Dmr is alternatively spliced, and three isoforms, Dmr a, b and c, are detected in the testis. Further, Dmr serves mainly as a 3' UTR, promotes trans-splicing and down-regulates the Dmrt1 protein. These results suggest that Dmr might play a negative regulatory role for Dmrt1 in male sexual development.
    Biochemical and Biophysical Research Communications 10/2010; 400(4):696-700. · 2.41 Impact Factor
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    ABSTRACT: The Argonaute proteins are recently identified and evolutionarily conserved family with two subfamilies Ago and Piwi, which play important roles in small RNA pathways. Most species have eight Argonaute members in their genomes, ranging from 1 to 27. Here we report identification of six Argonaute genes in pig, four members of the Ago subfamily (Ago1, Ago2, Ago3 and Ago4) and two members of the Piwi subfamily (Piwil1 and Piwil2), which were predicted to encode proteins of 857, 860, 860, 861, 861 and 985 amino acids, respectively. Phylogenetic analysis showed that the porcine Ago and Piwi genes were clustered into relevant branch of mammalian Argonaute members. The porcine Ago4- Ago1-Ago3 genes are linked together at the p12 of the chromosome 6, while Ago2 is located at the p15 of the chromosome 4. The porcine Piwil1 and Piwil2 are mapped together onto the chromosome 14, at the q14 and q11 respectively. Comparatively mapping of the Argonaute members on chromosomes showed that linkage group of the Ago4-Ago1-Ago3 and several neighborhood genes is evolutionarily conserved from chicken to mammals. The genes Piwil1 and Piwil2 are separated onto different chromosomes from fish to mammals, with exception to this tendency in both pig and stickleback, indicating an opposite tendency of recombination together or non-disjunction of these two genes during speciation. Further expression analysis showed an ubiquitous expression pattern of Ago members, oppositely a restricted expression pattern in gonads of the Piwi members, suggesting distinct potential roles of the porcine Argonaute genes.
    Genetica 07/2010; 138(7):805-12. · 1.68 Impact Factor
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    ABSTRACT: Role of apoptosis in gonadal transformation of the rice field eel remains unknown. Here we report characterization of apoptotic pattern of testis, ovary, and ovotestis of the rice field eel, a vertebrate with natural sex reversal characteristic. DNA laddering assay showed typical ladder with step around 200 bp in the gonads, especially in testis. Terminal transferase dUTP nick end labeling on gonads indicated obvious apoptotic signals in the seminiferous tubules. Western blot analysis revealed that pro-apoptotic genes, Caspase 9 and p53, were upregulated and anti-apoptotic factor Bcl2 was downregulated in testis compared with both ovary and ovotestis. These data indicated that sex reversal process is accompanied by gonadal apoptosis with the highest proportion of cell death in the testis. Furthermore, we identified the Hsp10 by differentially screening of testis, ovary, and ovotestis using microarray technique, which is evolutionarily conserved and differentially expressed during gonadal transformation. Downregulation of Hsp10 is consistent with high apoptosis during the gonadal transformation. Flow cytometry assay confirmed that Hsp10 inhibits the apoptosis in male gonadal cells. Moreover, upregulation and mis-localization at sub-cellular level of the HSP10 together with its partner HSP60 is associated with tumorigenesis in human testis. These results suggest that downregulation of Hsp10 would be one of the main causes of apoptosis in testis, overexpression of Hsp10 suppresses apoptosis, and potentially results in testis tumorigenesis, which provide clues for understanding the mechanisms of germ cell apoptosis. Development of Hsp10 as a diagnostic marker or even treatment target will be promising in testis cancer diagnosis and therapy.
    Journal of Experimental Zoology Part B Molecular and Developmental Evolution 06/2010; 314(4):257-66. · 2.12 Impact Factor
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    ABSTRACT: Dmrt7, a member of the Dmrt family of genes, is required for spermatogenesis. However, promoter functions of the gene Dmrt7 remain unknown. We have cloned and characterized the proximal promoter region of the mouse Dmrt7 gene. Functional analysis of the 5' flanking region by sequential deletion mutations revealed crucial positive elements between -60 and +1, in which two highly conserved and tandem CCAAT boxes: the CCAAT box1 (-48/-44) and the CCAAT box2 (-7/-3) are located. Site-directed mutagenesis studies demonstrated that both CCAAT boxes are indispensable to the promoter activity. Electrophoretic mobility shift assays (EMSAs) and gel-supershift assays indicated that transcription factor NF-Y binds to the promoter. Chromatin immunoprecipitation (ChIP) analysis demonstrated that NF-Y interacts in vivo with the promoter of the Dmrt7 gene in testis. Co-transfection and reporter analysis showed that over-expression of NF-Ys increased transcription of the Dmrt7-luc gene whereas expression of a dominant-negative NF-Ya decreased the transcription. This suggests that NF-Y can activate the Dmrt7 promoter. These results provide evidence of a transcription regulatory mechanism that controls Dmrt7 gene expression in mouse testis.
    International journal of biological sciences 01/2010; 6(7):655-64. · 3.17 Impact Factor
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    ABSTRACT: Post-transcriptional regulations play a crucial role during spermatogenesis of the vertebrates. Chromatoid body (CB) is a characteristic spermatid organelle that is supposed to exert its role in post-transcriptional processes, but its real functions remain largely unknown. Here we report identification of Mago from the rice field eel, and show its evolutionary conservation, differential expression and localization during gonadal transformation. The Mago interacts with Y14, which may facilitate nuclear export of both proteins in the Sertoli cells. Importantly, we have determined Mago as a novel component of the CB in the cytoplasm of the developing spermatid. Addition of Mago to the component list of the CB undoubtedly provides new clue as to the functions of the CB during spermatogenesis in the vertebrates.
    Journal of Experimental Zoology Part B Molecular and Developmental Evolution 11/2009; 314(3):232-41. · 2.12 Impact Factor
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    ABSTRACT: Alternative pre-mRNA splicing (AS) plays a central role in generating complex proteomes and influences development and disease. However, the regulation and etiology of AS in human tumorigenesis is not well understood. A Basic Local Alignment Search Tool database was constructed for the expressed sequence tags (ESTs) from all available databases of human cancer and normal tissues. An insertion or deletion in the alignment of EST/EST was used to identify alternatively spliced transcripts. Alignment of the ESTs with the genomic sequence was further used to confirm AS. Alternatively spliced transcripts in each tissue were then subtractively cross-screened to obtain tissue-specific variants. We systematically identified and characterized cancer/tissue-specific and alternatively spliced variants in the human genome based on a global view. We identified 15,093 cancer-specific variants of 9,989 genes from 27 types of human cancers and 14,376 normal tissue-specific variants of 7,240 genes from 35 normal tissues, which cover the main types of human tumors and normal tissues. Approximately 70% of these transcripts are novel. These data were integrated into a database HCSAS (http://202.114.72.39/database/human.html, pass:68756253). Moreover, we observed that the cancer-specific AS of both oncogenes and tumor suppressor genes are associated with specific cancer types. Cancer shows a preference in the selection of alternative splice-sites and utilization of alternative splicing types. These features of human cancer, together with the discovery of huge numbers of novel splice forms for cancer-associated genes, suggest an important and global role of cancer-specific AS during human tumorigenesis. We advise the use of cancer-specific alternative splicing as a potential source of new diagnostic, prognostic, predictive, and therapeutic tools for human cancer. The global view of cancer-specific AS is not only useful for exploring the complexity of the cancer transcriptome but also widens the eyeshot of clinical research.
    PLoS ONE 02/2009; 4(3):e4732. · 3.73 Impact Factor
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    ABSTRACT: The DM (doublesex and male aberrant-3) genes implicated in sexual development in diverse metazoan organisms have been proved to be involved in development of non-gonadal tissues. The aim of the present study was to identify and characterize Dmrt3 (DM-related transcription factor 3) of zebrafish. Zebrafish Dmrt3 has a conserved DMA domain, besides a common DM domain, which clustered it into the DMRTA subfamily. During embryogenesis, Dmrt3 expression increases gradually to a high level at pharyngula stage, which is restricted to the olfactory placode and the neural tube. In the juvenile zebrafish, the gene expression is first detected in undifferentiated gonad on 17 dpf (day post-fertilization). In adult, Dmrt3 is expressed only in the developing germ cells of both gonads, mainly in spermatogonia, spermatocytes and developing oocytes. The Dmrt3 has a functional NLS (nuclear localization signal) K(41)GHKR(45) within the DM domain, which ensures that Dmrt3 exerts its role in the nucleus. Moreover, EMSA (electrophoretic mobility-shift assay) indicates that the Dmrt3-derived DM polypeptide binds to similar sites of both targets of DSX (doublesex) and MAB-3 (male aberrant-3). These results suggest that as a DNA-binding protein, zebrafish Dmrt3 may function in the nucleus as a potential transcription factor to exert potential roles in the development of the olfactory placode, the neural tube and germ cells.
    Biology of the Cell 09/2008; 100(8):453-63. · 3.49 Impact Factor

Publication Stats

336 Citations
130 Downloads
4k Views
154.61 Total Impact Points

Institutions

  • 2001–2014
    • Wuhan University
      • • College of Life Sciences
      • • Department of Genetics
      Wu-han-shih, Hubei, China
  • 2013
    • Huazhong Agricultural University
      • College of Fisheries
      Wuhan, Hubei, China