Skip to main content
SpringerLink
Log in

The lgi-miR-2d is Potentially Involved in Shell Melanin Synthesis by Targeting mitf in Manila Clam Ruditapes philippinarum

  • Research
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Shell color as an important economic trait is also the crucial target trait for breeding and production. MicroRNA (miRNA) is an endogenous small non-coding RNA that can post-transcriptionally regulate the expression of target genes, it plays important roles in many life activities and physiological processes, such as shell color, stress response, and disease traits. In this study, we investigated the function of lgi-miR-2d in shell melanin formation and the expression patterns of lgi-miR-2d and target gene Rpmitf in Manila clam Ruditapes philippinarum. We further explored and verified the relationship between Rpmitf and lgi-miR-2d and identified the expression level of shell color-related gene changes by RNAi and injecting the antagomir of lgi-miR-2d, respectively. Our results indicated that lgi-miR-2d antagomir affected the expression of its target gene Rpmitf. In addition, the dual-luciferase reporter assay was conducted to confirm the direct interaction between lgi-miR-2d and Rpmitf. The results showed that the expression levels of melanin-related genes such as Rpmitf and tyr were significantly decreased in the positive treatment group compared with the blank control group after the Rpmitf dsRNA injection, indicating Rpmitf plays a crucial role in the melanin synthesis pathway. Taken together, we speculated that lgi-miR-2d might be negatively modulating Rpmitf, which might regulate other shell color-related genes, thereby affecting melanin synthesis in R. philippinarum.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Data Availability

The datasets generated in the current study are available from the corresponding author upon reasonable request.

References

  • Bai Z, Zheng H, Lin J, Wang G, Li J (2013) Comparative analysis of the transcriptome in tissues secreting purple and white nacre in the pearl mussel Hyriopsis cumingii. PLoS ONE 8:e53617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Budd A, McDougall C, Green K, Degnan BM (2014) Control of shell pigmentation by secretory tubules in the abalone mantle. Front Zool 11:62

    Article  Google Scholar 

  • Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136:642–655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen X, Liu X, Bai Z, Zhao L, Li J (2017) HcTyr and HcTyp-1 of Hyriopsis cumingii, novel tyrosinase and tyrosinase-related protein genes involved in nacre color formation. Comp Biochem Physiol B Biochem Mol Biol 204:1–8

    Article  CAS  PubMed  Google Scholar 

  • Chen X, Bai Z, Li J (2019a) The mantle exosome and microRNAs of Hyriopsis cumingii involved in nacre color formation. Mar Biotechnol (NY) 21:634–642

    Article  CAS  PubMed  Google Scholar 

  • Chen X, Zhang M, Zhang J, Bai Z, Li J (2019b) miR-4504 is involved in nacre color formation in Hyriopsis cumingii. Biochem Biophys Res Commun 517:210–215

    Article  CAS  PubMed  Google Scholar 

  • Deiana M, Dalle Carbonare L, Serena M, Cheri S, Parolini F, Gandini A, Marchetto G, Innamorati G, Manfredi M, Marengo E, Brandi J, Cecconi D, Mori A, Mina MM, Antoniazzi F, Mottes M, Tiso N, Malerba G, Zipeto D, Valenti MT (2018) New insights into the runt domain of RUNX2 in melanoma cell proliferation and migration. Cells 7:220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ding J, Wen Q, Huo Z, Nie H, Qin Y, Yan X (2021) Identification of shell-color-related microRNAs in the Manila clam Ruditapes philippinarum using high-throughput sequencing of small RNA transcriptomes. Sci Rep 11:8044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong C, Wang H, Xue L, Dong Y, Yang L, Fan R, Yu X, Tian X, Ma S, Smith GW (2012) Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model. RNA 18:1679–1686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • FAO (Food & Agriculture Organization) (2022) Food and Agriculture Organization of the United Nations, Rome

  • Feng D, Li Q, Yu H, Liu S, Kong L, Du S (2020) Integrated analysis of microRNA and mRNA expression profiles in Crassostrea gigas to reveal functional miRNA and miRNA-targets regulating shell pigmentation. Sci Rep 10:20238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guan Y, He M (2009) Progress on shell coloration polymorphism in seashells. Mar Sci Bull 28:108–114

    Google Scholar 

  • Guo J, Zhang JF, Wang WM, Cheung FW, Lu YF, Ng CF, Kung HF, Liu WK (2014) MicroRNA-218 inhibits melanogenesis by directly suppressing microphthalmia-associated transcription factor expression. RNA Biol 11:732–741

    Article  PubMed  PubMed Central  Google Scholar 

  • Hao S, Hou X, Sun D, Yu W, He C, Wang X (2015) Extraction and identification of the pigment in the Mytilus edulis shells and Rapana venosa operculum. Marine Sciences 11:49–53

    Google Scholar 

  • Heller J (1979) Visual versus non-visual selection of shell colour in an Israeli freshwater snail. Oecologia 44:98–104

    Article  PubMed  Google Scholar 

  • Huang S, Yoshitake K, Asaduzzaman M, Kinoshita S, Watabe S, Asakawa S (2020) Discovery and functional understanding of miRNAs in mollusks: a genome-wide profiling approach. RNA Biol 7196:1702–1715

    Google Scholar 

  • Lee JE (1997) Basic helix-loop-helix genes in neural development. Curr Opin Neurobiol 7:13–20

    Article  PubMed  Google Scholar 

  • Ling L, Ge X, Li Z, Zeng B, Xu J, Chen X, Shang P, James AA, Huang Y, Tan A (2015) MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori. RNA Biol 12:742–748

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu J, Nie H, Huo Z, Yan X (2023) Genome-wide identification and characterization of MITF genes in Ruditapes philippinarum and their involvement in the immune response to Vibrio anguillarum infection. Biochem Genet 61:2514–2530

    Article  CAS  PubMed  Google Scholar 

  • Luo M, Wang L, Zhu W, Fu J, Song F, Fang M, Dong J, Dong Z (2018) Identification and characterization of skin color microRNAs in Koi carp (Cyprinus carpio L.) by Illumina sequencing. BMC Genomics 19:779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ma Q, Jin Y, Jahan K, Jami MEH, Xu Q, Huo Z, Yang F, Yan X (2021) Effects of ammonia nitrogen on early growth and survival of the ‘zebra 2’ strain of the manila clam (Ruditapes philippinarum). Aquac Res 52(10):4767-4775

    Google Scholar 

  • Mao J, Zhang X, Zhang W, Tian Y, Wang X, Hao Z, Chang Y (2019) Genome-wide identification, characterization and expression analysis of the MITF gene in Yesso scallops (Patinopecten yessoensis) with different shell colors. Gene 688:155–162

    Article  CAS  PubMed  Google Scholar 

  • Mitton JB (1977) Shell color and pattern variation in Mytilus edulis and its adaptive significance. Chesap Sci 18:387–390

    Article  Google Scholar 

  • Nagai K, Yano M, Morimoto K, Miyamoto H (2007) Tyrosinase localization in mollusc shells. Comp Biochem Physiol B Biochem Mol Biol 146:207–214

    Article  PubMed  Google Scholar 

  • Nie H, Jiang K, Jiang L, Huo Z, Ding J, Yan X (2020a) Transcriptome analysis reveals the pigmentation related genes in four different shell color strains of the Manila clam Ruditapes philippinarum. Genomics 112:2011–2020

    Article  CAS  PubMed  Google Scholar 

  • Nie H, Jiang K, Li N, Jahan K, Jiang L, Huo Z, Yan X (2020b) Transcriptome analysis reveals the pigmentation-related genes in two shell color strains of the Manila clam Ruditapes philippinarum. Anim Biotechnol 32:439–450

    Article  PubMed  Google Scholar 

  • Qiu P, Zhang M, Wu Y, Liu Y, Wang Y, Zhang J, Song J, Feng S, Sun Y, Tan L, Yu X, Hu G, Wei M (2022) Cloning and characterization of microphthalmia-associated transcription factor-like gene provide insights into Cyclina sinensis clam shell melanin deposition. Aquac Res 53(4):1413-1423

    Google Scholar 

  • Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R (2004) Fast and effective prediction of microRNA/target duplexes. RNA 10:1507–1517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403:901–906

    Article  CAS  PubMed  Google Scholar 

  • Saito H, Yasumoto K-i, Takeda K, Takahashi K, Yamamoto H, Shibahara S (2003) Microphthalmia-associated transcription factor in the Wnt signaling pathway. Pigment Cell Res 16:261–265

    Article  CAS  PubMed  Google Scholar 

  • Sokolova IM, Berger VJ (2000) Physiological variation related to shell colour polymorphism in White Sea Littorina saxatilis. J Exp Mar Biol Ecol 245:1–23

    Article  Google Scholar 

  • Steingrímsson E, Tessarollo L, Pathak B, Hou L, Arnheiter H, Copeland NG, Jenkins NA (2002) Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development. Proc Natl Acad Sci USA 99:4477–4477

    Article  PubMed  PubMed Central  Google Scholar 

  • True JR (2003) Insect melanism: the molecules matter. Trends Ecol Evol 18:640–647

    Article  Google Scholar 

  • Vachtenheim J, Borovansky J (2010) “Transcription physiology” of pigment formation in melanocytes: central role of MITF. Exp Dermatol 19:617–627

    Article  CAS  PubMed  Google Scholar 

  • Varghese PK, Abu-Asab M, Dimitriadis EK, Dolinska MB, Morcos GP, Sergeev YV (2021) Tyrosinase Nanoparticles: understanding the melanogenesis pathway by isolating the products of tyrosinase enzymatic reaction. Int J Mol Sci 22:734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Y, Li SM, Huang J, Chen SY, Liu YP (2014) Mutations of TYR and MITF genes are associated with plumage colour phenotypes in Geese. Asian-Australas J Anim Sci 27:778–783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang P, Zhao Y, Fan R, Chen T, Dong C (2016) MicroRNA-21a-5p functions on the regulation of melanogenesis by targeting Sox5 in mouse skin melanocytes. Int J Mol Sci 17:959

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang L, Zhu W, Dong Z, Song F, Dong J, Fu J (2018) Comparative microRNA-seq analysis depicts candidate miRNAs involved in skin color differentiation in red tilapia. Int J Mol Sci 19:1209

    Article  PubMed  PubMed Central  Google Scholar 

  • Williams ST (2017) Molluscan shell colour. Biol Rev Camb Philos Soc 92:1039–1058

    Article  PubMed  Google Scholar 

  • Wu Z, Fu Y, Cao J, Yu M, Tang X, Zhao S (2014) Identification of differentially expressed miRNAs between white and black hair follicles by RNA-sequencing in the goat (Capra hircus). Int J Mol Sci 15:9531–9545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xing L, Sun L, Liu S, Wan Z, Li X, Miao T, Zhang L, Bai Y, Yang H (2018) Growth, histology, ultrastructure and expression of MITF and astacin in the pigmentation stages of green, white and purple morphs of the sea cucumber, Apostichopus japonicus. Aquac Res 49:177–187

    Article  CAS  Google Scholar 

  • Xu Q, Nie H, Yin Z, Zhang Y, Huo Z, Yan X (2021) MiRNA-mRNA integration analysis reveals the regulatory roles of MiRNAs in shell pigmentation of the Manila clam (Ruditapes philippinarum). Mar Biotechnol (NY) 23:976–993

    Article  CAS  PubMed  Google Scholar 

  • Yan X, Huo Z, Yang F, Zhang G (2015) Ruditapes philippinarum “zebra” clam. China fisheries 9:52

  • Yan X, Huo Z, Zhang Y, Yang F, Zhang G (2017) Ruditapes philippinarum “zebra 2” clam. China Fisheries 12:72–74

    Google Scholar 

  • Yan X, Nie H, Huo Z, Ding J, Li Z, Yan L, Jiang L, Mu Z, Wang H, Meng X, Chen P, Zhou M, Rbbani MG, Liu G, Li D (2019) Clam genome sequence clarifies the molecular basis of its benthic adaptation and extraordinary shell color diversity. iScience 19:1225–1237

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang J, Wang Y, Wang X, Bao Y (2020) Expression of MITF gene and its correlation with shell color in Crassostrea gigas. Journal of Fisheries of China 44:915–923

    Google Scholar 

  • Yin H, Luo M, Luo W, Wang L, Zhu W, Fu J, Dong Z (2020) miR-196a regulates the skin pigmentation of koi carp (Cyprinus carpio L.) by targeting transcription factor mitfa. Aquac Res 52:229–236

    Article  Google Scholar 

  • Yu F, Lu Y, Zhong Z, Qu B, Wang M, Yu X, Chen J (2021) Mitf involved in innate immunity by activating tyrosinase-mediated melanin synthesis in Pteria penguin. Front Immunol 12:626493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu W, He C, Wu C, Wang J, Li Z, Guo T, Li Y, Wang X (2015) Extraction and identification of melanin in shell and mantle of Pacific oyster Crassostrea gigas. Oceanologia Et Limnologia Sinica 46(4):909–914

    CAS  Google Scholar 

  • Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z, Que H, Xie Y, Holland PWH, Paps J, Zhu Y, Wu F, Chen Y, Wang J, Peng C, Meng J, Yang L, Liu J, Wen B, Zhang N, Huang Z, Zhu Q, Feng Y, Mount A, Hedgecock D, Xu Z, Liu Y, Domazet-Losˇo T, Du Y, Sun X, Zhang S, Liu B, Cheng P, Jiang X, Li J, Fan D, Wang W, Fu W, Wang T, Wang B, Zhang J, Peng Z, Li Y, Li N, Wang J, Chen M, He Y, Tan F, Song X, Zheng Q, Huang R, Yang H, Du X, Chen L, Yang M, Gaffney PM, Wang S, Luo L, She Z, Yao M, Huang W, Zhang S, Huang B, Zhang Y, Qu T, Ni P, Miao G, Wang J, Wang Q, Steinberg CEW, Wang H, Li N, Qian L, Zhang G, Li Y, Yang H, Liu X, Wang J, Yin Y, Wang J (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54

    Article  CAS  PubMed  Google Scholar 

  • Zhang S, Yue X, Jiang F, Wang H, Liu B (2017) Identification of an MITF gene and its polymorphisms associated with the Vibrio resistance trait in the clam Meretrix petechialis. Fish Shellfish Immunol 68:466–473

    Article  CAS  PubMed  Google Scholar 

  • Zhang S, Wang H, Yu J, Jiang F, Yue X, Liu B (2018) Identification of a gene encoding microphthalmia-associated transcription factor and its association with shell color in the clam Meretrix petechialis. Comp Biochem Physiol B Biochem Mol Biol 225:75–83

    Article  CAS  PubMed  Google Scholar 

  • Zheng H, Liu H, Zhang T, Wang S, Sun Z, Liu W, Li Y (2010) Total carotenoid differences in scallop tissues of Chlamys nobilis (Bivalve: Pectinidae) with regard to gender and shell colour. Food Chem 122:1164–1167

    Article  CAS  Google Scholar 

  • Zheng N, Xin Q, Zhu Z, Miao Z, Li L, Liu F, Huang Q (2015) cDNA cloning and expression of MITF gene and its effect on melanin deposition in silky fow. Scientia Agricultura Sinica 48:3711–3718

    CAS  Google Scholar 

  • Zhu Z, He J, Jia X, Jiang J, Bai R, Yu X, Lv L, Fan R, He X, Geng J, You R, Dong Y, Qiao D, Lee KB, Smith GW, Dong C (2010) MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in Alpaca (Lama pacos) skin melanocytes. Domest Anim Endocrinol 38:200–209

    Article  CAS  PubMed  Google Scholar 

  • Zhu Z, Ma Y, Li Y, Cheng Z, Li H, Zhang L, Xu D, Li P (2019) Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation. BMC Mol Biol 20:19

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Jinhong Bi from Rongcheng Marine Economic Development Center for their kind help in sample collection.

Funding

This work was supported by the Dalian Science and Technology Innovation Fund Project (2022JJ12SN054). The Project is sponsored by the Scientific Research Funding from Liaoning Provincial Department of Education (LJKZ0701; QL201703), the Inter-University Cooperation Project (Cooperation and Exchange, Collaborative Innovation) in Liaoning Province, the funds earmarked for Modern Agro-industry Technology Research System (CARS-49), and the Liaoning Postgraduate Education and Teaching Reform Project (LNYJG2022291).

Author information

Authors and Affiliations

  1. College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023, China

    Qiaoyue Xu, Hongtao Nie, Qianying Ma, Jiadi Wang, Zhongming Huo & Xiwu Yan

  2. Engineering and Technology Research Center of Shellfish Breeding in Liaoning Province, Dalian Ocean University, Dalian, 116023, China

    Qiaoyue Xu, Hongtao Nie, Qianying Ma, Jiadi Wang, Zhongming Huo & Xiwu Yan

Authors
  1. Qiaoyue Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongtao Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qianying Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jiadi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhongming Huo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiwu Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Qiaoyue Xu: data curation, data analysis, investigation, visualization, and writing—original draft preparation and revision. Hongtao Nie: conceptualization, methodology, supervision, and writing—revision, review, and editing. Qianying Ma: investigation. Jiadi Wang: investigation. Zhongming Huo: materials and resources. Xiwu Yan: conceptualization and resources.

Corresponding author

Correspondence to Hongtao Nie.

Ethics declarations

Ethics Approval and Consent to Participate

The R. philippinarum is neither an endangered nor protected species. All experiments in this study were conducted according to national and institutional guidelines.

Competing Interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 16 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, Q., Nie, H., Ma, Q. et al. The lgi-miR-2d is Potentially Involved in Shell Melanin Synthesis by Targeting mitf in Manila Clam Ruditapes philippinarum. Mar Biotechnol (2024). https://doi.org/10.1007/s10126-024-10307-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10126-024-10307-x

Keywords

Search

Navigation