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Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology

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Abstract

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

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Data Availability

The data analyzed in this study are presented in this article and the supplementary information file. The raw data supporting the conclusion can be shared by request.

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Funding

This work is financially supported by the Guangdong Provincial Key R&D Program (2021B0202020002), Science and Technology Planning Project of Guangzhou (202201011818), National Science Foundation of China (Nos. 32172968, 31972769, 32102788), Seed Industry Development Project of Agricultural and Rural Department of Guangdong Province (2022-SPY-00-012), the open project program of Yazhou Bay Innovation Institute of Hainan Tropical Ocean University (2022EHDKFXT05), and Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (311021006).

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Authors and Affiliations

  1. State Key Laboratory of Biocontrol and School of Life Sciences, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Guangdong Provincial Key Laboratory for Aquatic Economic Animals and Guangdong Provincial Engineering Technology Research Center for Healthy Breeding of Important Economic Fish, Sun Yat-Sen University, Xin Gang Xi Road, Haizhu District, 510275, Guangzhou, Guangdong Province, China

    Fengying Yan, Xinxun Xiao, Chen Long, Lin Tang, Chongwei Wang, Mingqing Zhang, Jin Zhang, Haoran Lin, Yong Zhang & Shuisheng Li

  2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, 266373, Qingdao, China

    Haoran Lin & Yong Zhang

  3. Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources of Ministry of Education, Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources, Yazhou Bay Innovation Institute, Hainan Tropical Ocean University, 572022, Sanya, China

    Hai Huang

Authors
  1. Fengying Yan
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  2. Xinxun Xiao
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  3. Chen Long
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  4. Lin Tang
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  7. Jin Zhang
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  9. Hai Huang
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  10. Yong Zhang
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  11. Shuisheng Li
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Contributions

Conception and design of the study (XX, SL, FY, YZ). Acquisition, analysis, and interpretation of data (FY, XX, CL, LT, WC, MZ, JZ, HH, SL). Writing the manuscript (FY, SL). All authors read and approved of the final article to be submitted.

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Correspondence to Yong Zhang or Shuisheng Li.

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Yan, F., Xiao, X., Long, C. et al. Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology. Mar Biotechnol 25, 388–402 (2023). https://doi.org/10.1007/s10126-023-10212-9

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