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<p><i>Ulva prolifera</i> is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of <i>U. prolifera</i> cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in <i>U. prolifera</i> based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (<i>aph7”</i>). Integration of <i>aph7”</i> in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of <i>aph7”</i>. The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of <i>aph7”</i> cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (<i>UpAPT</i>). <i>APT</i> gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance <i>U. prolifera</i> as a genetic platform, enabling functional research to elucidate <i>Ulva</i> biology, and to bring forth biotechnological utilization of algal resources.</p>

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Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera

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  • 发表日期：

    2025年10月6日

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  • Zheng Qin,
  • Whelver Surnido,
  • Hiroyuki Mizuta,
  • Toshiki Uji

摘要

Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7”). Integration of aph7” in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7”. The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7” cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.