Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

Abstract

The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.

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Funding

The present study was supported by the National Natural Science Foundation of China (41876188, 31972815), Major Basic Research Program of Shandong Province Natural Science Foundation (ZR2018ZB0210, ZR2019ZD17), theProject of Innovation & Development of Marine Economy (HHCL201803), Key Research and Development Program of Shandong Province (Food for Special Medical Purpose) (2018YYSP016), Key Research and Development Program of Shandong Province (2017CXGC0309), the Project of Shandong Province Higher Educational Science and Technology Program (J17KA132).

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Chunxiao Meng and Song Qin designed the experiments. Kang Wang and Yulin Cui analyzed the data and wrote the thesis with support from Tianzhong Liu. Zhengquan Gao and Yinchu Wang performed all experiments.

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Correspondence to Chunxiao Meng or Song Qin.

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Kang Wang and Yulin Cui are co-first authors

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Wang, K., Cui, Y., Wang, Y. et al. Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide. Mar Biotechnol (2020). https://doi.org/10.1007/s10126-020-09978-z

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Keywords

  • Haematococcus pluvialis
  • Chloroplast transformation
  • Antimicrobial peptide
  • Bar
  • Biolistic method