Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial
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ORIGINAL ARTICLE
Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide Kang Wang 1 & Yulin Cui 2,3 & Yinchu Wang 2,3 & Zhengquan Gao 1 & Tianzhong Liu 4 & Chunxiao Meng 1
&
Song Qin 2,3
Received: 25 February 2020 / Accepted: 14 May 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Haematococcus pluvialis . Chloroplast transformation . Antimicrobial peptide . Bar . Biolistic method
Introduction Haematococcus pluvialis is a freshwater planktonic singlecell green alga, belonging to Chlorophyceae of order
Kang Wang and Yulin Cui are co-first authors * Chunxiao Meng [email protected] * Song Qin [email protected] 1
School of Life Sciences, Shandong University of Technology, Zibo 255049, Shandong Province, China
2
Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
3
Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
4
Microalgal Biotechnology Group, CAS key Laboratory of Biofuels and Shandong Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and bioprocess technology, Chinese Academy of Sciences, Qingdao 266101, China
Volvocales (Hagen et al. 2002; Lee et al. 2016). It is capable of accumulating large amounts of astaxanthin (3, 3′-dihydroxy-diketo-β, β′-carotene-4, 4-dione), a liposoluble red ketocarotenoid with exceptional antioxidant properties (Galarza et al. 2018; Ma et al. 2018; Ota et al. 2018) and is considered to be the main source of natural astaxanthin (Ma et al. 2018; Ota et al. 2018). The application of astaxanthin in the field of human health has considerable potential (Liu 2018); in addition, it can also be used as a source of pigment and protein i
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