Abstract
Tisochrysis lutea is a haptophyte microalga commonly used as a commercial feed for juvenile fish and shellfish larvae. Genetic modification is of considerable importance for developing the potential economic value of T. lutea. However, the genetic transformation system of T. lutea has not yet been established, which limits functional genomic studies and strain improvement of this important microalgal species. In the current study, a chloroplast transformation vector harboring the phosphinothricin resistance gene (bar) as a selectable marker was established, and two short peptide-encoding genes (ant1 and ant2) driven by the endogenous psbA and rbcL promoters were cloned in this vector. The recombinant plasmid was transformed using a biolistic method into the trnI/trnA locus of the chloroplast genome via homologous recombination. After continuous selection on phosphinothricin, the integration of foreign genes and the expression of specific products in the transformants were detected using polymerase chain reaction (PCR), Southern blotting, and western blot analysis. This is the first report of establishment of a stable transformation system in T. lutea, which is a prerequisite for functional genomics and applied research on this species.
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References
Alkhamis Y, Qin JG (2016) Comparison of pigment and proximate compositions of Tisochrysis lutea in phototrophic and mixotrophic cultures. J Appl Phycol 28:35–42
Bendif EM, Probert I, Schroeder DC, de Vargas C (2013) On the description of Tisochrysis lutea gen. nov. sp. nov. and Isochrysis nuda sp. nov. in the Isochrysidales, and the transfer of Dicrateria to the Prymnesiales (Haptophyta). J Appl Phycol 25:1763–1776
Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolphanderson BL, Robertson D, Klein TM, Shark KB, Sanford JC (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538
Carrier G, Baroukh C, Rouxel C, Duboscq-Bidot L, Schreiber N, Bougaran G (2018) Draft genomes and phenotypic characterization of Tisochrysis lutea strains. Toward the production of domesticated strains with high added value. Algal Res 29:1–11
Carrier G, Garnier M, Le Cunff L, Probert I, De Vargas C, Corre E, Cadoret JP, Saint-Jean B (2014) Comparative transcriptome of wild type and selected strains of the microalgae Tisochrysis lutea provides insights into the genetic basis, lipid metabolism and the life cycle. PLoS One 9:e86889
Coll JM (2006) Methodologies for transferring DNA into eukaryotic microalgae. Span J Agric Res 4:316–330
Cosa BD, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74
Cui Y, Qin S, Jiang P (2014) Chloroplast transformation of Platymonas (Tetraselmis) subcordiformis with the bar gene as selectable marker. PLoS One 9:e98607
Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553
Dyo YM, Purton S (2018) The algal chloroplast as a synthetic biology platform for production of therapeutic proteins. Microbiology 164:113–121
Endo H, Hanawa Y, Araie H, Suzuki I, Shiraiwa Y (2018) Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme. Sci Rep 8:11230–11239
Esland L, Larrea-Alvarez M, Purton S (2018) Selectable markers and reporter genes for engineering the chloroplast of Chlamydomonas reinhardtii. Biology 7:26
Galarza JI, Gimpel JA, Rojas V, Arredondo-Vega BO, Henríquez V (2018) Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering. Algal Res 31:291–297
Green BR (2011) Chloroplast genomes of photosynthetic eukaryotes. Plant J 66:34–44
Guo SL, Zhao XQ, Tang Y, Wan C, Alam MA, Ho SH, Bai FW, Chang JS (2013) Establishment of an efficient genetic transformation system in Scenedesmus obliquus. J Biotechnol 163:61–68
Hu H, Ma LL, Shen XF, Li JY, Wang HF, Zeng RJ (2018) Effect of cultivation mode on the production of docosahexaenoic acid by Tisochrysis lutea. AMB Express 8:50–61
Lugo SK, Kunnimalaiyaan M, Singh NK, Nielsen BL (2004) Required sequence elements for chloroplast DNA replication activity in vitro and in electroporated chloroplasts. Plant Sci 166:151–161
Kang BR, Anderson AJ, Kim YC (2018) Hydrogen cyanide produced by Pseudomonas chlororaphis O6 exhibits nematicidal activity against Meloidogyne hapla. Plant Pathol J 34:35–43
Kang NK, Choi GG, Kim EK, Shin SE, Jeon S, Park MS, Jeong KJ, Jeong BR, Chang YK, Yang JW, Lee B (2015) Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina. Biotechnol Rep 8:10–15
Krichevsky A, Meyers B, Vainstein A, Maliga P, Citovsky V, Uversky V (2010) Autoluminescent plants. PLoS One 5:e1546
Melanie O, Ross IL, Ben H, Uversky VN (2014) Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae. PLoS One 9:e86841
Méndez-Leyva AB, Guo J, Mudd EA, Wong J, Schwartz JM, Day A (2019) The chloroplast genome of the marine microalga Tisochrysis lutea. Mitochondrial DNA B 4:253–255
Narra M, Kota S, Velivela Y, Ellendula R, Allini VR, Abbagani S (2018) Construction of chloroplast transformation vector and its functional evaluation in Momordica charantia L. 3 Biotech 8:140
Purton S, Szaub JB, Wannathong T, Young R, Economou CK (2013) Genetic engineering of algal chloroplasts: progress and prospects. Russ J Plant Physiol 60:491–499
Sandoval-Vargas JM, Macedo-Osorio KS, Durán-Figueroa NV, Garibay-Orijel C, Badillo-Corona JA (2018) Chloroplast engineering of Chlamydomonas reinhardtii to use phosphite as phosphorus source. Algal Res 33:291–297
Shi Q, Araie H, Bakku RK, Fukao Y, Rakwal R, Suzuki I, Shiraiwa Y (2015) Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea. Proteomics 15:4145–4158
Siddiqui A, Wei Z, Boehm M, Ahmad N (2020) Engineering microalgae through chloroplast transformation to produce high-value industrial products. Biotechnol Appl Biochem 67:30–40
Zhan J, Rong J, Wang Q (2017) Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect. Int J Hydrog Energy 42:8505–8517
Funding
This work was supported by National Natural Science Foundation of China (41876188), the Natural Science Foundation of Shandong Province, China (ZR2018ZB0210), and the Project of Innovation & Development of Marine Economy (HHCL201803).
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Bo, Y., Wang, K., Wu, Y. et al. Establishment of a chloroplast transformation system in Tisochrysis lutea. J Appl Phycol 32, 2959–2965 (2020). https://doi.org/10.1007/s10811-020-02159-4
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DOI: https://doi.org/10.1007/s10811-020-02159-4