Abstract
On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5′RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant Nos. 41406192, 41176144, and 41376139), the Science Foundation of the Chinese Academy of Sciences (Grant No. XDA1102040300).
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Yulin Cui and Jialin Zhao are co-first authors.
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Cui, Y., Zhao, J., Hou, S. et al. Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators. World J Microbiol Biotechnol 32, 83 (2016). https://doi.org/10.1007/s11274-016-2039-y
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DOI: https://doi.org/10.1007/s11274-016-2039-y