Abstract
Transgene silencing is a common phenomenon in higher plants and microalgae. In the present study, we analyzed transgene silencing in the macroalga Pyropia yezoensis. This is the first report of transgene silencing in macroalgae. Transgenic lines were generated by transformation with the PyGUS gene and a hygromycin resistance gene (PtAph7-1). Histochemical GUS staining detected PyGUS expression in only three out of 12 lines. Southern and northern blotting, polymerase chain reaction (PCR), and reverse transcription (RT)-PCR analyses indicated that the PyGUS gene was silenced in the other 9 lines. Interestingly, PyGUS gene silencing in transgenic line 7 was repeatedly initiated at a later stage, in the developmental process from monospore to thallus through asexual reproduction. We identified genes encoding Argonaute and Dicer-like protein, the major components of the RNA-silencing pathway, in the genome and transcripts of P. yezoensis. Interestingly, we found the DDA motif in the PIWI domain of the AGO, suggesting that translational repression may be the major gene-silencing pathway in P. yezoensis. In this study, the observation of PyGUS gene silencing and identification of RNA-silencing components indicate that the gene (transgene) silencing machinery functions actively in the macroalga P. yezoensis. These results will be useful to study the regulation of gene expression and RNA-silencing mechanisms in Pyropia species and related macroalgae.
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Acknowledgments
This work was supported by a Grant from the Golden Seed Project, Ministry of Agriculture, Food, and Rural Affairs (MAFRA), the Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA), a grant from the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2011-0031343), and a Grant from the KRIBB Research Initiative program.
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11816_2016_408_MOESM1_ESM.pptx
Supplementary Fig. 1 Nucleotide sequence of codon-optimized PtAph7-1. Supplementary Fig. 2 Confirmation of the transgene insertion in silence lines 11 and 12. a Schematic diagram of transformation vectors. P1, P2, and P3 indicate the regions used as probes for the southern analysis. b Southern blot with PtAph7-1 hybridization. c Southern blot with PyGUS hybridization. d Southern blot with CFP hybridization. Genomic DNA (30 μg) digested by Hind III was used for PtAph7-1 and PyGUS hybridizations. Genomic DNA (30 μg) digested by SacI was used for CFP hybridization. e Schematic diagrams and Nucleotide sequences of rescued plasmid from transgenic line 11. Many fragmented DNAs, including two fragmented plasmids (containing CFP and PtAph7-1), fragmented genomic sequences(contig38663, contig35739, and contig36680) and unsequenced region (black box) were rearranged and integrated into position 812 on the contig 38663 in Pyropia genome. The square box shows the nucleotide sequences of the junction region of the fragmented plasmid and genomic DNA in transgenic line 11. Red nuclotides indicate the NcoI restriction site on contig38663; black nucleotides and dots indicate flanking sequences of P. yezoensis; Blue capital nucleotides and dots indicate sequences of rearranged fragmented plasmids. Supplementary Fig. 3 Amino-acid sequences of PyDCL and PyAGO
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Shin, Y.J., Lim, JM., Park, J.H. et al. Characterization of PyGUS gene silencing in the red macroalga, Pyropia yezoensis . Plant Biotechnol Rep 10, 359–367 (2016). https://doi.org/10.1007/s11816-016-0408-5
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DOI: https://doi.org/10.1007/s11816-016-0408-5