TY - JOUR
T1 - Homologous sense and antisense expression of a gene in Dunaliella tertiolecta
AU - Zhang, Jing Yue
AU - Jeon, Hancheol
AU - Sim, Sang Jun
AU - Lee, Yew
AU - Jin, Eon Seon
N1 - Funding Information:
This work was supported by the Korea CCS R&D Center (KCRC) (NRF-2014M1A8A1049273) and NRF-2013R1A2A1A01015644 funded by the Korean Government (Ministry of Science, Ict. & Future Planning). This research was also supported by the Marine Biotechnology Program funded by the Ministry of Land, Transport, and Maritime Affairs of the Korean Government. This work was also supported by the Energy Efficiency and Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry and Energy, Republic of Korea. (No. 20142020200980).
Publisher Copyright:
© 2015, Springer-Verlag Berlin Heidelberg.
PY - 2015/10/10
Y1 - 2015/10/10
N2 - Main conclusion: Dunaliellatransformation using antisense and sense technology developed in this study will provide powerful tools for functional analysis and pathway-specific metabolic engineering inDunaliellafor industrial applications. Our aim was to investigate the potential of antisense expression and overexpression of a specific gene, the carotenoid biosynthesis-related (CBR) gene, in the microalgae Dunaliella. DNA amplified from sense and antisense vector constructs was used to transform Dunaliella tertiolecta. The Gateway vector for plant transformation was used to make an expression cassette, and the essential region for Dunaliella transformation was amplified by PCR and used for transformation. The transformation efficiency using a 3.2 kb PCR product was 130 transformants/μg DNA for both sense and antisense transformations. Among 200 BASTA-resistant colonies from the sense transformation and antisense transformation, separately, nine positive transformants for sense expression and five positive transformants for the antisense expression were obtained by genomic DNA PCR. The insertion was also verified by genomic Southern analysis. Among five positive sense transformants, one transformant was tested and verified for the overexpression of CBR-GFP fusion protein by Western blot analysis. One of the five antisense transformants showed almost complete inhibition of gene expression under light stress conditions (400 μmol photons m−2 s−1) as determined by quantitative RT-PCR and Western blot analysis. Although there was no difference in the growth patterns or photosynthetic parameters between the wild type (including the vector control) and transformants, the zeaxanthin content of the antisense CBR mutant was lowered under light stress conditions. Thus, we show that the sense and antisense RNA technology can be easily and strategically used for the functional analysis of interesting gene in D.tertiolecta.
AB - Main conclusion: Dunaliellatransformation using antisense and sense technology developed in this study will provide powerful tools for functional analysis and pathway-specific metabolic engineering inDunaliellafor industrial applications. Our aim was to investigate the potential of antisense expression and overexpression of a specific gene, the carotenoid biosynthesis-related (CBR) gene, in the microalgae Dunaliella. DNA amplified from sense and antisense vector constructs was used to transform Dunaliella tertiolecta. The Gateway vector for plant transformation was used to make an expression cassette, and the essential region for Dunaliella transformation was amplified by PCR and used for transformation. The transformation efficiency using a 3.2 kb PCR product was 130 transformants/μg DNA for both sense and antisense transformations. Among 200 BASTA-resistant colonies from the sense transformation and antisense transformation, separately, nine positive transformants for sense expression and five positive transformants for the antisense expression were obtained by genomic DNA PCR. The insertion was also verified by genomic Southern analysis. Among five positive sense transformants, one transformant was tested and verified for the overexpression of CBR-GFP fusion protein by Western blot analysis. One of the five antisense transformants showed almost complete inhibition of gene expression under light stress conditions (400 μmol photons m−2 s−1) as determined by quantitative RT-PCR and Western blot analysis. Although there was no difference in the growth patterns or photosynthetic parameters between the wild type (including the vector control) and transformants, the zeaxanthin content of the antisense CBR mutant was lowered under light stress conditions. Thus, we show that the sense and antisense RNA technology can be easily and strategically used for the functional analysis of interesting gene in D.tertiolecta.
KW - Antisense expression
KW - Carotenoid biosynthesis-related protein
KW - Dunaliella transformation
KW - Overexpression
UR - http://www.scopus.com/inward/record.url?scp=84941174894&partnerID=8YFLogxK
U2 - 10.1007/s00425-015-2369-2
DO - 10.1007/s00425-015-2369-2
M3 - Article
C2 - 26202735
AN - SCOPUS:84941174894
SN - 0032-0935
VL - 242
SP - 1051
EP - 1058
JO - Planta
JF - Planta
IS - 4
ER -