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Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Kasai Y, Oshima K, Ikeda F, Abe J, Yoshimitsu Y, Harayama S - Biotechnol Biofuels (2015)

Bottom Line: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system.These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates.Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.

ABSTRACT

Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.

Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.

Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

No MeSH data available.


Related in: MedlinePlus

PCR amplification of the pUT1 sequences integrated into the genomes of transgenic strains. a The pUT1 map: TUBP, the promoter region of PeTUBULIN1; cPeUMPS, the PeUMPS cDNA; ACTT, the terminator region of PeACT1. The positions of the five PCR primers and the PCR products amplified using the primers (1–5) are shown below the pUT1 map. b Agarose gel electrophoresis to verify the presence or absence of PCR products 1–5. M, DNA marker (λ-EcoT14 I digest); P, pUT1; A, 4 M-1A; B, 4 M-1B; C, 4 M-1C; D, 4 M-1D
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Fig2: PCR amplification of the pUT1 sequences integrated into the genomes of transgenic strains. a The pUT1 map: TUBP, the promoter region of PeTUBULIN1; cPeUMPS, the PeUMPS cDNA; ACTT, the terminator region of PeACT1. The positions of the five PCR primers and the PCR products amplified using the primers (1–5) are shown below the pUT1 map. b Agarose gel electrophoresis to verify the presence or absence of PCR products 1–5. M, DNA marker (λ-EcoT14 I digest); P, pUT1; A, 4 M-1A; B, 4 M-1B; C, 4 M-1C; D, 4 M-1D

Mentions: Six G418-resistant colonies were obtained, and the existence of pUT1 sequences in the transformants grown on MA5 minimal agar plates containing uracil was examined using PCR with two forward primers (one that hybridized with the 5′-ends of the PeTUBULIN1 promoter, and another that hybridized with the 5′-ends of the PeUMPS cDNA) and three reverse primers that hybridized the 3′-end of the PeUMPS cDNA, the middle part of the PeACTIN1 terminator, and the 3′-end of the PeACTIN1 terminator (Fig. 2). In the genomes of three out of the six transformants (named strains M4-1A, M4-1B, and M4-1C), the DNA region covering the PeTUBULIN1 promoter, PeUMPS cDNA, and the PeACTIN1 terminator was integrated. Conversely, the genome of the transformant named strain M4-1D contained a region covering the PeTUBULIN1 promoter, PeUMPS cDNA, and a 0.4-kb 5′-end of the PeACTIN1 terminator (Fig. 2). The PCR products amplified from the M4-1C genome using the primers Ptub_F and Tact_R were a mixture of four or more DNA fragments (Fig. 2b, lane 2_C), suggesting that the DNA region, the PeTUBULIN1 promoter–PeUMPS cDNA–the PeACTIN1 terminator, integrated in the genome of the transgenic M4-1C strain was unstable when grown in the presence of uracil.Fig. 2


Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Kasai Y, Oshima K, Ikeda F, Abe J, Yoshimitsu Y, Harayama S - Biotechnol Biofuels (2015)

PCR amplification of the pUT1 sequences integrated into the genomes of transgenic strains. a The pUT1 map: TUBP, the promoter region of PeTUBULIN1; cPeUMPS, the PeUMPS cDNA; ACTT, the terminator region of PeACT1. The positions of the five PCR primers and the PCR products amplified using the primers (1–5) are shown below the pUT1 map. b Agarose gel electrophoresis to verify the presence or absence of PCR products 1–5. M, DNA marker (λ-EcoT14 I digest); P, pUT1; A, 4 M-1A; B, 4 M-1B; C, 4 M-1C; D, 4 M-1D
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4489027&req=5

Fig2: PCR amplification of the pUT1 sequences integrated into the genomes of transgenic strains. a The pUT1 map: TUBP, the promoter region of PeTUBULIN1; cPeUMPS, the PeUMPS cDNA; ACTT, the terminator region of PeACT1. The positions of the five PCR primers and the PCR products amplified using the primers (1–5) are shown below the pUT1 map. b Agarose gel electrophoresis to verify the presence or absence of PCR products 1–5. M, DNA marker (λ-EcoT14 I digest); P, pUT1; A, 4 M-1A; B, 4 M-1B; C, 4 M-1C; D, 4 M-1D
Mentions: Six G418-resistant colonies were obtained, and the existence of pUT1 sequences in the transformants grown on MA5 minimal agar plates containing uracil was examined using PCR with two forward primers (one that hybridized with the 5′-ends of the PeTUBULIN1 promoter, and another that hybridized with the 5′-ends of the PeUMPS cDNA) and three reverse primers that hybridized the 3′-end of the PeUMPS cDNA, the middle part of the PeACTIN1 terminator, and the 3′-end of the PeACTIN1 terminator (Fig. 2). In the genomes of three out of the six transformants (named strains M4-1A, M4-1B, and M4-1C), the DNA region covering the PeTUBULIN1 promoter, PeUMPS cDNA, and the PeACTIN1 terminator was integrated. Conversely, the genome of the transformant named strain M4-1D contained a region covering the PeTUBULIN1 promoter, PeUMPS cDNA, and a 0.4-kb 5′-end of the PeACTIN1 terminator (Fig. 2). The PCR products amplified from the M4-1C genome using the primers Ptub_F and Tact_R were a mixture of four or more DNA fragments (Fig. 2b, lane 2_C), suggesting that the DNA region, the PeTUBULIN1 promoter–PeUMPS cDNA–the PeACTIN1 terminator, integrated in the genome of the transgenic M4-1C strain was unstable when grown in the presence of uracil.Fig. 2

Bottom Line: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system.These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates.Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.

ABSTRACT

Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.

Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.

Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

No MeSH data available.


Related in: MedlinePlus