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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

Real-time PCR analysis of the expression of PeUMPS in (a) pUT1 and (b) pUT2 transgenic strains. 18S rRNA was used as an internal control. Strain M4 is the parental strain of the transgenic strains. Real-time PCR detected expression from the defective PeUMPS in strain M4 in addition to the transgenic PeUMPS cDNA. The data are presented as fold changes in PeUMPS expression normalized to 18S rRNA expression in transgenic strains compared with strain M4. Bars represent the standard deviation of three replicates
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Fig3: Real-time PCR analysis of the expression of PeUMPS in (a) pUT1 and (b) pUT2 transgenic strains. 18S rRNA was used as an internal control. Strain M4 is the parental strain of the transgenic strains. Real-time PCR detected expression from the defective PeUMPS in strain M4 in addition to the transgenic PeUMPS cDNA. The data are presented as fold changes in PeUMPS expression normalized to 18S rRNA expression in transgenic strains compared with strain M4. Bars represent the standard deviation of three replicates

Mentions: To evaluate the correlation between the expression of PeUMPS cDNA and the growth speed, the expression of PeUMPS cDNA in the four transgenic strains was analyzed using real-time PCR and normalized against 18S rRNA levels (Fig. 3a). The PCR primers Umps_QRT_F and Umps_QRT_R were used to detect both the expression of transgenic PeUMPS cDNA and the expression of defective PeUMPS in the chromosome of strain M4. Strain M4-1C, which grew fastest (its colonies were visible in 6 weeks), exhibited a 1.5-fold higher expression than did strain M4. It suggested that the expression of the transgenic PeUMPS cDNA in strain M4-1C was 1.5−1-fold of the expression of the original PeUMPS. Similarly, the expression of the transgenic PeUMPS cDNA in strain M4-1D was approximately one quarter of the expression of the original PeUMPS. Strain M4-1D grew faster than strains M4-1A and M4-1B in the presence of orotate. In contrast, no transgene expression was detectable in strains M4-1A and M4-1B, which grew very slowly even in the presence of orotate. These results suggest that the slow growth of the transgenic strains on MA5 minimal agar plates was due to the low expression of the transgenic PeUMPS cDNA.Fig. 3


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)

Real-time PCR analysis of the expression of PeUMPS in (a) pUT1 and (b) pUT2 transgenic strains. 18S rRNA was used as an internal control. Strain M4 is the parental strain of the transgenic strains. Real-time PCR detected expression from the defective PeUMPS in strain M4 in addition to the transgenic PeUMPS cDNA. The data are presented as fold changes in PeUMPS expression normalized to 18S rRNA expression in transgenic strains compared with strain M4. Bars represent the standard deviation of three replicates
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Real-time PCR analysis of the expression of PeUMPS in (a) pUT1 and (b) pUT2 transgenic strains. 18S rRNA was used as an internal control. Strain M4 is the parental strain of the transgenic strains. Real-time PCR detected expression from the defective PeUMPS in strain M4 in addition to the transgenic PeUMPS cDNA. The data are presented as fold changes in PeUMPS expression normalized to 18S rRNA expression in transgenic strains compared with strain M4. Bars represent the standard deviation of three replicates
Mentions: To evaluate the correlation between the expression of PeUMPS cDNA and the growth speed, the expression of PeUMPS cDNA in the four transgenic strains was analyzed using real-time PCR and normalized against 18S rRNA levels (Fig. 3a). The PCR primers Umps_QRT_F and Umps_QRT_R were used to detect both the expression of transgenic PeUMPS cDNA and the expression of defective PeUMPS in the chromosome of strain M4. Strain M4-1C, which grew fastest (its colonies were visible in 6 weeks), exhibited a 1.5-fold higher expression than did strain M4. It suggested that the expression of the transgenic PeUMPS cDNA in strain M4-1C was 1.5−1-fold of the expression of the original PeUMPS. Similarly, the expression of the transgenic PeUMPS cDNA in strain M4-1D was approximately one quarter of the expression of the original PeUMPS. Strain M4-1D grew faster than strains M4-1A and M4-1B in the presence of orotate. In contrast, no transgene expression was detectable in strains M4-1A and M4-1B, which grew very slowly even in the presence of orotate. These results suggest that the slow growth of the transgenic strains on MA5 minimal agar plates was due to the low expression of the transgenic PeUMPS cDNA.Fig. 3

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