Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 47 (2014)

Mutation rates of Parachlorella kessleri by heavy-ion-beam irradiation andclassification of their genomic deletion types
T. Yamazaki,*1,*2 S. Ota,*1,*2 K. Oshima,*1 M. Hattori,*1 Y. Kazama,*3 T. Abe,*3,*4 S. Kawano,*1,*2
Consumption of fossil fuels such as petroleum oil not only
depletes a finite resource but also increases carbon dioxidelevels, which causes global warming. Because of carbon
fixation, plants can contribute to the reduction of carbondioxide levels. Moreover, biofuels from plant have been
considered as substitutes for fossil fuels. Microalgae, which
perform photosynthesis like terrestrial plants, have been
attracting attention as a feedstock of biodiesel production inrecent years. To increase the production of biomass andimprove the amount of biofuel from microalgae, thecommon process used is the isolation of strains with highbiomass productivity and/or high oil content from thenatural environment and optimization of culture conditions
for the isolated strains. However, this strategy does notnecessarily obtain the best strains. Therefore, we artificiallymodified the genome of microalgae by using theheavy-ion-beam irradiation. The heavy-ion beam isconsidered a mutagen that causes the partial deletion ofgenomic DNA very effectively, and it can also be used tobreed a variety of organisms, including flowering plants.
Because little is known about breeding of unicellular
microalgae by using heave-ion-beam irradiation, evaluationof its effectiveness is necessary.
In this study, we used a green microalga, Parachlorella
kessleri, the draft genome of which has been determined byour group. A search for the draft genome sequence revealed
that NR, NRT and NIR are single-copy genes, and NAR isduplicated into two genes (NAR1-1, NAR1-2). The
transcript of NAR1-1 was not detected in transcriptome data,suggesting that nitrate is metabolized to ammonia through asingle pathway (NRT-NR-NAR-NiR pathway) in P. kessleri(Fig. 1A). Therefore, a P. kesseleri mutant that can grow in
an ammonia-containing medium but not in anitrate-containing medium is considered to have a defectivein the nitrate assimilation pathway.
P. kessleri cells were irradiated by heavy-ion beams ofdifferent doses and nuclear species, and then screening wasperformed using following steps: (1) a single clone derived
from irradiated P. kessleri cells was inoculated in a TAP
medium containing ammonia1); (2) After the P. kessleri cells
were grown, they were inoculated in both TAP medium anda modified TAP medium containing only nitrate as anitrogen source; (3) a putative mutant grow in the TAP
medium, but not in the modified TAP medium. Doses andnuclear species used for the screening are carbon ions the*1

Department of Integrated Biosciences, Graduate School of Frontier
Sciences, University of Tokyo*2JST, CREST*3
RIKEN Innovation Center*4
RIKEN Nishina CenterA

B

Figure 1. (A) Nitrate assimilation pathway. NRT, nitratetransporter; NR, nitrate reductase; NAR, nitrite transporter;
NiR, nitrite reductase. (B) Phenotype of the mutant
requiring annmonium. Wild type and mutant P. kessleri
cells of wild type and mutant were grown on a medium
containing ammonium or nitrate. After one week, theircolonies were photographed.
(50 Gy, 25 Gy) and argon ions (25 Gy, 50 Gy). Fig. 1Bshows a representative phenotype of the mutant isolated
from the heavy-ion-beam irradiated mutant that requires
ammonia as a nitrogen source.
A large-scale of screening was performed, and the mutants
were isolated from about 4000 clones for 25 Gy and 50 Gyof argon-ion beams, and 25 Gy and 50 Gy carbon-ion
beams, respectively. As a result, the mutants requiringammonia from the individual doses and nuclear species
were obtained each 0, 4, 3 and 4, respectively. The
incidence in each experiment was 0.00, 0.10, 0.07, and0.10% (Table 1). In the future, we plan to calculate themutation rate of the irradiation by iron-ion beams.
Table 1. Mutation frequency by heavy-ion-beam irradiation
Nuclearspecies
CDose (Gy)

2550
Ar
2550
*Ota et al. (2013)

Isolated clone
numberPositive clone
numberFrequency(%)

Survival
rate (%)*

4005
4007
4007
4012

043
40.00
0.10
0.07
0.10

n.d.
59
50
28

Next, the PCR fragment analysis and restriction fragment
length polymorphism-PCR analysis were performed for theNR gene. However, there was no difference between thewild-type and all mutants, suggesting that the deletions ofgenomic DNA by heavy-ion-beam irradiation are very small
in microalgae. Currently, we are performing re-sequence
analysis using the next generation sequencer IonProton for
the 11 mutants, expecting a detailed comparison of thedeletion pattern of a set of genes involved in nitratemetabolism, including the NR gene.
This study was supported by JST, CREST (to SK).
References1) Ota et al. : Bioresour Technol. 149:432-438. (2013)- 301 -