JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

3-25
Analysis of Mutation Induced by Ion Beams and
Gamma-Rays in Vacuum-dried Conidia of
Aspergillus oryzae
K. Ito a) , Y. Hanya a) , K. Satoh b) and I. Narumi b)
a) R&D Division, Kikkoman Corp., b) Radiation-Applied Biology Division, QuBS, JAEA
Introduction
It is known that the mutation spectrum varies depending
on the mutation methods and conditions. We demonstrated
that ion-beam mutagenesis had a high mutation frequency
and a broad mutation spectrum compared to UV irradiation
1–2)
in Aspergillus species, previously . However, those
irradiation experiments were carried out against germinated
conidia growing on Malts agar plate, which generated easily
hydroxyl radicals that influence the mutation spectrum.
One way to maximize the characteristics of ion-beam
mutagenesis is to irradiate organisms in a dry form, by
which hydroxyl radicals generated by water radiolysis, is
kept to the minimum. In this report, we obtained 5FOA
(5-fluoroorotate) resistant mutants from vacuum-dried
conidia as a dry form to investigate the effect of irradiation
condition (irradiation to a wet form or to a dry form) on
mutation pattern. Moreover, we investigated mutagenesis
of vacuum-dried conidia that were irradiated with gamma
rays for a comparison purpose.
Materials and Methods
(1) Survival rate
Survival rate was calculated by counting the number of
colonies regenerated from constant number of conidia with
or without irradiation. The suspension containing 1 × 10 6
conidia (A. oryzae RIB40) was filtrated on a membrane filter
(pore size was 0.45 μm) and dried up in a vacuum desiccator.
The vacuum-dried conidia were irradiated with 12 C 5+
(220 MeV, 121.8 keV/μm) ion beams in a range of dose
from 100 to 400 Gy, or with gamma rays in a range from
100 to 1,200 Gy. Irradiated conidia were resuspended in
0.01% Tween80 solution. The suspension were diluted and
spread on Malts agar plate. The number of colonies was
counted after incubation at 30 °C for 3 days.
(2) Isolation of 5FOA resistant mutants
5FOA resistant mutants were isolated using the same
procedure previously described 2) .
(3) DNA analysis of 5FOA resistant mutants
The pyrG gene (orotidine 5’-phosphate decarboxylase)
and pyrF gene (orotate phosphoribosyl transferase) were
amplified from 5FOA resistant mutants by PCR. The
amplified fragments were cloned and sequenced to detect
mutation pattern.
Results and Discussion
(1) Survival rate and isolation 5FOA mutants from vacuum-
dried conidia irradiated with ion beams or gamma rays
Figure 1 shows the survival rate of vacuum-dried conidia
JAEA-Review 2010-065
- 81 -
irradiated with ion beams and gamma rays. The survival
rate of gamma irradiation was much higher than that of
ion-beam irradiation at the higher dose (over 300 Gy).
Twenty mutants that resisted to 5FOA with uracil
auxotrophy were obtained from ion-beam irradiated conidia.
On the other hand, no mutant was obtained from conidia
irradiated with gamma rays. In vacuum-dried conidia,
induction of mutation by ion beams might be more powerful
tool than gamma rays.
(2) Mutation analysis of 5FOA mutants from the
vacuum-dried conidia irradiated with ion beams
Among 20 mutants, either pyrG gene or pyrF gene was
not amplified in 8 mutants, and both of genes were not
amplified in 5 mutants. The large structural genomic
alteration, such as deletion, inversion or translocation, might
occurred around the region of each gene in these mutants.
Those mutants were not obtained from germinated conidia
that were irradiated with ion beams 2) . Although pyrG gene
and pyrF gene locates on different chromosome, it is
interesting that the mutants without amplification of both
genes were obtained. The spatial arrangement of each gene
might be close in nucleus. Among other 7 mutants, 5
mutants had deletion mutations (2 mutants were pyrG
deficient and 3 mutants were pyrF deficient). A lot of
mutants from germinated conidia with ion-beam irradiation
were single base substitution 2) , whereas only one mutant
from the vacuum-dried conidia was so.
In conclusion, the mutation pattern varied with
irradiation condition and complex structural genomic
alteration induced by ion-beam irradiation tended to occur in
the vacuum-dried conidia (a dry form) rather than
germinated conidia (a wet form).
Survival rate
E 0 0 200 400
Dose (Gy)
600 800 1000
gamma rays
ion beams
1200
E -1
(log)
E -2
E -3
Fig. 1 Survival rate rate of of vacuum-dried conidia conidia after after
irradiation irradiation with ion beams or and gamma rays rays in A. in oryze A.
RIB40. oryze RIB40.
References
1) K. Ito et al., JAEA Takasaki Ann. Rep. 2006 (2008) 94.
2) K. Ito et al., JAEA Takasaki Ann. Rep. 2007 (2008) 86.