全 文 ：
第 35卷第 6期 湖南农业大学学报(自然科学版) Vol.35 No.6
2009 年 12 月 Journal of Hunan Agricultural University (Natural Sciences) Dec．2009
文章编号：1007-1032(2009)06-0606-05
Mutation in Arabidopsis replication factor C Subunit 3 compromises
plant resistance to ultraviolet bombardment
XIA Shi-toua，b，CHENG Pinga，b，NI Jin-Kuib，YAN Dan-yangb，XUE Danb，LIANG Longb
(a.Hunan Provincial Key Laboratory of Phytohormones and Growth Development；b.College of Bioscience and
Biotechnology，HNAU，Changsha 410128，China)
Abstract：Replication factor C (RFC)，consisting of one large subunit and four small subunits，is an important factor
involved in DNA replication and repair mechanisms as well as cell proliferation. The subunit 3 of Arabidopsis RFC
(AtRFC3) is one of the 4 small subunits. In a former EMS (ethylmethane sulfonate) screening to find out more mutant
resistant to biotic and abiotic stresses，rfc3-1 was identified as a point mutant with G to A mutation occurred in the
second exon which compromised the resistance of rfc3-1 plants to ultraviolet. Transformation of wild type AtRFC3 gene
into rfc3-1 mutant reverted the mutant phenotypes，suggesting that AtRFC3 plays an important role in ultraviolet
resistance in Arabidopsis thaliana.
Key words：Arabidopsis thaliana；AtRFC3；complementation；ultraviolet resistance
拟南芥复制因子 C 亚基 3 基因突变削弱了
植物对紫外辐射的抗性
夏石头 a，b，成 平 a，b，倪金魁 b，颜丹阳 b，薛 丹 b，梁 龙 b
(湖南农业大学 a.湖南省植物激素与生长发育重点实验室；b.生物科学技术学院，湖南 长沙 410128)
摘 要：在前期以 EMS (甲基磺酸乙酯)筛选更多抗生物和非生物逆境的突变体试验中，发现了 1个从 G到 A的
点突变体 rfc3-1，该点突变削弱了植株抗紫外辐射的能力．将野生型拟南芥复制因子 C亚基 3基因转化到突变株
rfc3-1后恢复了突变株的野生型表型，证明紫外辐射抗性异常表型是由拟南芥复制因子 C亚基 3基因突变所引起
的，AtRFC3在拟南芥紫外辐射抗性中起重要作用．
关 键 词：拟南芥；复制因子 C亚基 3；遗传互补；紫外辐射抗性
中图分类号：Q754 文献标志码：A
Replication factor C(RFC) is a protein complex
with five subunits originally purified from the HeLa
cells as a host factor essential for the in vitro
replication of Simian virus 40(SV40) DNA[1-2].
Human RFC，also known as activator 1，can support
proliferating cell nuclear antigen (PCNA)-dependent
DNA synthesis by DNA polymerases δ and ε in the
presence of ATP ， and results in the efficient
elongation of DNA in the presence of human
single-stranded DNA-binding protein (hSSB). RFC
complex have been found in all eukaryotes and these
five different subunits were identified as one large
subunit (RFC140/RFC1) and four small subunits
(RFC37/RFC2 ， RFC36/RFC3 ， RFC40/RFC4 and
RFC38/RFC5)[3-7]．
In addition to its role in DNA replication，RFC
was reported to be involved in repair and check-point
control in cell cycle progression[8-10]. However，the
precise function of each subunit remains unclear，
especially in plants. In a former EMS screening to
收稿日期：2009-04-15
基金项目：国家自然科学基金项目(30970247)；湖南省
高校科学研究重点项目(09A037)；作物种质创新与资源利用
国家重点实验室培育基地开放项目；湖南农业大学稳定人才
基金(07WD14)
作者简介：夏石头(1971─), 男, 湖南衡阳人, 博士, 副
教授.
DOI:10.13331/j.cnki.jhau.2009.06.008
第 35卷第 6期 夏石头等 拟南芥复制因子C亚基 3基因突变削弱了植物对紫外辐射的抗性 607
find out more mutant resistant to biotic and abiotic
stresses，rfc3-1 was identified as a point mutant with
G to A mutation occurred in the second exon which
change the nonpolar aliphatic Gly-84 to a negative
charged Asp[11]. The mutant was also found to be less
resistant to ultraviolet， a common abiotic stress
besides its smaller configuration than that of the wild
type. This suggests an essential role of AtRFC3 in the
resistance to ultraviolet bombardment in Arabidopsis
thaliana.
1 Materials and methods
1.1 MS culture medium and MS plate
MS culture medium：4.4 g/L MS+0.5% sucrose+
0.8% agar ， pH 5.6~5.8. When the agar melted
completely，the liquid compound was moved out from
the microwave oven，cooled to 50~60 °C in the hood，
added with 100 μg/mL Amp and mixed well. Then the
medium was added to the sterile plastic or glass plate
to make MS plates. After the medium solidified in the
hood，the plates were then sealed in the sterile plastic
bag and stored at 4 °C for next step use.
1.2 Seed sterilization and plant growth
Seeds of the wild type and rfc3-1 mutant were
sterilized in sterilization solution (15% Chlorox+0.1%
Tween 20) for about 10 min，washed twice with sterile
water，resuspended in 0.1% sterile agar. To ensure the
same growth conditions ， the MS plates were
partitioned into two parts with mark pen，one half for
rfc3-1 mutants planting，and the other half for wild
type plants or the complementary plants (rfc3-1/rfc3-
1::RFC3) planting. Seeds were planted carefully and
evenly on MS plates in the hood， labelled， dried and
sealed with parafilm，then stored at 4 °C for vernaliz-
ation for 2~3 d. The plates were transferred to the
growth chamber for germination and growth under the
conditions of 16 h day (23 °C)/8 h night (21 °C).
1.3 Complementary construct reconstruct
The whole sequence of RFC3 genomic DNA was
divided into two fragments (fragment1 and fragment
2 )for PCR amplification using PhusionTM high-
fidelity PCR master mix (NEB) because of its large
size (about 4.9 kb). As there is an overlap area of 354
bp between fragment1 and fragment 2 and within
which the only one ClaⅠrestrict enzyme site is
located，it will not change the original nucleotide
sequece of RFC3 when the two fragments are digested
with ClaⅠand ligated into one fragment，thus the
right RFC3 gene could be cloned. PCR amplification
was carried out using the following conditions: 40
cycles of 10 s at 98 ºC，30 s at 55 ºC，3.5 min at 72 ºC
for fragment1(about 3.2 kb) and 40 cycles of 10 s at
98 ºC，30 s at 55 ºC，2.5 min at 72 ºC for fragment 2
(about 2.1 kb). The primer pairs used for amplification
of fragment 1 and fragment 2 sequences were
5′ cgcggatcccgtcctgcaaatgctgatga 3′ with 5′cgggagctc
acctatatgctcactgaagg 3′，and 5′cggggtaccacatggctgg
accagcagag 3′ with 5′cgcgtcgacagctcacgcccatcacaatg
3′. The PCR products were run on the gel，and the two
fragments were purified using QIAGEN gel extract kit
(QIAGEN Company) and digested with BamH andⅠ
ClaⅠ，or Kpn and Ⅰ Cla . After ligation into pG229 Ⅰ
vector (4.5 kp) digested with Kpn and Ⅰ BamHⅠ(all
of the restrict enzymes used here were from
Invitrogen Co.) ， the reconstructed construct was
transformed into Escherichia coli strain DH10B and
the positive clone was confirmed by restrict enzyme
digestion and DNA sequencing.
1.4 Transformation and transformant screen
The confirmed reconstructed construct was
transformed by electroporation into Agrobacterium
tumefaciens strain GV3101 and then transformed back
into RFC3 heterozygous plants， i.e. rfc3-1/RFC3
plants，using floral-dipping method. The transformed
plants were cultured in the growth chamber under the
conditions of 16 h day (23 °C)/8 h night (21°C) for 30
d before harvest. After drying for about one week
under room temperature，the seeds were sterilized and
sown in sterile cultural soil following the procedure of
section 1.1. The transformants were then screened by
spraying Basta and transferred to new cultural soil for
further assay. The primer pairs used for complem-
entary line check were designed as follows:5′
608 湖南农业大学学报(自然科学版) 2009年 12月
tatggtcctcccggtactcg3′ (wt) and 5′cactcctggacgaatat-
gtc3′ for wild type RFC3 check； 5′tatggtcctccc-
ggtactca3′ (mt) and 5’cactcctggacgaatatgtc3′ for rfc3-1
mutation site check. As there is only one base pair
mismatch when the wt primer pairs anneal to wild
type RFC3 sequence ， a fragment still can be
amplified，however，no fragment can be amplified
when the wt primer pairs anneal to the mutant rfc3-1
sequence because there are two base pairs mismatch.
To the contrary，a fragment will be amplified for the
mt primer pairs to the mutant rfc3-1 sequence，but no
fragment can be amplified for the mt primer pairs to
the wild type RFC3 sequence. As a result，if there is
only one band when use the wt or mt primer pairs，the
plant would be wild type or mutant homozygote，if
there are two bands，the plant should be heterozygote.
The identified rfc3-1 mutant homozygote with wild
type morphology must be the complementary plants
(rfc3-1/rfc3-1::RFC3).
1.5 Ultraviolet bombardment
After 4 days growth under the same conditions，
the wild type，rfc3-1 mutant and rfc3-1/rfc3-1::RFC3
complementary plants were treated with ultraviolet
(200~320 nm) for 0 min (0 kJ/m2)，20 min (12.96 kJ/
m2)，40 min (25.92 kJ/m2)，60 min (38.85 kJ/m2)， 80
min (51.84 kJ/m2) and 120 min (77.76 kJ/m2)，
respectively. Then the plates were sealed with
parafilm again and cultured in the growth chamber
under the conditions of 16 h day (23 °C)/8 h night
(21 °C) for another 5~7 d. Then the completely dead
and living seedling were counted respectively and the
survival rate of seedlings of the wild type and rfc3-1
mutant was calculated accordingly. This experiment
was repeated 4 times.
2 Results
2.1 PCR verification of rfc3-1 mutant
To study the roles of RFC3 in growth and
development，the rfc3-1 mutant were planted on soil
in the growth room under the conditions of 16 h day
(23 °C)/8 h night (21 °C). Two-week-old seedlings
were used for the extraction of DNA for mutation
verification. Fig.1 A shows some typical rfc3-1
mutant homozygote and wild type plants grown for
about 20 d. Specific PCR primers were designed to
check the presence of the mutation site (Fig.1 B).
Fig.1 B shows some typical PCR results of the
mutation verification. Because the mutation in rfc3-1
is recessive， the plant with typic specific rfc3-1
mutation band（Fig.1 B，a1~a7）but without the
corresponding wild type band（Fig.1B，b1~b7）in PCR
check should be rfc3-1 homozygote. The rfc3-1
homozygotes were found to have small configuration
with narrow and long leaves，comparing with the wild
type plants (Fig.1 A). And these homozygotes verified
were set for seeds for further assay.
A：20-d-old typical rfc3-1 mutant homozygote and wild type plants；
Wt：wild type plant，rfc3-1, mutant homozygote；B：PCR check of the rfc3-1
mutant. a1~a7：mt primer pairs check；b1~b7：wt primer pairs check；a1 and
b1 is the same seedling，and the same as the other 6 seedlings
Fig.1 Morphology and PCR check of the rfc3-1 mutant and wild type
plants
图1 rfc3-1突变体和野生型植株形态特征和PCR检验结果
2.2 Resistance to ultraviolet bombardment of
rfc3-1 mutant was compromised
For ultraviolet bombardment assay，the 4-day-old
plants of the wild type and rfc3-1 mutant were treated
with 0 min (0 kJ/m2)，20 min(12.96 kJ/m2)，40 min
(25.92 kJ/m2)，60 min (38.85 kJ/m2)，80 min (51.84
kJ/m2) and 120 min(77.76 kJ/m2) respectively，and the
survival rate of seedlings was counted(Fig.2). The rate
of rfc3-1 mutant began to decrease rapidly when the
wt
rfc3-1
A
B
a1 a2 a3 a4 a5 a6 a7 b1 b2 b3 b4 b5 b6 b7
第 35卷第 6期 夏石头等 拟南芥复制因子C亚基 3基因突变削弱了植物对紫外辐射的抗性 609
Wt：wild type；rfc3-1：mutant；rfc3-1/rfc3-1::RFC3：complementary
plants
Fig. 2 Seedlings survival rate under UV bombardment
图 2 紫外线照射处理后的幼苗成活率
seedlings were treated with ultraviolet for 40
min(25.92 kJ/m2). After being treated with ultraviolet
for 60 min (38.85 kJ/m2)，the survival rate of rfc3-1
mutant reduced to less than 60%，moreover，when the
treatment lasted for 120 min(77.76 kJ/m2) ， the
survival rate of rfc3-1 mutant was found to less than
40%. It is indicated that nearly 60% of the mutant
seedling died. However，the survival rate of wild type
plants were not affected for 40 min(25.92 kJ/m2)
treatment of ultraviolent bombardment.When seed-
lings were treated with ultraviolet for 60 min (38.85
kJ/m2)，the survival rate of wild type plants began to
decrease slowly. Impressively，when the ultraviolet
treatment lasted for 120 min(77.76 kJ/m2)，the wild
type plants still had 75% survival rate，and that was
35% higher than that of the rfc3-1 mutant plants under
the same conditions， indicating that RFC3 gene
mutation causes aberrant resistance to ultraviolet
bombardment.
2.3 Phenotypic complementation of rfc3-1 mutants
by wild type RFC3 transformation
To confirm whether the seedling resistance
defects in phenotype are caused by point mutation of
RFC3，the whole RFC1 genomic DNA together with
the native promoter (about 4.9 kb) was amplified and
verified (Fig.3A) by PCR. After retracted and purified
using QIAGEN gel extract kit (QIAGEN Company)，
the fragment was digested with KpnⅠand BamHⅠ
and fused into plant expression vector pG229 digested
with the same restrict emzymes. Fig.3 B shows that
the size of the positive reconstructed plasmid pG229-
RFC3 (about 9.4 kb) is right. To further confirm the
positive reconstructed plasmid，pG229-RFC3 plasmid
was digested with Hind Ⅲ，and the electrophotogram
A：PCR amplification of genomic RFC3；M：mark；1, 2, band of
genomic RFC3 ； B ： the reconstructed plasmid of pG229-RFC3 for
complementation. M：mark；1, 2, band of pG229-RFC3 plasmid；C：
pG229-RFC3 plasmid digestion with Hind Ⅲ；M：mark；1：positive clone；
2：negative control
Fig.3 Cloning of RFC3 gene
图3 RFC3基因的克隆
of the digestion products suggests that clone 1 is the
right positive clone (Fig.3 C). Then the positive
reconstructed plasmid was sequenced and transformed
back into the heterozygous plants with the RFC3
mutant allele，rfc3-1， by Agrobacterium tumefaciens
mediating floral-dipping method. After two rounds of
Basta screening and specific primer PCR validation
(details not shown) ， the complementary plants
(rfc3-1/rfc3-1::RFC3) were used for ultravilet
bombardment assay follow the same procedure of
section 1.5 (Fig.2). There no big difference was found
between rfc3-1/rfc3-1::RFC3 and wild type plants，
indicating that the mutant phenotype in ultraviolet
resistance was reverted by complementation.
3 Discussion
The roles of replication factor C as a whole (RFC)
in DNA replication ， repair and the cell cycle
progression and development of multicellular
organisms have been intensively studied in animals[1-5].
But the precise function of each subunit remains
unclear，especially in plants. Furukawa and colleagues
found by Northern blot analysis and in situ
hybridization that all the OsRFC subunits were
homogenieously expressed in meristematic tissues of
rice but the expression patterns differed between
M 1 2 1 2 M 1 2 M
A B C
0
20
40
60
80
100
120
wt
rfc3
rfc3-1/rfc3-1::RFC3
12080604020
Time (min)
Su
rv
iv
al
o
f s
ee
dl
in
gs
/%
610 湖南农业大学学报(自然科学版) 2009年 12月
different tissues[6-7]. However，the significance of this
difference to the biological function is so far unknown.
In a former study[12]，we used Arabidopsis as a model
system to understand the biological significance of the
subunits of RFC in higher plants，and found that
AtRFC1 ， the big subunit of RFC complex in
Arabidopsis thaliana，plays an important role in the
process of embryo development. RFC1 gene mutation
causes embryo development defect and embryo
abortion，and embryo abortion leads to seed abortion.
Here，rfc3-1 was identified as a point mutant with
G to A mutation located in exon 2 in a former EMS
screening to find out more mutant resistant to biotic
and abiotic stresses. The configuration of the mutant
was found to be smaller than that of the wild type.
Interestingly，the survival rate of rfc3-1 mutant under
ultraviolet bombardment was also found to decrease
rapidly when the treatment last for 40 min(25.92 kJ/
m2)， but the survival rate of the wild type plants were
not affected at this moment. When the treatment lasted
for 120 min (77.76 kJ/m2)，the survival rate of rfc3-1
mutant was less than 40 % and 35% lower than that of
the wild type plants under the same conditions. This
indicates that RFC3 gene mutation severely compro-
mised the plant resistance to ultraviolet bombardment.
After transforming the wild type RFC genomic DNA
back into the mutant，the complementary plants of
rfc3-1/rfc3-1::RFC3 showed the same resistance to
ultraviolet bombardment as the wild type plants under
the same conditions ， indicating that the mutant
phenotype in ultraviolet resistance was totally reverted
by complementation.This is consistent with the
important function of RFC in DNA replication，repair
and the cell cycle progression.Taken together，the
phenotype with ultraviolet resistance defect is caused
by the function loss of RFC3 protein and AtRFC3
plays an important role in the process of resistance to
ultraviolet bombardment. Further studies should be
focused on the understanding of the more detailed
mechanism of the biological function of AtRFC3 in
stress resistance in Arabidopsis.
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