全 文 ：　遗 传 学 报　Acta Genetica Sinica , April 2004 , 31 (4):335 ～ 339 ISSN 0379-4172
收稿日期:2003-06-03;修回日期:2003-11-03
基金项目:国家自然科学基金项目资助(编号:30070376)[ Supported by the National Natural Science Foundation of China(No.30070376)]
作者简介:韩永华(1971-),吉林双阳人 ,博士 ,专业方向:植物分子细胞遗传学。 E-mail:hanyhzhou@sohu.com;Tel:0773-5833710
①　通讯作者。 E-mail:songyc1936@yahoo.com.cn;Tel:027-87684505
Comparative Physical Localization of Maize mir1 Gene
in Zea mays L.and Coix lacryma-jobi L.
HAN Yong-Hua1 ,2 , WANG Xiao-Lan1 , LIU Li-Hua1 , SONG Yun-Chun1 , ①
(1.TheKey Laboratory of MOE for Plant Developmental Biology , Wuhan University , Wuhan　430072 , China;
2.College of Life Sciences , Guangxi Normal University , Guilin　541004 , China)
Abstract:The maize gene mir1 encoded a cystein proteinase which is resistant to fall armyworm.Previously , RFLP map indicated
that the mir1 was mapped on chromosome 6.However , physical location of the mir1 gene on chromosome 6 has not been reported.
In this study , the mir1 gene was physically located on the short arm of metaphase and pachytene chromosome 6 by dual-color fluo-
rescence in situ hybridization(FISH)with 45S rDNA as a reference marker.The results of Southern blotting suggested that there
were sequences homologous to mir1 in Coix lacryma-jobi L.genome.Then , the sequences were mapped on the distal region of long
arm of chromosome 7 in C.lacryma-jobi by FISH.The percent distance from the signal site to centromere was 73.33±0.15.
Key words:mir1 ;maize;Coix lacryma-jobi L.;fluorescence in situ hybridization(FISH)
玉米 mir1 基因在玉米和薏苡中的比较物理定位
韩永华1 , 2 , 王小兰1 , 刘立华1 , 宋运淳1 , ①
(1.武汉大学植物发育生物学教育部重点实验室 , 武汉　430072;
2.广西师范大学生命科学学院 , 桂林　541004)
摘　要:玉米基因 mir1 编码一种抗秋季黏虫的半胱氨酸蛋白酶。利用 RFLP作图 mir1 基因被定位在玉米第 6 号
染色体短臂上 ,但它在第 6 号染色体短臂上的物理位置还不知道。实验以 mir1 和 45S rDNA为探针 , 通过双色荧光
原位杂交技术确定了 mir1 基因在玉米细胞分裂中期和粗线期第 6 号染色体上的物理位置。 Southern 杂交结果表
明 ,在薏苡基因组中存在 mir1 基因的同源序列 , 进一步利用荧光原位杂交的方法确定 mir1 基因的同源序列定位于
薏苡第 7号染色体长臂的近末端 , 其信号与着丝粒的百分距离为 73.33±0.15。
关键词:mir1 ;玉米;薏苡;荧光原位杂交
中图分类号:Q343　　　文献标识码:A　　　文章编号:0379-4172(2004)04-0335-05
　　Comparative genetic studies have demonstrated that
gene content and orders are highly conserved within grass
family[ 1] .Even between different distant families , many
functional genes are highly conserved in structure and
function[ 2] .The comparative genetic maps have opened
the door to determine the relative order of homologous se-
quences along the chromosomes of distantly related species
with a level of detail and accuracy previously unattain-
able , and establish a framework for ultimately connecting
the genetics of different species
[ 2] .However ,genetic maps
could only show the relative positions and distances of
genes and molecular markers on chromosomes based on
the frequency of recombination.Therefore , a map position
gives only a rough estimate about the actual physical posi-
tion of a gene or marker on chromosomes.But physical
maps show real positions of genes or markers on the chro-
mosomes.The capability to physically localize a DNA-se-
quence and a gene will be valuable for estimating dis-
tances between markers and genes , analyzing the structure
of chromosomes and the mechanism of recombination.
Among the many economically important plants ,
maize is one of the most suitable materials for development
of a physical map by FISH because pachytene chromo-
somes of maize can be obtained and identified easily[ 3]
and have been widely used for cytogenetic analysis ,more-
over the genetic maps and YAC library are available.Up
to now , very few studies have been reported concerning
physical mapping of genes of agronomic interests in
maize
[ 4～ 6] .The maize mir1 (1 463 bp)gene encodes a
33 kDa cystein proteinase which is resistant to fall army-
worm.Southern analysis and RFLP mapping indicate the
mir1 is single-copy gene that maps on chromosome 6 at
bin 6.02 near umc59a[ 7] .However , the genetic analysis
isn t clear about the actual location of the mir1 gene on
chromosome 6.In present study , the mir1 was physically
mapped onto maize metaphase and pachytene chromosome
by dual-color fluorescence in situ hybridization (FISH)
with rDNA as a reference marker.We also analyzed its
homology and physical location in C.lacryma-jobi which
is a close relative of maize.
1　Material and Methods
1.1　Plant materials and chromosome preparation
Maize(Zea mays L.)inbred line Huangzao 4 , de-
rived from a native cultivar in China ,was donated by Pro-
fessor Song Jian-cheng (Shandong Agricultural Universi-
ty).Coix lacryma-jobi L.var.frumentacea Makino ,which
was a cultivated variety of C.lacryma-jobi ,was supplied
by professor Li jian-sheng (Department of Agronomy ,
Huazhong Agriculture University).
Mitotic metaphase and prometaphase chromosomes of
maize and C.lacryma-jobi were prepared using the proto-
plast technique as described by Song and Gustafson [ 8].
Pachytene chromosomes of maize were prepared from an-
thers at pachytene stage according to Zhong et al.[ 9] .
1.2　Probes and labelling
The mir1 cDNA cloned in pUC19(isolated from a
callus cDNA library from the maize inbred Mp708)and
45S rDNA cloned in the vector pUC18[ 10] were used.The
probes were labeled with biotin and digoxigenin , respec-
tively , following the nick translation protocol detailed in
the kit (Sino-American Biotechnology Company , Luoyang
City ,China).Dot blots were performed to detect the label-
ing efficiency.
1.3　Southern blotting
The DNA isolation of C.lacryma-jobi and the South-
ern blotting were performed as described by Doyle[ 11] and
Sambrook et al.[ 12] , respectively.The mir1 cDNA was la-
beled with 32P-dCTP followed the procedure of nick trans-
lation kit manual (Promaga , USA).The genomic DNA of
C.lacryma-jobi was digested with Hind Ⅲ and EcoRⅠ
respectively , fractionated by electrophoresis in 0.8% a-
garose gel , and transferred onto a nylon membrane (Hy-
bond N+).Hybridization was performed overnight in 6×
SSC ,5×Denhardt , 0.5%SDS , 100 μg/mL ssDNA)50
r/min at 62℃ for 18 h.Then the membrane was washed
with solution I(2×SSC and 0.1%SDS)for 3×10min ,
solution Ⅱ(0.1×SSC and 0.1% SDS)for 3×10 min
and solution Ⅲ(0.1×SSC)for 10 min , and then placed
for autoradiography for a week at -70℃.
1.4　In situ hybridization and fluorescence detection
　　Hybridization was performed as outlined by Song and
Gustafson[ 8] ,with an additional 5μg/mL pepsin treatment
for 10min.Biotinylated probes were detected with strepta-
vidin-Cy3 (Kirkegaard Perry Laboratories), followed by
biotinylated streptavidin(Vector Laboratories)and finally
with streptavidin-Cy3.Digoxigenin-labeled probes were
detected with anti-digoxigenin-FITC (fluorescein isothio-
cyanate;Boehringer Mannheim)and amplified by rabbit
anti-sheep-FITC (Vector Laboratories).Each detection
step took place at 37℃for 30min and slides were washed
with phosphate buffered saline between each step.After
immunological reactions , the slides were counterstained
with 1 μg/mL DAPI in 20% Vectashield.Chromosomes
were examined with an Olympus BX60 fluorescence mi-
croscope equipped with Sensys 1401E cooled CCD camer-
a.Red , green , and blue images were captured in black and
white with G ,B , and UV exciter filters , respectively.The
336 遗传学报　Acta Genetica Sinica　Vol.31　No.4　2004
images were combined and pseudo-coloured using software
V++(Digital Optics , Auckland , New Zealand).Pro-
cessing of images was accomplished using Adobe Photo-
shop 6.0.
2　Results
For establishing the physical position of mir1 in
maize by FISH , it was important to identify chromosome
6.The chromosome 6 could easily be recognized because
nucleolar organizer region(NOR)locates on the short arm
of chromosome 6(at bin 6.01)and NOR which is com-
posed of tandem arrayed 45S rDNA is a good recognition
marker for chromosome 6.Fig.1 ,A ～ B showed the physi-
cal locations of mir1 (red signals)and 45S rDNA(green
signals) on the maize metaphase (Fig.1 , A) and
pachytene (Fig.1 , B) chromosome 6 by double-color
FISH.The results showed that the mir1 andNOR were al-
most overlapping on the mitotic metaphase chromosome 6.
They were separated at certain distance on pachytene
chromosome.
Fig.1　FISH results of maize mir1 gene on Zea mays L.and Coix lacryma-jobi L.
Chromosomes A , B:Hybridization signals of mir1 (red signals , indicated by arrows)and 45S rDNA(green signals , indicated by
arrowheads)were detected simultaneously in maize metaphase and pachytene chromosome 6;C ,D:Hybridizat ion signals of mir1
(red signals , indicated by arrows)were detected on the long arm of chromosome 7 of C.lacryma-jobi.
337HAN Yong-Hua et al.:Comparative Physical Localization of Maize mir1 Gene in Zea mays L.and Coix lacryma-jobi L.
　　When mir1 was hybridized to C.lacryma-jobi total
DNA digested with HindⅢ and EcoRⅠ , three bands and
two bands had been detected respectively (Fig.2:A ,B).
The result of Southern blotting confirmed that there existed
sequences homologous to maize gene mir1 in C.lacryma-
jobi.It provided a foundation for further mapping the ho-
mologous sequences on C.lacryma-jobi chromosomes with
FISH.
Fig.2　The results of Southern hybridization of
mir1 with genomic DNA in C.lacryma-jobi
Digestedwith HindⅢ(A)and EcoRⅠ(B).
The homologous sequences of mir1 were repro-
ducibly detected on distal region of long arm of one ho-
mologous chromosome pair in C.lacryma-jobi (Fig.1:C ,
D).For C.lacryma-jobi , the recognition of the metaphase
chromosome was difficult due to chromosomal similarity in
morphology.However , prometaphase chromosomes of C.
lacryma-jobi showed obvious DAPI differential staining.
Based on the DAPI fluorescence banding patterns ,we have
constructed a quantitative chromosome map of C.lacry-
ma-jobi[ 13] .Based on the map with chromosomal relative
length and arm ratio , we confirmed that the mir1 was
mapped on the long arm of chromosome 7 of C.lacryma-
jobi.The arm ratio of chromosomes with mir1 signal was
1.17±0.05 ,which agrees with the arm ratio of C.lacry-
ma-jobi chromosome 7 on quantitative chromosome map.
The chromosome 7 with intense DAPI fluorescence on the
whole chromosome was also easily distinguished from some
chromosomes , which showed faint DAPI fluorescence on
the terminal regions.The percent distance from the signal
site to centromere was 73.33 ±0.15.The hybridization
signals were simultaneously detected on two sister chro-
matids of two members of the homologous chromosomes 7
but the case was very infrequent.The hybridization signals
usually appeared on one chromatid or two sister chro-
matids of one member of the homologous chromosome
pair.
The mir1 and NOR locate on the same chromosome
in Zea mays L., but they are on different chromosomes
and NOR locates on the short arm of chromosome 2 in C.
lacryma-jobi[ 13] .This shows there are quite difference on
genes arrangement between genomes of Zea mays L.and
C.lacryma-jobi although they are close relatives.
3　Discussion
The integration of physical and genetic map is a goal
for maize , as well as for other crop plants.Fluorescence in
situ hybridization is a rapid and reliable method for physi-
cally mapping DNA sequences.Using this method , a com-
prehensive map of maize may be generated by integrating
the physical map with the genetic map.Such a map will
be extremely valuable for isolating genes as well as for un-
derstanding events of recombination and rearrangement.In
contrast to the relatively easy localization of repetitive se-
quences on chromosomes in many plant species ,unequivo-
cal mapping of single-copy sequences to plant chromo-
somes has proven considerably difficult[ 14 , 15] .Undoubted-
ly ,FISH with heterologous genes as probes is more diffi-
cult.It is hard to achieve a satisfied experimental result
without the probes with enough length and high conserva-
tion , and the support of perfect technology in this field.
Moreover , the ultimate shortcoming of FISH is that the flu-
orescence signal is easily quenched and it is difficult to
catch.The signal might quench even before observed.This
is one of the essential factors causing lower rate of detec-
tion.In our experiments , appropriate anti-quenching agent
Vectashield ,multiple cascade amplification of signals and
sensitive cooled charge-coupled device (CCD) camera
were being applied which were helpful to detect and catch
weaker signals.Therefore ,we succeeded to locate the mir1
on maize and C.lacryma-jobi chromosomes.
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339HAN Yong-Hua et al.:Comparative Physical Localization of Maize mir1 Gene in Zea mays L.and Coix lacryma-jobi L.