全 文 ：Construction and Characterization of a Bacterial Artificial Chromosome
Library for Triticum boeoticum
CHEN Fan_Guo1 ,2 , ZHANG Xue_Yong1＊ , XIA Guang_Min2 , JIA Ji_Zeng1
(1.Key Laboratory of Crop Germplasm and Biotechnology , Ministry of Agriculture , Institute of Crop Germplasm Resources , Chinese Academy of
Agricultural Sciences , Beijing 100081 , China;2.Life Science School , Shandong University , Jinan 250100 , China)
Abstract: A bacterial artificial chromosome library has been constructed for Triticum boeoticum Boiss
(AbAb)using the bacterial artificial chromosome (BAC)vector pECBAC1.The library consists of about
170 000 clones.A random sampling analysis of 200 BAC clones indicates that the average insert size is 104
kb.Based on the genome size of T.boeoticum , the library is about three times as large as T .boeoticum hap-
loid genome(5 600 Mb).Screening the BAC library with cpDNA sequence psbA gene and mtDNA sequence
atp6 gene as probe shows that contamination of the library with chloroplast and mitochondrial clones is less
than 1%.The library will be a useful platform in gene clone and genomic research of wheat.
Key words: Triticum boeoticum;BAC library;genome
Recently , bacterial artificial chromosome (BAC)li-
braries have been developed rapidly[ 1-6] .They have been
crucial resources for genome physical mapping and map_
based cloning of genes[ 7-9] .However , it is very difficult
to construct BAC libraries for species with large genomes
like wheat.The genome size of common wheat(Triticum
aestivum 2n=6x=42 , AABBDD) is extremely large
(16 700 Mb/1C)[ 10] .It comprises a large number of
repetitive sequences and three similar genomes A , B and
D.Although we can construct the library of common
wheat , it is difficult to locate a BAC clone in specific
chromosome because of similarity of the three genomes.
Therefore, it is very difficult to carry out positional
cloning and chromosome walking in common wheat.Be-
cause of the complexity of its genome , common wheat
genome has lagged behind studies of model plants with
smaller genomes like rice and Arabidopsis thaliana
[ 11] .In
order to overcome these difficulties , one of the possible
alternatives is to construct BAC libraries for the diploid
progenitors of common wheat.In fact , BAC libraries for
the A and D genome donors have been constructed by
American and Australian scientists respectively recent-
ly
[ 11 ,12] .The A genome library (T.monococcum AmAm)
consists of 276 480 clones with an average insert size of
115 kb.These libraries will undoubtedly contribute con-
siderably to hexaploid wheat genomics research.It has
been tried to use rice BAC libraries as a bridge for posi-
tional cloning of wheat and barley genes , which opens a
new pathway for solving this problem though failed[ 13 ,14] .
We have constructed libraries for T .boeoticum
Boiss(2n=14 , AbAb), one diploid progenitor of com-
mon wheat , which makes gene cloning of wheat possible
because of single genome.However , the genome of T .
boeoticum is still 13_fold larger than that of rice (430
Mb/1C), which is probably due to the additional DNA
with more than 85% repetitive sequences[ 15] .
1 Materials and Methods
1.1 Materials
Triticum boeoticum Boiss with powder mildew resis-
tance.
1.2 Methods
1.2.1 Preparation of BAC vector The BAC vector
pECBAC1
[ 14]
was isolated and purified using Qiagen kit(QIAGEN Germany).The purified vector was digested to
completion with BamH Ⅰ (New England Bio_lab)and
dephosphorylated with HK (Epicenter Technologies
USA).The quality vector was portioned out , each of 10
ng/μL , and stored at -80 ℃ in aliquots of 10μL.
1.2.2 High_molecular_weight (HMW)DNA isola-
tion Twenty grams of green leaves were collected from 3_
week_old plants grown in pots.Agarose plugs containing
HMW DNA were prepared based on the methods in refer-
ences [ 4] , [ 14] and [ 16] , with a little modification.
Briefly , leaves were ground in liquid nitrogen with a mor-
tar and a pestle for about 0.5 -1 h and immediately
transferred into an ice_cold beaker containing 200 mL ice_
cold 1×HB (homogenization buffer , 0.01 mol/L Tris_
HCl , 0.08 mol/L KCl , 0.1 mol/L EDTA , 1 mmol/L
spermine ,1 mmol/L spermidine , 0.5 mol/L sucrose , pH
9.4-9.5)plus 0.15%(V/V)β_mercaptoethanol.The
contents were gently swirled with a bar for 20 min on ice ,
and then filtered through two layers of cheesecloth and one
layer of miracloth or four layers of miracloth.The nuclei
were centrifuged with 1 800g for 20 min at 4 ℃.The
pellet was rinsed 2 -3 times in wash buffer (1×HB
buffer plus 0.15%(V/V)β_mercaptoethanol and 0.5%
(V/V)Triton X_100), resuspended in 1-2 mL 1×HB
buffer without β_mercaptoethanol , adjusted the nuclear
concentration to 5×107 nuclei/mL -7×107/mL , and
Received:2001-06-21 Accepted:2001-11-06
Supported by the National Natural Science Foundation of China(39870494)and National Transgenic Plant Project(J99_A_018).
＊Author for correspondence.E_mail:
植 物 学 报
Acta Botanica Sinica 2002 , 44(4):451-456
embedded in equal volume of 1% low melt_point agarose
plugs.The plugs were obtained and incubated in lysis
buffer(0.5 mol/L EDTA , 1% sodium lauryl sarcosine ,
0.2 mg/mL proteinase K , pH 9.0-9.3)at 50 ℃for 24
to 48 h.The plugs were rinsed in 0.5mol/L EDTA(pH
9.0-9.3)at 50 ℃for 1 h , and washed for another 1 h
in 0.05 mol/L EDTA(pH 8.0)at 4 ℃.Agarose plugs
were dialyzed against sterile 0.5×TBE buffer for 3 h ,
and then run in a 1% agarose gel using CHEF apparatus(Bio_RAD)with 0.5×TBE buffer at 12.5 ℃ and 4
V/cm for 6 h with a 5 s pulse time and 120°angle.The
plugs were collected from slot and stored in 0.5 mol/L
EDTA(pH 8.0)at 4 ℃ for later use.
1.2.3 Preparation and size selection of BamH Ⅰ
partially digested HMW DNA (1)Pre_treatment:The
plugs were dialyzed three times on ice against 50 volumes
of 1×TE (10 mmol/L Tris_HCl (pH 8.0);1 mmol/L
EDTA (pH 8.0))plus 0.1 mmol/L PMSF (phenyl-
methyl sulfonyl fluoride)for 1 h each time , followed by
washing three times on ice with 50 volumes of 1 ×TE
without PMSF for 1 h each time.(2)The selection of de-
sirable partial digestion condition and large scale partial
digestion:After pre_treatment , the selection of desirable
partial digestion condition was performed with BamH Ⅰ
as described previously[ 14] .The desirable partial diges-
tion condition that produced a major DNA fragment rang-
ing from 150 kb to 400 kb was the optimal condition and
used for large scale partial digestion.(3)Size selection:
The restricted large fragments of HMW DNA were size_se-
lected as described by Kazutoyo et al[ 16] .Briefly , the
size selection was performed in a CHEF apparatus , and
separation was carried out in three steps , all with pulse
direction at a 120°angle.(a)The initial direction of the
field allowed the DNA to migrate from the well toward the
nearest gel edge in 0.5×TBE buffer at 12.5 ℃ and 5
V/cm for 6 h with a 15 s pulse time.(b)The same con-
ditions were used after the direction of the gel was
changed , to bring all fragments remaining in the gel back
to the original starting wells.Prior to step 3 , a new DNA
marker was applied to the flanking wells which had not
been used previously.(c)HMW fragments were then re-
solved at 6 V/cm for 16 h with a 0.1 to 40 s pulse time.
After electrophoresis , the flanking marker lane was ex-
cised from the gel and stained with ethidium bromide to
indicate the location of the size landers.Gel slices were
then cut f rom the HMW DNA lanes at 0.5 cm interval to
obtain gel slices in the range of 100 kb to 350 kb.In or-
der to recover HMW DNA , the electro_elution procedure
was employed using dialysis tube (3/4 diameter , Life
Technologies)in 0.5×TBE at 12.5 ℃ and 6V/cm for 3
h with 30 s pulse time at a 120°angle.After electro_elu-
tion , the dialysis tube was dialyzed against 1×TE (pH
8.0)at 4 ℃ three times , at least 1 h for each time.At
last , the DNA concentration was examined via elec-
trophoresis withλDNA as standard.Afterwards , the DNA
was stored at 4 ℃and should be used within 10 d to pre-
vent degradation.
1.2.4 BAC library construction Ligation was per-
formed in 100 mL reaction solution containing 100 ng of
partially digested DNA , 25 ng of the linearized and de-
phosphorylated pECBAC1 BAC vector and 4 U of T4 DNA
ligase(New England Bio_lab)at 16 ℃ overnight.After
ligation , the reaction was desalted and concentrated by
drop_dialysis against 0.5×TE(pH 8.0)with 30%PEG
8000 for 30 min on ice according to Kazutoyo et al[ 16] .
Two microlitres of ligation solution were used to electro_
porate 20 μL of E.coli cells(DH10B , Gibco , BRL)by
a BRL cell_porator system according to the manufacture s
instruction.After transformations , the cells were re_sus-
pended in 1 mL SOC medium (2% Bacto tryptone ,
0.15% Bacto Yeast Extract , 10 mmol/L NaCl , 10
mmol/L MgCl2 , 10 mmol/L MgSO4 , 20 mmol/L Glu-
cose , pH 7.0)and incubated for 1 h at 37 ℃with shak-
ing at 225 r/min.The SOC medium with the cells was
plated on LB solid medium containing 12.5 μg/mL chlo-
ramphenicol , 50μg/mL X_gal and 25 μg/mL IPTG(iso-
propyl thiogalactoside), and incubated at 37 ℃ for 18 h
to 24 h.White recombinant colonies were picked and
transferred to 384_well plates(Genetix)containing 80 μL
LB freezing buffer according to Woo et al[ 2] .The plates
were incubated 8 h at 37 ℃, duplicated , and stored re-
spectively in two -80 ℃ ice boxes.
1.2.5 BAC library characterization (1)Insert_size
determination and Southern blot hybridization:To esti-
mate insert size of clones and library capacity , a total of
200 BAC clones were selected randomly throughout the li-
brary.BAC DNA was isolated from 5 mL cultures grown
overnight in LB medium containing 12.5μg/mL chloram-
phenicol by the alkaline lysis procedure[ 17] .Then the
BAC DNA was digested with Not Ⅰ (New England Bio_
lab)and analyzed by pulsed_field electrophoresis in 1%
agarose gel(0.5×TBE buffer , 6 V/cm , 5-15 s switch
time , 16 h run time , 12.5 ℃, 120°angle).Southern
blots of size_separated BAC inserts were carried out using
standard protocols[ 17] .T.boeoticum genomic DNA la-
beled by
32
P via random priming techniques
[ 17]
was used
as probe.(2)Cytoplast organelles DNA contamination:
To determine the percentage of contamination with cyto-
plast organelles DNA , we randomly transferred 382 BAC
clones to a Hybond N+ filter (Amersham).The mixture
of barley chloroplast psbA gene and maize mitochondrial
atp6 gene was dotted onto filter as positive control.A
blue clone DNA was dotted on it as negative control.The
filter was placed on LB agar plate containing 12.5μg/mL
chloramphenicol , and incubated at 37 ℃ for 15 h.The
mixture of 32P_labelled psbA and atp6 DNA was used as
probe.Transferring filter and hybridization procedures fol-
lowed the methods of Sambrook et al
[ 17] .(3)Stability of
BAC clones:The stability of the BAC clones was checked
by culturing three random clones about 100 generations.
Samples of the cultures were taken at approximately 0 , 50
and 100 generations of culturing.Clone culturing , plas-
mid extraction and electrophoresing methods were the
same as the above.
452 植物学报 Acta Botanica Sinica Vol.44 No.4 2002
2 Results and Discussion
2.1 Vector preparation
The suitability of a BAC library for positional cloning
depends on its adequate genome coverage , appropriate in-
sert size , a low percentage of chloroplast and mitochondri-
al DNA contamination , and low frequency of empty
clones.In order to obtain a good library , it is necessary
to optimize every step in the construction of the library.
There are two crucial steps , vector preparation and prepa-
ration of HMW DNA.
The vector quality influences percentage of empty
clones and library quality.To optimize vector prepara-
tion , three steps were used.(1)After digestion and alka-
line phosphatase treatment , self_ligation and pulsed_field
electrophoresis were used to remove circular and dimeric
vectors away from linear vectors.(2)The ready vector
should be used as soon as possible because vector dephos-
phorylation will lead to a rapid degradation of vector even
stored at -80 ℃[ 9] .(3)The vector was stored at -80℃with glycerol in aliquot of 10 μL.It is important to
minimize vector degradation during storage
[ 17] .To mini-
mize the influence of vector degradation , the fresh vector
should be used in construction of BAC library.Re_freez-
ing and thawing glycerol stock should be avoided.It was
also suggested that two sequential cesium chloride_ultra_
centrifugation steps instead of the column purification pro-
cedure be used in construction for many plant BAC li-
braries to enhance quality of the libraries
[ 9 , 18 ,19] .
2.2 Preparation of partially digested HMW DNA
Quality and quantity of partially digested HMW DNA
are as important as that of the vector for the construction
of quality BAC library[ 1 ,3 ,11] .
2.2.1 Grinding time and washing nuclei Grinding
instead of cutting leaves can prevent DNA from physical
shearing.Enough nuclei can be obtained if fewer leaves
are ground for longer time.For example , we can obtain 1
-2 mL nuclei solution with concentration of 5×107/mL-7×107/mL if we grind 20 g leaves for 30 min.At the
same time , we can obtain the same amount if we grind 10
g leaves for 1 h , which suggests that grinding for longer
time is necessary.Zhang et al[ 1] reported that the addi-
tional three washing steps for the nuclei purification yield-
ed good quality and stable HMW DNA , and also resulted
in a very low percentage of cytoplast organelles contamina-
tion in the nuclei (<1.4%)[ 1] .The concentration of
nuclei embedded in agarose had direct effects on the di-
gestion and ligation.Too high concentration will cause
difficulty in DNA digestion and too low concentration will
decrease ligation efficiency[ 2 , 3] .Agarose concentration
used to embed nuclei is also very important.Too high
concentration will cause difficulty in DNA partial diges-
tion
[ 6] .
2.2.2 Determination of optimal partial digestion
conditions and size selection We tested the conditions
for partial digestion by varying the amount of restriction
enzyme.After extraction of the HMW DNA , serial
digestion tests were carried out with 0 , 0.3 , 0.6 , 1.2 ,
2.4 and 4.8 U of BamHⅠ respectively for per 1/3 plug
to optimize the condition of the partial digestions.In-
creasing amounts of restriction enzyme showed a clear pat-
tern of increasing digestion , with an optimum at 0.6 and
1.2 U of BamHⅠ (Fig.1).
Fig.1. DNA fragment size after being partially digested in agarose
by six different concentrations of BamHⅠ .
The results indicate that 0.6 U(3)and 1.2 U(4)are suitable for
construction of BAC library.M , the molecular weight standard
(Lambda ladder);1-6 , DNA digested with 0 , 0.3 , 0.6 , 1.2 ,
2.4 and 4.8 U of BamHⅠ respectively.
Therefore , we used 0.6 , 0.9 and 1.2 U of
BamH Ⅰ to digest HMW DNA in a large scale.Partially
digested DNA fragments ranging from 100 kb to 150 kb ,
150 kb to 200 kb , 200 kb to 250 kb , 250 kb to 300 kb ,
300 kb to 350 kb were collected respectively.Then liga-
tion and transformation were carried out.After isolation of
plasmid , purification , BAC DNA digestion and PFGE
analysis , it was indicated that the larger the partial diges-
tion DNA size was , the smaller the actual average insert_
size DNA was(Fig.2 and Table 1).So we used recov-
ered and partially digested DNA size ranging from 100 kb
to 200 kb for ligation and transformation(Table 1).The
white clones were transferred into 384_well plates.About
170 thousand clones were picked and pooled in 6 384_well
plates.This made store and exchange of the library rela-
tively easy.One hundred possibly different clones were
stored in one well.The result indicated that even for T.
boeoticum , with a large genome , only 6 384_well plates
were sufficient for a BAC library.It would dramatically
reduce the cost and storage space needed.
We used both two_step method[ 1] and three_step
method[ 16] to separate partially digestedHMW DNA in our
experiment , and found that the latter was better.The
yield and concentration of selected DNA using the former
CHEN Fan_Guo et al:Construction and Characterization of a Bacterial Artificial Chromosome Library for Triticum boeoticum 453
Fig.2. DNA size effects on the transformation frequency.
The transformation frequency decreased significantly when the target DNA size was raised.The target DNA sizes were 250-300 kb(A)and
100-150 kb(B)respectively.
Table 1 The relationship between target DNA size and the rate of
transformation and the size of inserted DNA fragments
Target DNA
size(kb) 100-150 150-200 200-250 250-300 300-350
Blue and white
clones
730 725 645 357 120
White clones 712 704 400 128 24
Rate of clones
without inserts
(%)
0.83 2.5 33.3 50 58.3
Size of inserted
DNA fragments
(kb)
107 102 85 70 70
Ligation solution was not dialyzed.The number of recombination clones was
the average of three electro_transformations.Rate of clones wi thout inserts and
size of inserted DNA fragments were the average assay in three times, in
which 40 BAC clones were analyzed each time.
method were often very low though its quality was very
good.It would not enhance the efficiency of ligation and
transformation even if the selected DNA was dialyzed after
electroelution according to Wang et al[ 6] .We obtained
enough quality DNA using the three-step method , and
found that the effect was very good in using DNA frag-
ments ranging from 100 kb to 200 kb for ligation and
transformation.The result was the same as that of Wang
et al[ 6] .
2.3 Ligation and transformation
A major limitation in constructing BAC vector_based
libraries for large and complex genomes is the transforma-
tion efficiency required to create enough recombination
clones.It can be solved through enhancing ligation con-
centration.For an optimal ligation condition , we tested
various molar ratios of the vector to the selected DNA
fragments ranging from 20∶1 to 1∶1.We found that liga-
tion with a ratio 3.5∶1 could generate more white clones
than that of other ratios.0.5×TE buffer containing 30%
PEG8000 was used as dialysis buffer to concentrate the
ligation mixture.Under optimal conditions , as many as
5 000-10 000 clones per 2 μL of dialyzed ligation mix-
ture could be obtained.Qu et al[ 4] used agar instead of
PEG to dialyze ligation , and obtained a similar result.
We used electroporator instead of heat_shock , and the
transformation efficiency was enhanced notably .
2.4 Library characterization
To test the quality of the BAC library , we analyzed
the sizes of the inserts , the contamination of cytoplast or-
ganelles DNA , and the clone stability of the library.
2.4.1 Insert size determination and Southern blot
hybridization Inserted DNA was isolated from 200 ran-
dom clones.The recombination vectors were digested to
completion with Not Ⅰ , and fractionated in 1% agarose
CHEF gels.It was indicated that the insert size ranged
from 35 kb to 260 kb , with an average size of 104 kb.
When less than 2% of the clones were without inserts ,
more than 75% of BAC clones in the library have insert
sizes ranging from 80 kb to 140 kb(Fig.3A).Based on a
genome size of T.boeoticum , 5 600Mb , the library cov-
erage is about three times of haploid genome equivalents ,
which can provide 90%[ 18] possibility to isolate some in-
teresting genes or sequences of T .boeoticum in the li-
brary.Figure 3B shows a Southern blot hybridization of
the gel in Fig.3A probed with T .boeoticum genomic
DNA.It was indicated that the cloned DNA fragments
were originated from T.boeoticum.The strongly hy-
bridizing signals indicated that many of the BACs con-
tained highly repetitive DNA.Weakly bands possibly in-
dicated low_copy DNA.This result is in accordance with
the actual theory that wheat genome contains more than
85% repetitive sequences in their genomes[ 15] .
454 植物学报 Acta Botanica Sinica Vol.44 No.4 2002
Fig.3. Evaluation of the insert size.
A.Most of the inserts are about 100 kb.B.Southern hybridization
with Triticum boeoticum genomic DNA as probe indicates the inserts
are from nuclear.M , DNA size ladder , 1-20 , BAC clones digest-
ed with NotⅠ respectively.
2.4.2 Cytoplast organelle contamination Nuclei
DNA library contaminated by cytoplast organelle DNA not
only decreased the capacity of library , but also caused er-
ror of chromosome walking
[ 13 ,14] .To avoid cytoplast or-
ganelle contamination , we used PFGE to separate HMW
DNA , which could remove most of cytoplast organelle
DNA[ 4] .To estimate the representation of cytoplast or-
ganelle DNA in the library , filter binding clones were
probed with a barley chloroplast gene psbA and a maize
mitochondrial gene atp6.The result of this screening
showed that less than 1% of the library sequences were
cytoplast DNA (Fig.4).The low percentage of contami-
nation indicated that the library was suitable for cloning
desirable genes.
Fig.4. Dot hybridization with chloroplast psbA and atp6 indicated
that less than 1% of the white clones were with cytoplasm DNA in-
serts(Dots with arrow are the positive control).
2.4.3 Library stability analysis The stability of the
BAC clones was investigated by culturing three random
clones for 50 and 100 generations.The result showed that
the banding patterns of the plasmids from three different
stages were consistent.No absence and recombination oc-
curred , indicating that the BAC clones were stable.The
result was the same as the previous
[ 2 ,4] .
In this paper , we have described construction and
characterization of the first quality BAC library for T.
boeoticum using pECBAC1 vector in China.Although the
abundance of repetitive sequences of wheat causes addi-
tional problems for chromosome walking and positional
cloning , they can be overcome through the selection of
single or low copy_number sub_clones from BAC ends as
described by Woo et al[ 2] .This large insert library pro-
vides an important platform for positional cloning of inter-
esting genes and genomic research of wheat.
Fig.5. After 50 and 100 generation s culture respectively , the
clone still conveys the big inserts steadily.
Lanes 1 , 4 , 7 , first generation;Lanes 2 , 5 , 8 , fiftieth generation;
Lanes 3 , 6 , 9 , one hundredth generation.
Acknowledgements We are grateful to JIAN Tao , LIU
Yue , PANG Bing_Shuang for their help during the con-
struction of the library.Thanks are extended to Dr.
WANG Bin and Dr.LUO Meng for cytoplasm organelle
probes.
References:
[ 1] Zhang H B , Zhao X P , Ding X L , Paterson A H , Wing R
A.Preparation of megabase DNA from plant nuclei.Plant
J , 1995 , 7:175-184.
[ 2 ] Woo S S , Jiang J , Gill B S , Paterson A H , Wing R A.
Construction and characterization of a bacterial artificial
chromosome library of Sorghum bicolor.Nucl Acids Res ,
1994 , 22:4922-4931.[ 3] Frijters A C J , Zhang Z , Damme M V , Wang G L , Ronald
P C , Michemore R W.Construction of a bacterial artificial
chromosome library containing large EcoRⅠ and Hind Ⅲ
genomic fragments of lettuce.Theor Appl Genet , 1997 , 94:
390-399.
[ 4 ] Qu X_P (曲雪萍), Fu J_M (伏建民), Li C_Y(李传
友), Jia J_H (贾建航), Jin D_M(金德敏), Wan Q(王
倩), Yang R_C(杨仁崔), Wang B(王斌).Construction
and characterization of a bacterial artificial chromosome li-
brary of rice 5460F.Chin Sci Bull(科学通报), 1999 ,
44:1532-1537.(in Chinese)
CHEN Fan_Guo et al:Construction and Characterization of a Bacterial Artificial Chromosome Library for Triticum boeoticum 455
[ 5] Vinatzer B A , Zhang H B , Sansavini S.Construction and
characterization of a bacterial artificial chromosome library of
apple.Theor Appl Genet , 1998 , 97:1183-1190.[ 6 ] Wang W_M(王文明), Jiang G_H (江光怀),Wang S_Q(王世全), Zhu L_H (朱立煌), Zhai W_X (翟文学).
Construction of a deep coverage rice BAC library and identi-
fication of clones associated with disease_resistant genes.
Acta Genet Sin(遗传学报), 2001 , 28:120-128.[ 7 ] Brommonschenkel S H , Tanksley S D.Map_based cloning
of the tomato genomic region that spans the Sw_5 tospovirus
resistance gene in tomato.Mol Gen Genet , 1997 , 256:121-126.[ 8 ] Chen M , Sanmiguel P , Oliveria A C , Woo S S , Zhang H ,
Wing R A , Bennetzen J L.Microcolinearity in sh2_homolo-
gous regions of the maize , rice and sorghum genomes.Natl
Acad Sci USA , 1997 , 94:3431-3435.
[ 9 ] Danash D , Penuela S , Mudge J , Denny R L , Nordstrom
H , Martinez J P , Young A D.A bacterial artificial chromo-
some library of soybean and identification of clones near a
major cyst nematode resistance gene.Theor Appl Genet ,
1998 , 96:196-202.[ 10] Bennett M D, Leitch I J.Nuclear DNA amounts in an-
giosperms.Ann Bot , 1995 , 76:461-462.
[ 11] Lijavetzky D, Muzzi G , Wicker T , Keller B , Wing R ,
Dobcovsky J.Construction and characterization of a bacteri-
al artificial chromosome library for A genome of wheat.
Genome , 1999 , 42:1176-1182.
[ 12] Moullet O , Zhang H B , Lagudah E S.Construction and
characterization of a large DNA insert library from the D
genome of wheat.Theor Appl Genet , 1999 , 99:919-928.
[ 13] Wang G L, Hosten T E , Song W Y, Wang H P , Ronald P
C.Construction of a rice bacterial artificial chromosome li-
brary and identification of clones linked to Xa_21 disease
resistance locus.Plant J , 1995 , 7:525-533.
[ 14] Zhang H B , Choi S , Woo S S , Jiang J , Li Z , Wing R A.
Construction and characterization of two rice bacterial artifi-
cial chromosome libraries from parents of a permanent re-
combinant inbred mapping population. Mol Breeding ,
1996 , 2:11-24.[ 15] Flavell R B , Odell M.Ribosomal RNA genes on homolo-
gous chromosomes of group 5 and 6 in hexaploid wheat.
Heredity , 1976 , 37:377-385.[ 16] Kazutoyo O , Peng Y W , Zhao B , Frengen E , Minako
Tateno M , Catanese J J , Jong P J.An improved approach
for construction of bacterial artificial chromosome libraries.
Genomics , 1998 , 52:1-8.[ 17] Sambrook J , Fritsch E F , Maniatis T.Molecular Cloning:
A Laboratory Manual.2nd ed.New York:Cold Spring
Harbor Laboratory Press , 1989.[ 18] Clarke L , Carbon J.A clony bank containing synthetic
ColE1 hybrid plasmids representative of the entire E.coli
genome.Cell , 1976 , 9:91-100.[ 19] Mozo T , Fischer S , Meier_Ewert S , Lehrach H , Altmann
T.Use of the IGF BAC library for physical mapping of the
Arabidopsis thaliana genome.Plant J , 1998 , 16:377 -
384.
野生一粒小麦 BAC文库的构建和鉴定
陈凡国1 ,2 张学勇1＊ 夏光敏2 贾继增1
(1.中国农业科学院品种资源研究所 ,农业部作物种质资源与生物技术重点实验室 , 北京 100081;2.山东大学生命学院 , 济南 250100)
摘要: 以细菌人工染色体 pECBAC1 为载体 ,构建了野生一粒小麦(Triticum boeoticum Boiss)的基因组 BAC 文库。该
文库共包含约 17万个克隆 , 平均插入片段长度为 104 kb ,按野生一粒小麦基因组为5 600Mb 计算 , 文库覆盖了约 3
倍的该物种基因组。用大麦叶绿体 psbA 基因和玉米线粒体atp6 基因作混合探针 ,检测发现该文库中含细胞器基因
组同源序列的克隆数小于 1% 。该文库的建成 , 为小麦基因的克隆及基因组学研究提供了技术平台。
关键词: 野生一粒小麦;BAC 文库;基因组
中图分类号:Q943 文献标识码:A 文章编号:0577-7496(2002)04-0451-06
收稿日期:2001-06-21 接收日期:2001-11-06
基金项目:国家自然科学基金(39870494);国家转基因植物及产业化专项基金(J99_A_018)。
＊通讯作者。E_mai l:
(责任编辑:谢 巍)
456 植物学报 Acta Botanica Sinica Vol.44 No.4 2002