全 文 ：Vol. 29 , No. 4
pp. 486～490 　July , 2003
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 29 卷 第 4 期
2003 年 7 月 　486～490 页
Construction of a Brassica napus Bacterial Artif icial Chromosome Library and Iden2
tif ication of Clones Linked to Boron Efficiency Gene
HU Zheng 　XU Fang2Sen 　ZHAO Jian2Wei 　MENGJin2Ling Ξ
( National Key Laboratory of Crop Genetic Improvement , National Molecular Breeding Center , Huazhong Agricultural University , Wuhan , Hubei 430070 , China)
Abstract 　A bacterial artificial chromosome (BAC) library was constructed for a Brassica napus cultivar : Ning RS21. The
library contains 82944 clones with 80 kb DNA insert size in average representing equivalents of 511 Brassica napus haploid
genome. The BAC DNA can maintain stably in E. coli after about 100 generations of culture. pa28 , an Arabidopsis EST
clone which linked to a boron efficiency gene B E1 in B . napus , was used as probe to screen the library : consequently , 13
positive clones were identified. These positive clones were divided into four groups. Three of 13 clones were identified to
link to the B E1 locus by Southern analysis.
Key words 　Brassica napus ; BAC library ; Boron efficiency gene
甘蓝型油菜基因组文库的构建及与硼高效基因相连锁克隆的筛选
胡　正　徐芳森　赵建伟　孟金陵 3
(华中农业大学作物遗传改良国家重点实验室 ,国家分子育种中心 ,湖北武汉 430070)
摘 　要 　以甘蓝型油菜宁 RS21 为材料 ,构建了含有 82944 个克隆的甘蓝型油菜的 BAC基因组文库。从文库中随机挑取
克隆进行 DNA 长度检测 ,BAC克隆平均插入片段大小为 80 kb ,覆盖甘蓝型油菜基因组的 511 倍。随机挑取 108 克隆进
行继代培养 100 代 ,分离质粒酶切检测表明不存在插入片段丢失现象 ,表明该文库的克隆在大肠杆菌中稳定存在 ;以与
硼高效基因相连锁的分子标记拟南芥 cDNA 克隆 pa28 为探针对文库进行了筛选 ,共获得 13 个阳性克隆 ,进一步研究表
明 ,3 个克隆 13E3、32G8、186G20 与 B E1 基因相连锁。
关键词 　甘蓝型油菜 ;BAC文库 ;硼高效基因
中图分类号 : S565 　　　文献标识码 : A
　　There were two basic vectors for construction of large
DNA fragment insert library : the yeast artificial chromosome
( YAC) vector[1 ] and the bacterial artificial chromosome
(BAC) vector[2 ] . The BAC vector had more advantages than
the yeast artificial chromosome ( YAC) vector in library’s
construction. The simply manipulation of DNA isolation and
purification , the DNA maintain stably and low chimerism in
E. coli made the BAC vector used widely since it was devel2
oped. In plant , BAC library were constructed for genome se2
quencing in Arabidopsis and rice[3 ,4 ] , and genomic analysis
and gene cloning in a range of crops and model plants[5～7] .
Boron was one of the essential micronutrients in the
growth and development of plant[8 ] . Rapeseed ( B . napus) is
one of the most important crops in China as well as in the
world. It has been proved that Ning RS21 , a cultivar of B .
napus , has Sclerotinia resistant and boron high efficiency
characteristic[9 ,10] . A boron efficiency gene , named B E1 in
B . napus , was mapped on the linkage group 9 flanked the
marker pa28c and tg2h10b with a 018 cM distance to the for2
mer
[11]
. To construct an BAC library with the genomic DNA
of NingRS21 would be a fundamental work for genomic studies
and gene isolation of B E1 , Sclerotinia resistant gene and oth2ΞFoundation item :The National 863 High Technology Program of China (2001AA222311) and National Special Foundation for Transgenic Plant (J00A20082
01) .
Biography :HU Zheng (1977 - ) ; Male ;Zhejiang Province ; Master. 　3 Corresponding author :MENGJin2Ling , E2mail :jmeng @mail . hzau. cn
Received (收稿日期) :2002209206 ;Accepted(接受日期) :2003201213.

ers related to agronomic important characteristics in B . na2
pus . In this article , we reported the construction and charac2
terization of a BAC library with the variety Ning RS21 and the
identification of three clones linked to the B E1 .
1 　Material and methods
1. 1 　Vector preparation
　　The pBeloBAC11 plasmid was kindly provided by Dr
Hong2bin Zhang (Texas A &M University , USA) . The vec2
tor DNA was isolated by alkaline lysis method and was digest2
ed completely with BamH Ⅰ. The digested vector DNA was
purified according to the method described by Osoegawa[12] .
The vector DNA was dissolved in ddH2O at 10 ng/μL for liga2
tion.
1. 2 　Preparation of high2molecular2weight DNA, partial
digestion and size selection
High2molecular2weight ( HMW) DNA was extracted from
young leaf tissue as described by Zhang[13] . The HMW DNA
was digested partially with BamHⅠ. The DNA fragment from
100 kb to 200 kb was recovered from gel using two2size selec2
tion according to Woo[7 ] . After lysis , the recovered DNA was
adjusted to a concentration of 1 ng/μL.
1. 3 　Ligation and transformation
The ligation was performed at a molar ratio of 1 ¬ 5
between the insert DNA and the vector at 16 ℃for overnight .
The ligation product was transformed into the Electro MAX
DH10B cells (BRL , Gaithersburg , Md. ) by electrophoresis
using a BioRad Gene Pulser. The transformation condition
were 100 Ω , 1. 8 kV and 25μF. After transformation , the
cells were collected into 1 mL SOC medium and incubated in
shaker at 37 ℃for 1 h at 200 r/ min. Cells was then plated on
LB plates containing chloramphenicol (1215μg/ mL) , IPTG
(15μg/ mL) and X2gal (60μg/ mL) and incubated at 37 ℃
for 18 h. White clones growing from the culture were picked
out into 3842well microtiter plates containing 90μL LB freez2
ing buffer (36 mmol/ L K2HPO4 , 1312 mmol/ L KH2PO4 , 117
mmol/ L sodium citrate , 014 mmol/ L MgSO4 , 618 mmol/ L
(NH4) 2SO4 ,414 % glycerol , LB) [7 ] . The plates were incu2
bated at 37 ℃for 12 h and stored at - 80 ℃.
1. 4 　Characterization of the BAC library
BAC clones were selected randomly to analysis the size
of insert DNA fragment . The BAC DNA was isolated by alka2
line lysis method and was digested completely with Not Ⅰ.
The BAC DNA was electrophoresed in a pulsed2field gel elec2
trophoresis ( PFGE) using 015 ×TBE buffer at 11 ℃ and 6
V/ s for 12 h with 5 s initial and 15 s final pulse time at 120
angle. After electrophoresis , the gel was stained with ethidi2
um bromide and photographed.
Two clones were randomly selected to analysis their ge2
netic stability in E. coli . The clones were inoculated in LB
culture at 37 ℃ according to the method described by
Zhang[14] . BAC DNA samples were isolated from the first day
culture and last day culture and digested completely with
Hind Ⅲ. DNA digested samples were electrophoresis at
018 % agrose gel and stained with ethidium bromide.
1. 5 　Preparation of high2density filters and the library
screening
High2density colony filters for library screening were
prepared using the Biomeck 2000 Robotics Workstation. BAC
clones were gridded in double spots using a 3 ×3 matrix with
no clone in the center. This pattern allowed 3072 clones in
each filter. Colony filters was performed for hybridization ac2
cording to Zhang[15] .
1. 6 　Detection of the chromosome structure in B E1 re2
gion between Qingyou 10 and Ning RS21
The total DNA was extracted from the B2efficiency culti2
vars (Qingyou 10 and Ning RS21) and one low B2efficiency
cultivar ( Bakow) . The DNA was digested complete with
EcoR Ⅰand transformed to Nylon + . Hybridization was taken
with the pa28 probe to analysis the structure in the region of
B E1 between different cultivars.
1. 7 　Identification of clones linked to Boron efficiency
gene
The Arabidopsis EST clone , pa28 which was tightly
linked to the boron efficiency gene B E1 , was selected as a
probe to screen the library following Wu’s procedure[15] .
BAC clones hybrided with the probe were picked out from the
library. DNA samples from the positive clones were digested
with EcoR Ⅰ, and transferred to the Nylon + . Southern blot
was taken with pa28 probe to identify the clones linked to
B E1.
2 　Results
2. 1 　Characterization of the BAC library
　　The library constructed from the B E1 containing cultivar
of B . napus , consists of 82944 clones stored in 3842well mi2
crotiter plates. One hundred and eight clones were randomly
picked out for analyzing the fragment of insert DNA fragment .
784　4 期 HU Zheng et al . : Construction of a Brassica napus Bacterial Artificial Chromosome Library and Identification . . . 　 　

Five of them had no insert DNA fragment . The insert size was
about 80 kb in average varied from 55 kb to 230 kb (Fig. 1) .
Based on an estimated genome size of 1235 Mb for Brassica
napus [17] , the library represents about 511 equivalents of the
genome.
Fig. 1 Analysis the size of 14 random BAC clones by PFGE. Lane12lane 14 were BAC clones , lane 15 was the vector ,
the common band in all lanes was the vector DNA.
　　The stability of BAC clones in E. coli is important for
genome research. The restriction analysis was taken between
generation 1 and 100 cultures of two BAC clones. It showed
that the different generation BAC clone shared the same
bands , which suggested the BAC clones can be maintained
stably in E. coli (Fig. 2) .
Fig. 2 Clone digested with Hind Ⅲ. Lane 1 : BAC 12I12 ; lane 2 :
BAC 12I12 after cultured for 100 generations ; Lane 3 :BAC 59I18 ; lane
4 :BAC 59I18 after cultured for 100 generations ; lane 5 : 1kb Ladder
2. 2 　Detection of the chromosome structure in B E1 re2
gion between different cultivars
A major gene for high B2efficiency (B2deficient toler2
ance , B E1) was detected flanked by pa28c and tg2h10b
marker and the genetic distance was 018 cM and 616 cM
(Fig. 3) [11] . This map was developed using RFLP and AFLP
markers in a F2 population from a cross between one high B2
efficiency cultivar ( Qingyou 10) and one low B2efficiency
cultivar (Bakow) .
Fig. 3 Genetic linkage maps of the B E1 region in B . napus
Although genetic analysis showed that two high B2effi2
ciency cultivars Ning RS21 and Qingyou10 shared the same
allele of high B E1 gene , it is better to verify whether they
have similar DNA in the B E1 region before applying the clone
pa28 , which was closely linked to B E1 in the map construct2
ed with Qingyou 10 , as probe to screen the BAC library con2
structed with genomic DNA of Ning RS21. The pa28c marker
was mapped according to the results of hybridization to the
EcoR Ⅰ2digested parents genomic DNA with pa28 probe
884 　　　作 　　物 　　学 　　报 29 卷 　

( Fig. 4) . It showed a band of around 11 kb was the polymor2
phism band and the Ning RS21 shared the same bands with
the Qingyou 10. The result suggested that there was the same
chromosome structure in B E1 region between Qingyou 10 and
Ning RS21 and the size of polymorphism band was about 11
kb. So it is reasonable to identify the clones linked to B E1
using pa28 probe to screen the library.
Fig. 4 The results of hybridization with the EcoR Ⅰ2digested
plant DNA. 1 :Qingyou10 , 2 :Bakow ,3 :Ning2RS21
2. 3 　Identification of clones linked to the boron efficien2
Fig. 5 Positive clones by screening the library with the
pa28 probe. Arrows indicated one positive clone
cy gene
Total 13 positive clones were obtained by screening the
BAC library with pa28 probe ( Fig. 5) . Fig. 4 showed that
pa28 probe detected four loci in Ning RS21. To identify the
clone linked to the B E1 locus , these positive clones were fur2
ther analyzed by Southern analysis using pa28 probe. The re2
sult of Southern bolt showed the positive clones were divided
into four groups ( Table 1) . From Table 1 , three clones
(13E3 32G8 186G20) had a band of around 11 kb by South2
ern blot . It was suggested that these three clones were linked
to the B E1 .
Table 1 The positive clones detected by pa28 markers
The size of band (kb) BAC clone
2. 5 163D6
2. 7 12I12 40P7 43D2 59I18
62C9 113K6 139B17
4. 2 77D5 215O20
11 13E3 32G8 186G20
3 　Discussion
To construct a good bacterial artificial chromosome
(BAC) library is a foundation to physical mapping , genome
sequencing , map2based gene cloning and so on. The library
contains 82944 clones with an average insert size of 80 kb.
416 % clones have no insert DNA , based on the B . napus
genome size of 1235 Mb , there is a 9915 % chance to detect
a specific clone if the chloroplast fraction is subtracted out .
This was reflected when the library was screened with pa28
probe. Although the average size of insert DNA was not large
enough , but this BAC library should be very useful for B .
napus genome research.
As a micronutrient , boron has an important role in plant
growth and development[8 ] . Boron deficiency would decrease
significantly the number of pollen[16 ,17] . Boron also obviously
affects seed yield and quality of rapeseed[18 ,19] . Brassica na2
pus , one of the main oil crops in China , usually requires
more boron[20] . Isolating boron2efficiency gene is valuable for
B . napus and other sensitive crops to boron deficiency.
The Brassica napus genetic map is 1832. 9 cM over 19
linkage groups[11] and the haploid genome size is 1235 Mb ,
which yield an average of 67318 kb/ cM. It is difficult to
clone the B E1 in B . napus as the genetic distance between
pa28c and B E1 is 018 cM. Comparative genetic showed that
map2based cloning of genes in large genome species could be
taken in small genome species when they have enough colin2
earity[21] . Rice genetic maps and molecular markers were ap2
plied to saturate the Rpg1 and Rpg4 region in barley based
on the synteny among them[22～24] and the physical mapping of
them was constructed using rice BAC. This strategy also can
be applied in B . napus using Arabidopsis thaliana messages ,
as the Brassica species are closely related to the model plant
A. thaliana. The pa28c and homologous fragment of tg2h10b
984　4 期 HU Zheng et al . : Construction of a Brassica napus Bacterial Artificial Chromosome Library and Identification . . . 　 　

was located in chromosome 1 of A. thaliana with 614 cM of
genetic distance[25] . Mapping a Brassica gene to an interval
of less than 10 cM is often likely to identify the homologous
collinear region in A. thaliana[26] , Which suggested that the
region of pa28c and tg2h10b remained collinear between B .
napus and A. thaliana. To saturate the region of B E1 in B .
napus , it is possible to select the DNA markers between the
pa28 and tg2h10b in A. thaliana and map them in B . na2
pus . Once the markers cosegregated with B E1 are founded in
A. thaliana , the candidate gene would be selected to identify
the function as the A. thaliana genome was entirely se2
quenced[27] . In the other hands , if the candidate gene cannot
find in A. thaliana , the saturated markers in the B E1 region
will make the physical mapping easy using the B . napus BAC
library we constructed.
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094 　　　作 　　物 　　学 　　报 29 卷