全 文 ：广 西 植 物 Guihaia Jul． 2013，33(4) :456－459 http:/ / journal． gxzw． gxib． cn
DOI:10． 3969 / j． issn． 1000-3142． 2013． 04． 005
张俊娥，李芬，孙蒙祥． 从烟草 EST微卫星标记中筛选合子胚中优势表达的基因［J］． 广西植物，2013，33(4) :456－459
Zhang JE，Li F，Sun MX． Polymorphic microsatellite markers from expressed sequences tag in Nicotiana tabacum for obtaining the genes of preponderant ex-
pression in zygotes［J］． Guihaia，2013，33(4) :456－459
Polymorphic microsatellite markers from expressed
sequences tag in Nicotiana tabacum for obtaining
the genes of preponderant expression in zygotes
ZHANG Jun-E1，LI Fen2，SUN Meng-Xiang3
(1． College of Life Sciences，Jiangxi Normal University，Nanchang 330022，China;2． College of Life Sciences，
Henan Normal University，Xinxiang 453007，China;3． Key Laboratory of Ministry of Education for
Plant Development Biology，College of Life Sciences，Wuhan University，Wuhan 430072，China )
Abstract:In order to obtain the genes of preponderant expression in zygote cells of tobacco，CAP3 program was used to
assemble ESTs from GenBank，and MISA program were used to screen microsatellites． Then polymorphic microsatellites
were amplified in cDNA of tobacco zygotes library． The results showed that sixteen polymorphic SSＲ markers from tobac-
co ESTs deposited in a public sequence database were developed． Of these，nine genes could be successfully amplified in
cDNA of tobacco zygotes library． The study would provide new channel to screen genes of preponderant expression in to-
bacco zygotes．
Key words:EST(expressed sequence tag) ;SSＲ(simple sequence repeats) ;genes of preponderant expression;
zygotes;tobacco
中图分类号:Q948 文献标识码:A 文章编号:1000-3142(2013)04-0456-04
从烟草 EST微卫星标记中筛选合子胚中优势表达的基因
张俊娥1，李 芬2，孙蒙祥3
(1． 江西师范大学 生命科学学院，南昌 330022;2． 河南师范大学 生命科学学院，河南 新乡
453007;3． 武汉大学 生命科学学院 植物发育生物学教育部重点实验室，武汉 430072 )
摘 要:为获得烟草合子胚中的优势表达基因，利用 CAP3程序对来自烟草公共数据库的 EST序列进行组装，
利用 MISA程序从组装后的 EST中筛选 SSＲ位点，将多态性的 SSＲ位点在烟草合子文库中进行扩增并对等位
基因进行分析。结果表明:具有多态性的 16个 SSＲ标记中，有 9 个基因能从烟草的合子库中成功扩增得到。
该研究为筛选烟草合子胚中优势表达基因提供新的途径。
关键词:EST;SSＲ;优势表达基因;合子;烟草
Tobacco(Nicotiana tabacum)is an important agri-
cultural crop that plays a significant role in the
economies of many countries(Davis ＆ Nielsen，1999)．
Furthermore，tobacco is considered to be one of the most
important model systems in biotechnology． Despite
being an inbreeding，highly homozygous plant，as well as
being of high-economic and scientific importance，it is
surprising that the genetic analysis of tobacco is still in
收稿日期:2012-11-06 修回日期:2013-02-10
基金项目:江西省自然科学基金(2010GZN0109);江西省教育厅基金(GTT11386)
作者简介:张俊娥，(1972-)，女，山西榆社人，博士，副教授，研究方向为植物生物技术与遗传育种，(E-mail)zhangjune2001@ aliyun． com。
its infancy． Although 684 microsatellite markers were
screened from public domain databanks by Bindler et
al．(2007)for a linkage map of tobacco，more microsat-
ellite markers need to be developed to complement the
studies． No markers can currently be used to study gene
expression in early embryogenesis of tobacco．
Simple sequence repeats(SSＲs)are multiallelic in
nature，have abundant polymorphisms，high-information
content，codominant inheritance，can be simply manipu-
lated，and are easily detected by PCＲ． SSＲs markers，
therefore，have been widely used in genetic studies(Bin-
dler et al．，2007;Morgante ＆ Oivieri，1993;Powell et
al．，1996;Smulders et al．，1997;He et al．，2003)． So
far，a number of SSＲ markers have been identified from
public EST databases and used for applications in eco-
nomic plants，such as potato(Milbourne et al．，1998) ，
grape(Scott et al．，2000) ，citrus(Jiang et al．，2006) ，
and tobacco (Bindler et al．，2007 )． The 684
microsatellite markers of tobacco developed by Bindler
et al． were predominantly derived from genomic se-
quences of the Tobacco Genome Initiative (TGI) ，
through bioinformatics screening for microsatellite
motifs． We searched GenBank to screen for another set
of polymorphic microsatellite markers in order to obtain
useful genes of preponderant expression in zygotes for
studying gene expression in early embryogenesis of to-
bacco．
1 Materials and Methods
1． 1 Plant materials
Twelve tobacco cultivars，“Burley 21”，“Taiyan
No． 7”，“KY14”，“Samsun”，“Yunyan No． 87”，
“Souther Ｒhodesia”，“SＲ1”，“Smoke HAMA”，“Neph-
rite”，“Virginia331”，“W38”and“Gexin No． 1”were
used in present work． The plants were grown in a green-
house at 25 ℃ with a 16 h light period．
1． 2 Search for SSＲ loci
A total of 133 609 ESTs derived from tobacco were
obtained by searching GenBank (http:/ /www． ncbi．
nlm． nih． gov /)． These ESTs were assembled with
CAP3(Huang ＆ Madan，1999)． The nonredundant
dataset was used for screening microsatellites using the
MISA program (http:/ /pgrc． ipk-gatersleben． de /
misa /)． We adopted a criteria of searching for SSＲ loci
with a minimum of 12-bp repeat length．
1． 3 The primer pairs designed，polymorphism
analysis and genes amplification from tobacco
zygotes libraryTwenty primer pairs flanking the SSＲ loci were
screened for functionality． The primer pairs were de-
signed using the software Primer3(http:/ / redb． ncpgr．
cn /modules / redbtools /primer3． php) ，and were synthe-
sized for detecting polymorphisms among tobacco culti-
vars． ESTs containing polymorphic microsatellites were
searched against the GenBank nonredundant database u-
sing BLASTX algorithms(http:/ /www． ncbi． nlm． nih．
gov /BLAST)with expected value ＜ 10-7 for annotation of
putative functions．
Twelve tobacco cultivars were employed for poly-
morphism evaluation． PCＲ conditions were as follows，
20－30 ng temple DNA，0． 2 μmol·L-1 of each primer，
0． 2 mmol· L-1 dNTP，10 mmol · L-1 Tris-Cl (pH
8． 3) ，50 mmol·L-1 KCl，3 mmol·L-1 MgCl2，1 unit of
Taq DNA polymerase in a volume of 20 μL． PCＲ was
performed on PTC-100 Peltier thermal cycler with a pro-
gram of 94 ℃ for 3 min，32 cycles of 30 s at 94 ℃，30 s
at appropriate annealing temperature(Table 1) ，30 s at
72 ℃，and a final extension of 4 min at 72 ℃． The PCＲ
products were detected on ethidium bromide-stained 1．
5% agarose gels，and then on denatured PAGE by silver
staining．
Using the twenty primer pairs synthesized to
amplify genes from tobacco zygotes library(The zygotes
library from Key Laboratory of Ministry of Education for
Plant Development Biology，College of Life Sciences，
Wuhan University) ，PCＲ conditions of the amplification
genes were as above． The PCＲ products were detected
on ethidium bromide-stained 1． 5% agarose gels．
2 Ｒesults and Analysis
A total of 133 609 ESTs derived from tobacco Gen-
Bank were assembled with CAP3，resulting in 64 732
unigenes (including 18 039 contigs and 46 693
singlets)． The nonredundant dataset was analyzed for
screening microsatellites using the MISA program． A
7544期 张俊娥等:从烟草 EST微卫星标记中筛选合子胚中优势表达的基因
total of 40 894 SSＲ loci were identified in the unigenes．
Twenty primer pairs flanking the SSＲ loci were screened
and designed using the software Primer3，and were syn-
thesized for detecting polymorphisms among tobacco cul-
tivars．
Tobacco was an inbreeding，highly homozygous
plant，so there were few polymorphisms among the
tobacco cultivars． Twelve tobacco cultivars were
employed for polymorphism evaluation for further
screening out cross-parents with polymorphisms．
SSＲ markers were defined as either functional or
non-functional (Bindler et al．，2007)． Of the 20
screened potential SSＲ markers，17 primer pairs were
functional． For the functional primers，16 had polymor-
phisms among the tested tobacco cultivars，which were
BP750276， EB445309， EB683490， EB683247，
EB683081， EB683181， EB681632， EB682048，
EB681345， EB679090， EB680649， EB683715，
EB698114，EB690260，DW003872，DW004801． Of
them，polymorphisms of DW004801 among the tested to-
bacco cultivars were shown in Fig． 1． Functions for the
polymorphic SSＲ associated ESTs were determined by
BLASTX in GenBank，and 11 SSＲ loci showed
significant similarities to known genes(Table 1)．
Fig． 1 PCＲ products of DW004801 on denatured
PAGE by silver staining 1． Burley 21;2． Taiyan No． 7;
3． KY14;4． Samsun;5． Yunyan No． 87;6． Souther Ｒhodesia;
7． SＲ1;8． Smoke HAMA;9． Nephrite;10． Virginia331;
11． W38;12． Gexin No． 1．
After screening out the genes with polymorphisms，
cross parents were confirmed for further studying gene
expression in early embryogenesis of tobacco． The
sixteen genes with polymorphisms were amplified from
tobacco zygotes library． Nine genes (EB445309，
EB683490， EB683181， EB681632， EB682048，
EB683715，EB698114，EB690260，DW003872)could be
successfully amplified in cDNA of tobacco zygotes
library(Fig． 2)． Their length was about 200 bp． Of the
nine genes，six genes(EB683490，EB681632，EB682048，
EB683715，EB698114，DW003872)showed significant
similarities to known genes．
Fig． 2 Genes amplified from cDNA of tobacco
zygotes library (two repeats) A． DW003872;
B． EB683490;M． 50 bp DNA ladder．
3 Conclusion and Discussion
In order to study the expression pattern of genes
during early embryogenesis in plants，enough viable and
healthy cells of early embryogenesis must be isolated
from them． In addition，appropriate markers must be ob-
tained． We selected tobacco as the experimental
material to study the expression pattern of genes during
early embryogenesis in plants，as at present we have de-
veloped reliable technique to isolate healthy cells of
early embryogenesis (including zygote，two-cell，four-
cell，eight-cell，16-cell，32-cell，and so on) from
tobacco． The technique resolves one main difficulty of
above-mentioned，and it is also crucial to reliability of
the experimental results． However，obtaining appropriate
markers are also difficult，because polymorphism is few
due to self-pollinated characteristic of tobacco． Twelve
tobacco cultivars and twenty primer pairs flanking the
SSＲ loci were choosed to select polymorphic parents for
further studying the expression pattern of genes during
early embryogenesis． The results showed that sixteen
polymorphic SSＲ markers from tobacco ESTs deposited
in a public sequence database were developed． After se-
lecting out polymorphic SSＲ markers，they must be am-
plified in cDNA of tobacco zygotes library for further
confirming whether or not expression in zygote cells．
The results showed that nine genes can be successfully
amplified in cDNA of tobacco zygotes library． By above
854 广 西 植 物 33卷
Table 1 Characteristics of 16 polymorphic EST-derived SSＲs for tobacco
Locus
(Accession No．)
Size range
(bp)
Ｒepeat unit
Primers sequence
(5 / －3 /)
Ta
(℃)
No． of
allele
Protein homologue
(Accession No．)
E-value
BP750276 189－239 (TTA)16 F:TCTCCACTTCCTTGCTCCA
Ｒ:CGCATGAAAAGAGACGTCAA
53 5 Pepsin A［Arabidopsis thali-
ana］．(NP_188636)
1e-28
EB445309 198－235 (GCCAAT)3 F:AAGAAGGCAGAAGCACAACC
Ｒ:TTCTCTTCCATGCTGTGGTG
53 3 No significant similarity —
EB683490 203－241 (CAA)6 F:AATCATTCGAACGCCAACTC
Ｒ:GCGGACTATTGTCCTGCTTT
60 4 Avr9 /Cf-9 rapidly elicited
protein 194［Nicotiana taba-
cum］．(AAG43553)
7e-55
EB683247 195－249 (GA)25 F:CCTTCTCGGAACCAAACAGA
Ｒ:GGCCAGCCACTTCAGTAATTT
53 5 No significant similarity —
EB683081 193－241 (TGG)6 F:GCACCTGAAAAAGGAAGTCG
Ｒ:TATCATCTCTCCGCCACCA
60 3 hypothetical protein［Nicotiana
tabacum］．(BAC53934)
2e-22
EB683181 204－233 (TCA)5 F:GGGGCTAGAATCGGAACAA
Ｒ:CCACTGTCCCTGATTTCCAT
53 3 Os05g0270400［Oryza sativa
(japonica cultivar-group) ］．
(NP_001055058)
6e-09
EB681632 193－229 (CTT)5 F:ATCTCCGGTTCGTTGTTCAC
Ｒ:ACGACGTCGGAGAACTCATC
53 2 Thioredoxin x ［Arabidopsis
thaliana］(AAF15952)
8e-46
EB682048 183－231 (AAG)6 F:GAGATAATGGCGAAGGTGGA
Ｒ:AGGGAGAGGCATAGGGTTGT
54 3 forever young oxidoreductase
［Solanum bulbocastanum］．
(AAL60069)
2e-113
EB681345 194－237 (TCT)6 F:CGACCAAACTTGGGTTCAGT
Ｒ:GTGGCCTTAGGGGAGAGAAC
55 3 unknown protein［Arabidopsis
thaliana］．(NP_192933)
4e-91
EB679090 189－241 (ACA)6 F:GATGCAGCCAGGAACACATA
Ｒ:TGTGAAGTCCACTGCTTCCA
53 2 AN3(ANGUSITFOLIA3) ［Ar-
abidopsis thaliana］． (NP _
198216)
2e-23
EB680649 195－236 (CTG)6 F:TCACCTTCAGCTTTGTCGTG
Ｒ:TTGTTTGCGGAAGCATACTG
52 3 No significant similarity —
EB683715 172－203 (TTG)6 F:CACCAGGTGCACAGAGACAT
Ｒ:CAATCCGTTCCACATGTTTG
53 4 Os09g0493800［Oryza sativa
(japonica cultivar-group) ］．
(NP_001063548)
2e-07
EB698114 201－258 (AAC)6 F:CCAGCAGATAAGGCAAATGA
Ｒ:TCGCAATTGGTAAATCAGCA
50 6 PnFL-2 ［ Ipomoea nil ］
(AAG49896)
1e-30
EB690260 206－232 (AG)9 F:GGGGAGGAAGATCTGATGAA
Ｒ:CCCTTCTGGAAGGAGAAGAGA
54 2 No significant similarity —
DW003872 190－212 (GAA)6 F:AATCAATTCCAGGCTCATCG
Ｒ:TTTTTCCCCTCTTATGCCACT
50 2 No significant similarity —
DW004801 189－248 (CGC)6 F:GACGCAGGAGGTGATGAAAT
Ｒ:TATTGAGAAGCAACGCCACA
52 4 unknown protein［Arabidopsis
thaliana］．(NP_565494)
9e-19
Notes:Ta ． Annealing temperatures，putative homology acquired by searching the NCBI nonredundant database with BLASTX with an expected value
＜ 10-7 ．
work，a foundation of further studying the expression
pattern of genes during early embryogenesis was laid．
It is obvious that the genes of preponderant expres-
sion in zygote cells can be found from tobacco public
database，which will establish the foundation for further
studying the genes expression of early embryogenesis of
tobacco． These informative EST-SSＲ markers will be
useful in studying gene expression in early
embryogenesis of tobacco．
However，the technique of EST-SSＲ exists its limi-
tation of few polymorphism while comparing it with the
traditional SSＲ technique． For instance，low density is
in constructing genetic map． It is thus clear that tobacco
EST database for gene mapping and marker selection is
relatively limited． The study used only the data for em-
bryogenesis development，so polymorphism was not
high． Therefore，the selected markers for the genes of
preponderant expression in zygotes were not comprehen-
sive． But，the method was still a very effective to obtain
a part of genes of preponderant expression in zygotes．
(下转第 555页 Continue on page 555 )
9544期 张俊娥等:从烟草 EST微卫星标记中筛选合子胚中优势表达的基因
现明显正相关，高浓度反而会削弱镇痛活性，由此可
见，当油樟叶挥发油作为中枢镇痛药物时，其对浓度
依赖性小。醋酸扭体试验是化学刺激剂致痛，引起
的是内脏痛，主要是用于筛选外周镇痛药物。给予
不同剂量的油樟叶挥发油的小鼠扭体次数均低于对
照组，尤其是高剂量组和中剂量与溶剂对照组和油
樟叶低剂量组有显著差异，提示油樟叶挥发油具有
良好的外周镇痛活性，该生物学活性可能与 1，8-桉
叶油素相关(Le et al．，2001)。
参考文献:
A dzu B，Amos S，Kapu SD，et al． 2003． Anti-inflammatory and
anti-nociceptive effects of Sphaeranthus senegalensis［J］． J Eth-
nopharmacol，84(2－3) :169－173
Elisabetsky E，Amador TA，Albuquerque ＲＲ，et al． 1995． Analge-
sic activity of Psychotria colorata (Willd． ex Ｒ． ＆ S．)Muell．
Arg． alkaloids［J］． J Ethnopharmacol，48(2) :77－83
Le Bars D，Gozariu M，Cadden SW． 2001． Animal models of noci-
ception［J］． Pharmacol Ｒev，53(4) :597－652
Lee HJ，Hyun EA， Yoon WJ，et al． 2006． In vitro anti-
inflammatory and anti-oxidative effects of Cinnamomum camphora
extracts［J］． J Ethnopharmacol，103(2) :208－216
Ling J，Liu WY． 1996． Cytotoxicity of two new ribosome-
inactivating proteins，cinnamomin and camphorin，to carcinoma
cells［J］． Cell Biochem Funct，14(3) :157－161
Luo ZJ(罗中杰) ，Li WY(李维一) ，Wei Q(魏琴) ，et al． 2001．
The status Quo and future of Cinnamomum longepaniculatum in
Yibin(宜宾油樟的现状及未来) ［J］． J Sichuan Norm Univ (四
川师范大学学报)． 24(3) :317－319
Santos FA，Ｒao VS． 2000． Antiinflammatory and antinociceptive
effects of 1，8-cineole a terpenoid oxide present in many plant es-
sential oils［J］． Phytother Ｒes，14(4) :240－244
Wei Q(魏琴) ，Li Q(李群) ，Luo Y(罗扬) ，et al． 2006． Anti-
fungal activity of leaf essential oil from Cinnamomum longepanic-
ulatum(油樟油对植物病原真菌活性的抑制作用) ［J］． Chin J
Oil Crop Sci (中国油料作物学报) ，28(1) :63－66
Wei Q(魏琴) ，Zhou YK(周宇科) ，Zhou LJ(周黎军) ，et al．
2009． Inhibitive activity of Cinnamomum oil against bacteria(油
樟油抑制细菌生长的活性试验) ［J］． Chin J Trop Agric (热带
农业科学) ，29 (1) :5－7
Xu H，Liu WY． 2004． Cinnamomin-a versatile type II ribosome-in-
activating protein［J］． Acta Biochim Biophys Sin，36(3) :169
－176
檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿
(上接第 459页 Continue from page 459 )
Nine genes had been screened by this method and had
been used in studying gene expression in early embryo-
genesis of tobacco． They had high similarity to known
database． Now，we are carrying out verification of the
genes screened to determine their biological function． In
addition，we will also compare the screened genes with
tobacco genome which have been publicized to open
function in March 2012 in Nicotiana tomentosiformis
Goodspeed genome and Nicotiana sylvestris genome for
further obtaining some valuable information．
The study would provide an effective and applied
way to find molecular markers for preponderant
expression in zygotes．
Ｒeferences:
Bindler G，Hoeven Ｒ，Irfan GI． 2007． A microsatellite marker based
linkage map of tobacco［J］． Theoret Appl Gen，114:341－349
Davis DL，Nielsen MT． 1999． Tobacco-Production，Chemistry and
Technology［M］． Oxford:Blackwell
He C，Poysa V，Yu K． 2003． Development and characterization of
simple sequence repeat (SSＲ)markers and their use in
determining relationships among Lycopersicon esculentum cultivars
［J］． Theoret Appl Gen，106:363－373
Huang X，Madan A． 1999． CAP3:a DNA sequence assembly
program［J］． Gen Ｒesh，9:868－877
Jiang D，Zhang GY，Hong QB． 2006． Analysis of microsatellites in
Citrus unigenes［J］． Acta Gen Sin，33(4) :345－353
Milbourne D，Meyer ＲC，Collins AJ，et al． 1998． Isolation，charac-
terization and mapping of simple sequences repeat loci in potato
［J］． Mol ＆ Gen Gen，259:233－245
Morgante M，Oivieri AM． 1993． PCＲ-amplified microsatellites as
markers in plant genetics［J］． Plant J，3:175－182
Powell W，Machray GC，Provan J． 1996． Polymorphism revealed by
simple sequence repeats［J］． Trends Plant Sci，1:215－222
Smulders MJM，Bredemeijer G，Ｒus-Kortekaas W，et al． 1997． Use
of short microsatellites from database sequences to generate poly-
morphisms among Lycopersicon esculentum cultivars and accessions
of other Lycopersicon species［J］． Theoret Appl Gen，94:264－272
Scott KD，Eggler P，Seaton G，et al． 2000． Analysis of SSＲs derived
from grape ESTs［J］． Theoret Appl Gen，100:723－726
5554 期 曹玫等:油樟叶挥发油的镇痛活性研究