全 文 ：高山植物黄管秦艽 cDNA 文库构建与
表达序列标签 (EST) 分析?
尹红菊 , 王留阳 , 高 茜 , 高大海 , 张东远 , 刘建全
(兰州大学干旱与草地生态教育部重点实验室分子生态研究所 , 甘肃 兰州 730000)
摘要 : 黄管秦艽 ( Gentiana officinalis) 是一种重要的藏药高山植物 , 本研究构建了该物种开花期的 cDNA 文
库。经检测达到中等 cDNA 文库水平 , 文库滴度为 1 .2×107 pfu?ml , 重组率 95 .9% , 插入片段平均长度大
于 500 bp。对 343 个随机挑选的重组克隆进行部分测序 , 获得的 ESTs经编辑后共有 181 条有效序列。经生物信
息学方法分析 181 条表达序列标签 (EST) 代表 144 个单克隆序列 , 其中 55 个与已鉴定的基因同源 , 35 个序列与
未鉴定的 EST 匹配 , 54 个未找到同源序列 ; 后两者共有 89个 EST 序列未发现功能相似的蛋白。对已鉴定的 EST
进行功能分析发现 , 相关基因主要编码以下蛋白 : 与蛋白表达相关的占 35% ; 光合作用相关的占 22% ; 新陈代
谢相关的占 18% ; 抗性相关的占 11% ; 质膜运输和细胞分裂相关的分别占 5% ; 染色体变化和细胞信号转导的
分别占 2%。根据有效 EST序列设计引物 , 通过 RT-PCR 进一验证了所得 EST 的准确性。这些研究结果为将来
研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。
关键词 : 黄管秦艽 ; 基因组学 ; cDNA 文库 ; 表达序列标签 ; 龙胆属
中图分类号 : Q 943 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 02 - 146 - 07
cDNA Library Construction and Expressed Sequence Tags
(ESTs) Analyses of an Alpine Plant Species
Gentiana officinalis (Gentianaceae)
YIN Hong-Ju, WANG Liu-Yang, GAO Qian, GAO Da-Hai ,
ZHANG Dong-Yuan, LIU J ian-Quan
( Instituteof Molecular Ecology, Key Laboratory of Arid andGrassland Ecology, School of LifeSciences,
Lanzhou University, Lanzhou 730000 , China)
Abstract: Gentiana officinalis, an alpine plant, one of thewidely usedTibetan traditional medicines . In this study, total
RNA was extracted fromthe whole plant of flowering individuals of this species, and a cDNA expression library was con-
structed usingCreatorTM SMARTTM cDNA Library ConstructionKit . The resultsshowedthat thetiter of thecDNA expression
library was 1.2×107 pfu?ml and the efficiency of recombinationwas 95 .9% . The average length of insert fragments in the
librarywas longer than 500 bp . A total of 181 valid ESTs were obtained from random sequencing of 343 clones . Further
bioinformatic analyses suggestedthey represented144 unique clonal sequences in which55 sequencesshowed high homolo-
gy to previously identified genes in Gentianaceae or other plants, 35 sequencesmatched toother uncharacterized expressed
sequence tags (ESTs) , and 54 sequences showed no well matches to available sequences in DNA databases . No protein
matched to the latter two sorts of ESTs ( 89) . Fifty-five ESTs with matched proteins were involved in a series of diverse
functions: protein expression (35% ) , photosynthesis (22% ) , metabolism ( 18 % ) , defense (11 % ) , membranetransport
(5 % ) , cell division (5% ) , chromosome metabolism ( 2% ) and signaling components (2 % ) . At last, RT-PCR primers
were designed according to the effectiveESTs to amplified the cDNAs of G. officinalis, whichfurther verified the accuracy
云 南 植 物 研 究 2009 , 31 (2) : 146～152
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.08181
? ?Received date: 2008 - 09 - 24 , Accepted date: 2008 - 11 - 20
作者简介 : 尹红菊 (1985 - ) 女 , 硕士 , 主要从事植物功能基因的研究。
of theESTs . This cDNA library provided acritical basis for further analyses of functional genes and gene expression in this
alpine species . Inaddition, these ESTs could be used to design functional nuclear primers for studying population genetics
of this species and closely related species .
Key words: Gentiana officinalis; Genomic Research; cDNA library; Expressed SequenceTags; Gentiana
Gentiana ( Smith, 1936 ) , a large genus in the
Gentianaceae, is widely distributed in the high moun-
tains in the temperate regions of the world ( Ho and
Liu, 2001 ) . Many species of this genus have been
used as traditional Chinese medicines and?or Tibetan
medicines ( Pharmacopoeia commission of PRC,
2000) , for example,“Long Dan”and“QingJ iao”.
These medicines were used to stimulate digestion and
appetite and relieve heartburn and stomach ( Van der
Sluis et al. , 1983; Tang and Eisenbrand, 1992) . The
main chemical constituents in these plants are comprise
gentiopicroside and swertiamarin ( Skrzypczak et al. ,
1993) . Despite the diverse researches on the resource,
taxonomy and evolution of the genus ( Adams, 1995;
Ho, 1985 , 1988; Ho and Liu, 2001) , nuclear genes of
thegenus received little attention . A cDNA library and
expressed sequence tags (ESTs) , through sequencing of
randomly selected cDNA clones fromcDNA library, pro-
vide an important basis for further functional analyses of
nuclear genes within one species (Ewing et al. , 1999 ,
2000; Nelson et al. , 2000 ) . In addition, EST-derived
nuclear markers can be developed from such ESTs and
used to evaluategenetic diversity, interspecific relation-
shipsof plants (Ma et al. , 2006; Zhou et al. , 2007) .
In this study, we aimed to construct cDNA library
and analyze ESTs characteristics of G. officinalis . This
species occurs in the high altitude region and has been
used as one of traditional Tibetan medicine . To our
knowledge, this is the first time to construct a cDNA li-
brary and sequence ESTs for one alpinegentiana species .
1 Materials and Methods
1 .1 Plant materials
Theflowering individualsof G. officinaliswere usedto exact
the total RNA . Fresh leaves, flowers androotswerecollected to-
gether and dehydrated in liquid nitrogen, then stored at - 80℃
for further RNA extraction .
1 . 2 RNA extraction and cDNA library construction
Total RNA was extracted usingTrizol Reagent Kit (Molecu-
lar Research Center, Inc . USA) . The following procedure was
performed accordingto themanufacturer′s recommendation of the
CreatorTM SMARTTM cDNA Library Construction Kit ( Clontech,
Mountain View, CA) . At first, using SMART technique, CDS
III?3’primer was usedtosynthesizethefirst-strandcDNA . Long
distancepolymerase chain reaction ( LD PCR) was used to syn-
thesize the double-strand cDNA that was then digested by SfiI
and fractionated by CHROMA SP IN-400 Column . The cDNAs
are longer than 0 .5 kb were collected and ligated to pBNR-LIB
vector . The recombinant plasmids transformed into E . coli-
DH5α . The quality of the cDNA library was strictly checked by
conventional titer determination . Twenty-one plaques were ran-
domly picked and tested using PCR with universal primers-M13
derived fromthe sequence flanking of the vector .
1 . 3 Sequence assembly , alignment and analysis
Clones for sequencing were selected randomly from the cD-
NA library, and eachclonewas incubateat 37℃ in1 .5 ml of LB
broth overnight with shaking . Plasmid DNAs of cDNA clones for
sequencing were isolatedwith the standard alkaline lyses protocol
using the Mini-plasmid kit ( U-gene) . The cDNA inserts were
subjected to single-pass partial sequencing from the 5′end by
employing the 5′end sequencing primer and ABI chemicals on
ABI 3730 DNA sequencers (Shanghai Bioasia, PRC) . We used
to MEGA4.0 ( Borland, America) to analyze the EST sequence .
Each EST was firstly analyzed using a multimodule custom pipe-
line which linked sequencebackup, basecalling, theelimination
of sequences shorter than 100 bp ( and low-quality sequences) ,
vector trimming, and sequence assembly . The resulting
unisequenceswere compared against the nonredundant (nr) pro-
tein database at the protein level by using BLASTx with default
parameters . In general , similarities with E-values < 10 - 5 were
considered significant . Unisequences displaying no significant
similarity to known genes ( BLASTx E-values > 10 - 5 ) were
searched against the dbESTest-others ( non-mouse, non-human)
using thetBLASTx algorithm .
1 . 4 RT-PCR confirmation
Reverse transcription ( RT) was performed based on 2μg of
thepreviously isolated total RNAs of G. officinalis . The cDNAs
were synthsized accroding to the Reverse Transcriptase M-MLV
(RNase H - ) Kit (TakaraBiotechnologyCo ., Ltd) . To validate
theaccuracyof the ESTs, RT-PCR was performed using cDNAs
as templates and a specific pair of primers designed for each se-
lected gene . The amplification conditionswere 1 cycle for 2 min
7412 期 YIN Hong-J u et al. : cDNA Library Construction and Expressed Sequence Tags ( ESTs) Analyses of an . . .
at 94℃ , and 25 cycles of 94℃ for 30 s, 52℃ for 1 min, and
72℃ for 1 min 30 s . PCR products were electrophoresed on a
1 .1% agarose?EtBr gel .
2 Results and Discussions
2 .1 Extraction and purification of total RNA
The ratio of OD260?OD280 of total RNA was
1 .98 . Then, the integrity of the total RNA was ana-
lyzed by agarosegel electrophoresis ( Fig . 1) . Accord-
ing to Fig . 1 , the bands of 28S and 18S were obvious
and the brightness of the band of the 28S was about
twice of the 18S .
Fig . 1 Total RNA of Gentiana officinalis
2 .2 cDNA synthesis
Using 2μg RNAs, first-strand cDNAs were syn-
thesized . Then, 1?5 of the1st cDNA wereused tosyn-
thesize the ds cDNAs . After 22 thermal cycles, 5μl of
100 μl was analyzed by agarose gel electrophoresis
(Fig . 2) . The bands of ds cDNAs were dispersed and
the length of ds cDNA was mainly bounded on 500 -
5000 bp, indicating the well quantity of the ds cDNA .
This method was also previously showed to obtain a
higher percentageof full-length cDNA .
Fig . 2 ds cDNA synthesized using theSMART control reagents
2 .3 The quantity of the cDNA library
The library titer was measured as 1 .2×107 pfu?
ml . In order to analyze the size of the constructed li-
brary and the diversity of cDNA inserts, 21 plaques
were randomly selected, and amplified with primers-
M13F?R (synthesised by TaKaRa Company, sense se-
quence: 5′- G TAAAACGACGGCCAGT - 3′? anti-
sense 5′- AACAGCTATGACCATG - 3′) followingthe
program: 94℃ 5 min; 94℃ 30 s, 52℃ 30 s, 72℃ 1
min for 35 cycles; 72℃ 10 min . PCR products were
checkedby the DNA markers ( Fig . 3 ) . The percent-
ageof recombinants from the library was 95 .9% . The
average length of the inserts was 900 bp . All these
analyses indicate that RNAs in the cDNA library are
well represented .
2 . 4 General characteristics of ESTs
A total of 343 cDNA cloneswereselected randomly
fromthe library and single-pass sequencesweregenerat-
ed . After excluding those poorly sequenced and?or with
less than 100 bases, a total of 181 ESTs wereobtained .
Contigs that consist of one sequence were considered
singletons, while contigs comprised of two or more
sequences were classified as redundant ESTs or contigs .
Fig . 3 Composition of cDNA fragments in the libraries
841 云 南 植 物 研 究 31 卷
TheseEST sequences were clustered in 20 contigs and
124 singletons . Since the cDNA library has not been
amplified and the clones for sequencing have not been
subtracted, the number of ESTs basically reflect the
prevalenceof the correspondingmRNA (Fig . 4) .
Based onmatches with available data (Table 1) ,
among 144 unique sequences, 55 showed homology to
previously identified genes in Gentianaceae or other
plants, 35 matchedother uncharacterized expressed se-
quence tags ( ESTs) , and 54 showed no significant
matches to sequences present in DNA databases . The
latter two classes of ESTs ( together 89 sequences) also
showed no correspondingproteinmatch . Further analy-
ses of theESTs with the matched proteins showed they
Fig . 4 Prevelence distribution of identified ESTs
( number of singletons is 124 ; numbers of contigs with two, three, four,
five and six sequences are 12 , 2 , 2 , 1 , 2 , respectively)
Table 1 Database match of Gentiana officinalis ESTs to the genes of the other organisms
GenBank
accessions
Function
Score
(bits) E-Value Closest Species Copies
Cell division
AY611040 ?NAM protein 284 3 .00 E-75 Picea glauca 1 }
AY639034 ?auxin-induced putative CP12 domain-containing protein 72 .1 1 .00 E-10 Arachis hypogaea 1 }
BK000123 ?putative phytosulfokinepeptide precursor 95 4 .00 E-17 Solanumlycopersicum 1 }
Chromosome metabolism
EF520004 ?sister chromatid cohesion 2 104 6 .00 E-20 Arabidopsis thaliana 1 }
Defense
AY173073 ?catalase 408 3 .00E-128 Hypericumperforatum 1 }
DQ444292 ?metallothionein- like protein 61 .6 6 .00 E-10 Camellia sinensis 2 }
DQ497591 ?Gentiana siphonantha isolate 1 trnS-trnG intergenic spacer 74 .8 6 .00 E-20 Gentiana siphonantha 1 }
EU271754 ?osmotin 81 .7 1 .00 E-13 Piper colubrinum 1 }
NM—111462 VRCI2 ?A ( race-cold-inducable 2A ) 122 3 .00 E-25 Arabidopsis thaliana 1 }
NM—114701 Vhaloacid dehalogenase- likehydrolase family protein 286 ?1 .00 E-74 Arabidopsis thaliana 1 }
Membrane transport
AF003347 ATP phosphoribosyltransferase 162 ?2 .00 E-37 Thlaspi goesingense 1 }
AF127442 ATAF1 2-like protein 250 ?1 .00 E-69 Picea abies 1 }
AF051222 ATAF1 2-like protein 297 ?3 .00E-108 Picea mariana 1 }
Metabolism
AB027191 ?isopentenyl pyrophosphate isomerase 389 8 .00E-106 Gentiana lutea 2 }
AB281494 ?alpha?beta hydrolase fold superfamily 301 2 .00 E-92 Gentiana triflora 2 }
AF367442 NAD-dependent malate dehydrogenase 144 ?4 .00 E-32 Prunus persica 1 }
AJ 251269 ?geraniol 10-hydroxylase 143 1 .00 E-31 Catharanthus roseus 1 }
AM269122 ?putative glycogenin 383 5 .00E-104 Picea abies 1 }
AM269142 ?putative homeodomain leucine zipper protein 377 4 .00E-102 Picea abies 1 }
AM269256 ?monooxygenase 325 3 .00 E-87 Picea abies 1 }
AM269265 ?monooxygenase 225 2 .00 E-56 Picea abies 1 }
EU344848 ?plastidic aldolase 209 5 .00 E-64 Solanumtuberosum 1 }
LAUMTNADH NADH dehydrogenase subunit 4 &483 ?9 .00E-134 Lactuca sativa 1 }
Photosynthesis
AB017366 ?phytoene cyclase 532 2 .00E-154 Gentiana lutea 2 }
AB027191 ?isopentenyl pyrophosphate isomerase 389 8 .00E-106 Gentiana lutea 2 }
AB236868 ?ribulose-1 , 5-bisphosphate carboxylase?oxygenase small subunit 325 2 .00 E-89 Panax ginseng 2 }
AF034631 chlorophyll a?b binding protein LHCII type I precursor 223 ?8 .00 E-58 Panax ginseng 1 }
AJ 577578 ?PSII K protein 116 4 .00 E-44 Olea europaea 1 }
DQ781306 ?chloroplast photosystem II light- inducibleprotein 80 .3 1 .00 E-19 Pachysandra terminalis 1 }
DQ887080 ?photosystem I psaH protein 232 2 .00 E-58 Arachis hypogaea 4 }
EF203260 ?phytoene synthase 3 385 3 .00E-104 Gentiana lutea 2 }
EU308517 ?ribulose-1 , 5-bisphosphate carboxylase?oxygenase large subunit 175 3 .00 E-41 Silene aegyptiaca 1 }
9412 期 YIN Hong-J u et al. : cDNA Library Construction and Expressed Sequence Tags ( ESTs) Analyses of an . . .
Continue table 1
GenBank
accessions
Function
Score
(bits) E-Value Closest Species Copies
X66727 ?P . taeda gene for protochlorophyllide reductase 85 .8 8 .00 E-43 Pinus taeda 1 }
X95987 ?PSII polypeptide 184 4 .00 E-49 Solanumlycopersicum 1 }
Protein expression
AB236868 ?ribulose-1 , 5-bisphosphate carboxylase?oxygenase small subunit 325 2 .00 E-89 Panax ginseng 1 }
AB236868 ?ribulose-1 , 5-bisphosphate carboxylase?oxygenase small subunit 332 2 .00 E-88 Panax ginseng 1 }
AB237912 ?ribosomal protein S12 153 3 .00 E-36 Nicotiana sylvestris 2 }
AF127593 putative 60 ?S ribosomal protein L13a 412 4 .00E-122 Picea abies 6 }
AF479180 26 ?S ribosomal RNA 189 1 .00 E-45 Exacumaffine 1 }
AF479180 26 ?S ribosomal RNA 245 2 .00 E-62 Exacumaffine 1 }
AJ 316582 ?ribosomal protein S12 135 7 .00 E-36 Atropa belladonna 1 }
AM111313 ?Plantago major mRNA for histin H3 293 9 .00 E-77 Plantago major 1 }
AP009123 Ribosomal protein S12 ?259 9 .00 E-74 Gossypiumbarbadense 5 }
AP009374 ribosomal protein S12 ?319 1 .00 E-88 Lepidium virginicum 1 }
DQ176643 ?Vitis pseudoreticulata clone EST-423 23S ribosomal RNA 132 1 .00 E-29 Vitis pseudoreticulata 4 }
DQ673255 ?ribosomal protein S12 116 1 .00 E-36 J asminum nudiflorum 1 }
DQ673255 ?ribosomal protein S12 116 7 .00 E-39 J asminum nudiflorum 1 }
DQ629362 ?large subunit ribosomal RNA 348 2 .00 E-93 Sabia sp . Qiu 91025 $1 }
EF207443 ?ribosomal protein S19 250 2 .00 E-64 Cercidiphyllumjaponicum 1 }
EF207453 ?ribosomal protein L2 347 4 .00 E-93 Peridiscus lucidus 1 }
EU118126 ?ribosomal protein S12 182 4 .00 E-44 Ipomoea purpurea 1 }
EU301782 ?Daucus carota 26 eS ribosomal RNA gene 192 5 .00 E-47 Daucus carota 1 }
EU431223 ?ribosomal protein S12 349 1 .00 E-93 Carica papaya 1 }
Signaling components
AY936336 ?NdhC 261 5 .00 E-67 Operculina aequisepala 1 }
Unclassified
AC139600 ?unknown 244 4 .00 E-62 Medicago truncatula 1 }
AC187538 ?unknown 329 1 .00 E-87 Solanumlycopersicum 1 }
AK224216 ?unknown 309 2 .00 E-81 Oryza punctata 1 }
AK224613 ?unknown 137 5 .00 E-30 Solanumlycopersicum 1 }
AK246203 ?unknown 159 2 .00 E-36 Solanumlycopersicum 1 }
AK246262 ?unknown 280 2 .00 E-75 Solanumlycopersicum 1 }
AK246799 ?unknown 230 1 .00 E-61 Solanumlycopersicum 1 }
AK246799 ?unknown 229 5 .00 E-61 Solanumlycopersicum 1 }
AK251177 ?unknown 242 5 .00 E-62 Hordeum vulgare 1 }
AL606457 unknown 105 ?5 .00 E-49 Oryza sativa JaponicaGroup 1 }
AM425978 ?unknown 241 5 .00 E-61 Vitis vinifera 1 }
AM454485 ?unknown 183 1 .00 E-43 Vitis vinifera 1 }
AM462697 ?unknown 48 .7 3 .00 E-27 Vitis vinifera 1 }
AM482227 ?unknown 61 .1 4 .00 E-12 Vitis vinifera 1 }
AP004898 unknown 199 ?3 .00 E-54 Lotus japonicus 2 }
AP008212 unknown 155 ?6 .00 E-59 Oryza sativa JaponicaGroup 1 }
AP008218 unknown 115 ?4 .00 E-23 Oryza sativa JaponicaGroup 1 }
AY142543 ?unknown 238 2 .00 E-66 Arabidopsis thaliana 1 }
AY142543 ?unknown 238 2 .00 E-66 Arabidopsis thaliana 1 }
CT831892 ?unknown 376 1 .00E-101 Oryza sativa Indica Group 1 }
CU223189 ?unknown 177 2 .00 E-49 Populus tremula 1 }
CU224065 ?unknown 143 2 .00 E-31 Populus tremula 1 }
CU224528 ?unknown 54 .7 3 .00 E-22 Populus tremula 1 }
DQ226906 ?unknown 79 2 .00 E-12 Boechera divaricarpa 1 }
EF085796 ?unknown 199 2 .00 E-49 Picea sitchensis 1 }
EF087806 ?unknown 69 .3 4 .00 E-09 Picea sitchensis 1 }
EF146998 ?unknown 90 .9 1 .00 E-36 Populus trichocarpa 1 }
EF147066 ?unknown 72 .5 3 .00 E-17 Populus trichocarpa 1 }
EF148586 ?unknown 142 3 .00 E-31 Populus trichocarpa 1 }
EF534108 ?unknown 105 2 .00 E-22 Beta vulgaris 1 }
NM—001050318 ?unknown 113 6 .00 E-23 Oryza sativa JaponicaGroup 1 }
NM—001050861 ?unknown 91 .3 4 .00 E-27 Oryza sativa JaponicaGroup 1 }
NM—111205 Vunknown 139 ?6 .00 E-39 Arabidopsis thaliana 1 }
NM—124490 Vunknown 65 ?. 7 2 .00 E-22 Arabidopsis thaliana 1 }
Y08501 ?unknown 62 .5 7 .00 E-14 Arabidopsis thaliana 1 }
051 云 南 植 物 研 究 31 卷
are involved in protein expression ( 35% ) , photosyn-
thesis (22% ) , metabolism (18% ) , defense ( 11% ) ,
membrane transport (5% ) , cell division (5% ) , chro-
mosome metabolism ( 2% ) and signaling components
(2% ) (Fig . 5) .
2.5 Verification of the ESTs′accuracy using RT-PCR
To verified the accuracy of ESTs, we designed 12
pairsof RT-PCR primers (Table2 ) accroding to the ef-
fective EST sequences selected randomly fromTable 1 .
These primers were used to amplify the cDNAs . The
expected products wereobtained for eachpairs of prim-
ers (Fig . 6) .
In conclusion, we successfully constructed the
cDNA library of G. officinalis . This librarywill provide
a basis for cloning functional genes of this species in
the future . In addition, ESTs can be further used to
design a series of primers to amplify nuclear fragments
of this species and closely related species duringstudy-
ing populationgenetics of thesespecies and interspecif-
ic relationships at genomic level .
Fig . 5 Functional classification of ESTs according to the tBLASTx search results
( 1 . Protein expression 35 % ; 2 . Photosynthesis 22 % ; 3 . Metabolism 18 % ; 4 . Defense 11 % ; 5 . Membrane transport 5 % ;
6 . Cell division 5 % ; 7 . Chromosome Metabolism 2 % ; 8 . Signaling components 2 % )
Table 2 Semi-quantitative RT-PCR primers designed according to the G. officinalis ESTs
Primers Match Accessions Product length Sequences of primers
G1 fAB281494 &632 ?
S 5 #′CGTTATGGAAGAGTACACAGCA 3′
A 5′TCACTCCCCTCAAACATAACAC 3′
G2 fEF520004 !1230 #
S 5 #′CCTCACATTACACCTTGCTACA 3′
A 5′GCGAATAAGCAAGCCAAGAC 3′
G3 fDQ226906 (1047 #
S 5 #′AATGGTTCACCGATTCCCCC 3′
A 5′CTTTACATAAACTCGACAGG 3′
G4 fAF051222 #900 ?
S 5 #′TCGTCAAACAAGTCAATCCG 3′
A 5′GCTTTCCAGTATCCAGAACCAG 3′
G5 fAF003347 #1163
S 5 #′CAAATACCATTTTACAAAATTC 3′
A 5′GGCACAAGGCAAGAAGGCGA 3′
G6 fAB027191 &755 ?
S 5 #′TTTGCTGCATAGAGCGTTTAGC 3′
A 5′ATGTCAGTAGCTTCTTGGAGGG 3′
G7 fEF203260 !692 ?
S 5 #′GCGTCACACATAAATCCAACCG 3′
A 5′ACTCCTTTCTCTGCATCATCATAG 3′
G8 fAB017366 &975 ?
S 5 #′GTGCGTGCGCTGAGATATTA 3′
A 5′CACGCTCCATAGAATCGTCATC 3′
G9 fAB017366 &1145 #
S 5 #′GTGCGTGCGCTGAGATATTA 3′
A 5′CACGCTCCATAGAATCGTCATC 3′
G10 rAF034631 #601 ?
S 5 #′GTCTCTTCTTCCTTCTCAGT 3′
A 5′TTTATTTGATGGAGTTCGAA 3′
G11 rEU853017 &985 ?
S 5 #′GC TACGCCAGGTATTACCCA 3′
A 5′GA TTTCCCCGTTCTCTCTCC 3′
G12 rEU344848 &1167 #
S 5 #′AATGGTTCACCGATTCCCCC 3′
A 5′CTTTACATAAACTCGACAGG 3′
1512 期 YIN Hong-J u et al. : cDNA Library Construction and Expressed Sequence Tags ( ESTs) Analyses of an . . .
Fig . 6 RT-PCR Results
( fromright to left, are 2000 marker and the products by G1 to G12 pairs of primers)
Reference :
Adam ?sLG, 1995 . Chionogentias ( Gentianaceae) , a new generic name
for the Australasian“snow gentians,”and a revision of the Austra-
lian species [ J ] . Australian Systematic Botany, 8 : 935—1011
Ewin (g R , Olivier P, Jean MC , 1999 - 2000 . Comparative analysis of the
Arabidopsisand rice expressed sequence tag (EST) sets [ J ] . In Sil-
ico Biology, 1 : 197—213
Ho T /N , Liu SW, 2001 . A worldwide monograph of Gentiana [ M] . Bei-
jing: Science Press, 480—481
Ho T .N , 1985 . A study on thegenus Gentiana of China ( 1 ) [ J ] . Acta
Phytotaxonomica Sinica, 23 : 43—52
Ho T -N , 1988 . Gentianaceae [A ] . In: Wu ZY , Raven PH eds . Floraof
China [M ] . Beijing: Science Press, 62 : 67—70
MaX ;F , Szmidt AE , Wang XR , 2006 . Genetic Structure and Evolution-
ary History of aDiploid Hybrid Pine Pinus densata Inferred from the
Nucleotide Variation at Seven Gene Loci [ J ] . Moleculer Biology
and Evolution , 23 : 807—816
Nels ?on PS, Han D, Rochon Y et al. , 2000 . Comprehensive analyses of
prostate gene expression: convergenceof expressed sequence tag data-
bases, transcript profiling and proteomics [ J ] . Electrophoresis, 21 :
1823—1831
Skrz ?ypczak L , Wesolowska M, Skrzypczak E, 1993 . Gentiana species
XII : In vitro culture, regeneration, and production of secoiridoid glu-
cosides [ A ] . In: Bajaj Y PS ed . Biotechnology in Agriculture and
Forestry [M ] . Berlin: Springer-Verlag Press, 21 : 172—186
Smit ?h H , 1936 . Gentianaceae in Handel-Mazzetti [ J ] . Symbolae Sini-
cae, 7 : 979
Tang ?W, Eisenbrand G, 1992 . Gentiana spp [ A ] . In: Tang W, Eisen-
brand G eds . Chinese drugs of plant origin, chemistry, pharmacolo-
gy, and use in traditional and modern medicine [M ] . Berlin: Spri-
nger-Verlag Press, 549—553
Van ?Der Sluis WG, Van Der Nat JM, Labadie RP, 1983 . Thin-layer
chromatographic bioassay of iridoid and secoiridoid glucosides with a
fungitoxic aglucone moiety using b-glucosidase and the fungus Peni-
cilliumexpansum as a test organism [ J ] . J ournal of Chromatogra-
phy, 259: 522—526
Zhou RC , Zeng K , Wu W et al. , 2007 . Population Genetics of Specia-
tion in Nonmodel Organisms: I . Ancestral Polymorphism in Man-
groves [ J ] . Moleculer Ecology and Evoluion, 24 : 2746—2754
251 云 南 植 物 研 究 31 卷