全 文 ：Vol. 31 , No. 1
pp. 24 - 28 　Jan. , 2005
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 31 卷 第 1 期
2005 年 1 月 　24～28 页
Cloning and Characterization of Novel Tobacco Genes Induced by Ethephon Using
Improved Differential Display Method
YU Tao , YI Ping , ZHI Li2Feng , LI Yang2Sheng , ZHU Ying2Guo 3
( Key Laboratory of Ministry of Education for Plant Developmental Biology , College of Life Sciences , Wuhan University , Wuhan 430072 , Hubei , China)
Abstract :An improved differential display approach was used to identify novel genes induced by the ethylene generator
ethephon from tobacco leaves1 Total 24 cDNA fragments that show different expression level between ethephon2treated and
control plants were isolated1 Dot blotting indicated that 17 of them were positive1 Sequence analysis revealed that four of
them ( DF6 , DF8 , DF9 , DF13) represented novel genes1 One of them ( DF8) was an unknown gene , the other three
were new cDNA fragments with homology with some known genes1 RT2PCR analysis of the four novel cDNA clones revealed
that three of them ( DF6 , DF8 , DF13) were expressed constitutively in untreated control plants , and had improved mRNA
transcript level in ethephon treated tobacco plants1 While DF9 mRNA occurred only in the treated plants1
Key words :DDRT2PCR ; Ethylene ; Tobacco
利用改进的差异显示技术分离烟草中受乙烯利诱导的新基因
余　涛　易　平　支立峰　李阳生　朱英国 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 ,湖北武汉 430072)
摘 　要 :利用改进的差异显示技术从烟草中分离受乙烯利调控的新基因。在分离到的 24 个差异片段中 ,通过点杂交验
证 ,表明其中 17 个为阳性。对这些阳性片段的序列分析表明有 4 个新基因 ( DF6 , DF8 , DF9 , DF13) 。其中 DF8 是一个
未知基因 ,其他 3 个通过 GenBank 搜索可以找到一些同源的已知基因。RT2PCR 分析表明 , DF6、DF8、DF13 在对照中也
有表达 ,受乙烯利诱导其表达水平上调。而 DF9 则仅在处理植株中表达。
关键词 :DDRT2PCR ;乙烯 ;烟草
中图分类号 : S572
　　Endogenous plant hormone ethylene plays an impor2
tant role in plant growth and development , including ger2
mination , flower and leaf senescence , fruit ripening , leaf
abscission , cell2fate determination in the root epidermis ,
root modulation , sex determination , programmed cell
death , and responsiveness to stress pathogen attack[1 ,2 ] 1
How this simple olefin evokes such a diverse array of
physiology processes has been a central question in ethy2
lene research[3 ] 1 Elucidation of the mechanism of ethylene
perception and signal transduction began with the isolation
of mutants that had defective ethylene responses1 By this
way , more than a dozen genes have been implicated in the
ethylene2signaling pathway[2 ] 1 An alternative approach is to identify ethylene2re2sponsible genes to define the regulatory DNA elementsthat mediate transcriptional activation and subsequently toprovide tools to link ethylene signal transduction pathwayto gene transcription1 Many ethylene2responsive geneshave been isolated , such as tomato fruit E4 gene[4 ,5 ] , thecarnation senescence — related glutathione S2transferase( GST1) gene[6 ] and the tobacco defense genes[7 ,8 ] 1 Theysuggested that it is very useful in exploring the ethylene2signaling pathway to isolate ethylene2responsible genes1In this paper , we used ethylene generator , ethephonto induce ethylene response in tobacco plants and de2scribed the isolation of four cDNA clones , corresponding
繱基金项目 : 国家 973 基金项目 (2001CB1088)资助。
作者简介 : 余涛 (1977 - ) ,男 ,武汉大学生命科学学院博士研究生 ,主要从事植物发育遗传学研究。 3 通讯作者 :朱英国。E2mail :
zhuyg @public1wh1hb1cn
Received(收稿日期) :2003210224 ,Accepted(接受日期) : 20032122261

to novel ethephon2inducible genes by an improved cDNA
DDRT2PCR method1 They were all reported first time ,
and might be useful for the research of the function and
signal pathway of the plant hormone ethylene1
1 　Materials and Methods
111 　Plant materials and ethephon treatment
　　Tobacco ( Nicotiana tabacum cv1 SRI) plants were
grown in soil under standard greenhouse conditions1 Six
weeks old tobacco plants were used in the experiments1
For observing the effect of ethephon , three fully expanded
tobacco leaves were cut from the plant and floated on 50
mL solution of ethephon ( ET , 100 mmolΠL) in Petri dish2
es with diameter of 15 cm[9 ] , tobacco leaves floated on
Milli2Q (MQ) water served as an appropriate control1 In2
cubation was done under continuous white fluorescent light
(150μmol·m - 2·s - 1 ) 1 Leaves were collected at the time
indicated and used immediately for RNA extraction1
112 　Differential display screening
Total RNA from leaf materials was extracted using
TRIzol ( Invitrogen) and used for first strand cDNA syn2
thesis1 Five micrograms of total RNA were heated at 72 ℃
for 10 min , cooled on ice for 5 min and then mixed with 2
μL 25 mmolΠL dNTPs , 4μL 011 molΠL DTT , 2μL pri2
mer XSCVN ( 5′2GACTCGAGTCG ACATCGAGCT17 VN2
3′) , 8μL of 5 ×first2strand synthesis buffer (Promega) ,
1μL RNasin ribonuclease inhibitor (Biostar International ,
Canada) , 12 μL of water and 1 μL of reverse tran2
scriptase (Promega) 1 The resulting solution was incubated
for 2 h at 42 ℃1 After adding 2μL of RNase H (AP Bio2
tech) the mix was incubated for 20 min at 37 ℃1 The re2
action was terminated by heating the sample at 75 ℃for
15 min1
Differential display ( DD ) PCR amplification of
cDNA was performed following a modified protocol from
Hoeberichts et al1 (2001) [10 ] 1 cDNA (2μL) was ampli2
fied with XSC (5′2GACTCGAGTCGACATCGAGC23′) as
a reverse primer , and arbitrary 18 - 25 bp oligonucleoti2
des as forward primers1 Reaction mixtures were prepared
with 215μL of 10 ×PCR buffer , 115μL of 25 mmolΠL
MgCl2 , 1μL of 011 molΠL dNTPs , 015μL of 10μmolΠL
XSC primer , 2μL of 10μmolΠL arbitrary primers , 15μL
of H2O and 015μL Taq DNA polymerase1 PCR condi2
tions were : 4 min at 94 ℃; 2 min at 45 ℃; 5 min at
72 ℃; 5 cycles of 30 s at 94 ℃, 30 s at 52 ℃and 50 s at
72 ℃; 35 cycles of 30 s at 94 ℃, 30 s at 56 ℃and 50 s
at 72 ℃; 5 min at 72 ℃1 Reaction products were separat2
ed by denatured PAGE and stained with silver1 Differen2
tial fragments were re2amplified , recovered and then
cloned with the p GEM2T Easy Vector System (Promega) 1
113 　Dot blot analysis
Total 100 ng of each differential fragments DNA were
loaded on a small strip of nylon membrane positively
charged1 The same quantity of tobacco actin gene DNA
was also applied as a reference1 The total RNA of ethep2
hon treated and control tobacco plant were used to be la2
beled with 32 P and hybridize with the membrane1
114 　DNA sequence analysis
The differential fragments clones were delivered to
Shanghai Casarray Co1 , Ltd1 for sequencing1 Database
searches and comparison with published sequences were
carried out using the BLAST program[11 ] 1
115 　RT2PCR analysis
The RT2PCR analysis was carried out as the method
of Zegzouti et al1 (1999) [12 ] 1 As an internal control , a
fragment of the endogenous tobacco actin cDNA was am2
plified concomitantly with the DF ( differential fragment)
clone by adding 2 μmolΠL of actin2specific primers
( ACT1 : 5′2TGCTGGCCGTGACCTAACTG23′; ACT2 :
5′2TGCTAAGGGATGCGAGGATG23′) to the PCR reac2
tion buffer1 The PCR products were separated on a 114 %
agarose gel , transferred to a nylon membrane and hybri2
dized with a mixture of 32 P2labeled DF (differential frag2
ment) and actin probes1
2 　Results
211 　Isolation of ethephon2regulated cDNA fragments
　　The RNA from tobacco leaves treated with ethephon
for 4 h and control were analyzed1 After separated by de2
natured PAGE , total 24 differential fragments ( DF1224) ,
varying in length from 300 bp to 115 kb showing either an
increase or decrease in gene expression during ethephon
treatment were isolated1 Some of them were shown in
Fig111
52　第 1 期 YU Tao et al . :Cloning and Characterization of Novel Tobacco Genes Induced by Ethephon Using. . . 　　　

Fig11 Some differential fragments exhibited
by improved DDRT2PCR
ET: Ethephon2treated plant1 C: Non2treated control1 Arrows indicated
some of the cDNA fragments expressed differently in ET treated plants
and control1 1 ,2 ,7 indicated the differential fragments expressed special2
ly in control plants ; 3 ,4 ,5 ,6 indicated the differential fragments ex2
pressed specially in ethephon2treated plants1
Fig12 Verify the differential fragments by dot blotting
ET: Ethephon2treated plant1 C:Non2treated control1 Arrows indicated the
hybrid signal of tobacco actin as a referance1 A1 , B1 , C1 , D1 , E1 ,
D2 , A3 , C3 , A4 , B4 , D4 , E4 indicated the differential fragments
highly expressed in ethephon2treated plants ; A2 , B2 , C2 , E2 , A5 indi2
cated the differential fragments highly expressed in control plants ; B3 ,
D3 , E3 , C4 , B5 , C5 , D5 showed no different expression pattern
between control and ethephon2treated plants and were proved to be false
positive1
212 　Verify the differential fragments by dot blotting
In order to identify the authenticity of these differen2
tial fragments , dot blots analysis was carried out1 As
shown in Fig12 , 17 of them showed different expression
level between control and ethephon2treated ( ET) plants
and were confirmed to be true differential fragments1 One
of them had no hybrid signals in both control and ET
plants , which might be the result of the low expression
level1 The other 6 failed to show differential expression
level between control and ET plants and were considered
to be false positive1
213 　Sequence analysis of the four novel cDNA clones
In order to explore what these fragments represent ,
those true positive fragments were cloned into p GEM2T
easy vector for sequencing , and the sequencing results
were compared in GenBank to search homongous
sequences1 Sequence analysis revealed that four of them
( DF6 , DF8 , DF9 and DF13) were novel gene frag2
ments ( Table 1) , the other fragments were some known
genes such as tobacco class Ⅰchitinase B gene ( DF2) ,
tobacco beta21 , 32glucanase gene ( DF18 ) which had
been reported to be ethylene2responsible[13 ] 1 DNA se2
quence analysis of the four novel cDNA fragments revealed
that DF6 shares 87 % similarity with S1 tuberosum mRNA
which encode a stolon tip protein induced during the early
stages of tuberisation[14 ] and a ripening2related mRNA
( ERT 13) in L1 esculentum which was induced by ethyl2
ene treatment [15 ] 1
The DF8 cDNA fragment is 750 bp long1 A BLAST
nucleotide search with this fragment did not show any sig2
nificant homology1
The DF9 comprises 700 bp and is most similar to
many kinds of auxin2induced protein genes pCNT103 ,
pCNT110 , pCNT111 , pGNT1 and pGNT35 (with a gene2
ral designation 1032type genes) in tobacco1 It also shows
some sequence identity with a glutathione S2transferase
gene in tomato and a pathogenesis2related protein ( PR1)
gene in potato1
The last fragment DF13 is about 500 bp long1 A
BLAST nucleotide search revealed that this fragment is
82 % homologous to tobacco glycine2rich protein precursor
( GRP) gene that was induced by tobacco mosaic virus
(TMV) infection or salicylic acid treatment[16 ] 1 It also
shares 84 % nucleotide sequence identity with a cell wall
protein (TLRP) gene in tobacco1
The four cDNA fragments which represent novel
genes were delivered into GenBank database1 The acces2
sion numbers were DF6 : AY491535 ; DF8 : AY491537 ;
DF9 : AY491536 ; DF13 : AY4915381
214 　RT2PCR analysis of the novel cDNA clones
The RT2PCR analysis of the four differential frag2
ments representing novel cDNAs was shown in Fig131 The
results suggested that they were all up2regulated by ethe2
phon1 For DF6 , DF9 and DF13 , the mRNA level in2
62 　　　　作 　　物 　　学 　　报 第 31 卷 　

creased after 4 h of ethephon treatment1 Until 24 h after
treatment , there were still high transcription level of DF6
and DF131 While for DF8 , the transcription level was
abundant in 4 h of treatment , but decreased to the initial
level in 24 h of treatment1 In the four genes , only DF9
had almost no transcription in non2treated control , but the
other three showed certain mRNA expression in control
plants1
Table 1 The novel ethephon2regulated cDNA fragments isolated from tobacco by improved DDRT2PCR
Differential
fragment Size Homologous sequence
Nucleotide acid
identity
GenBank accession
number 3
DF6 - 400 bp A stolon tip protein gene in S1 tuberosum 87 % Z11679
A ripening2related gene ( ERT13) in L1esculentum 85 % X72731
DF8 - 750 bp Unknown
DF9 - 700 bp Auxin2induced 1032type genes in tobacco 75 % X56263
Glutathione S2transferase gene in tomato 72 % AJ250001
A pathogenesis2related protein (PR1) gene in potato 70 % J03679
DF13 - 500 bp Tobacco glycine2rich protein precursor ( GRP) gene 82 % M37152
Cell wall protein (TLRP) gene in tobacco 84 % Y19032
　　Note : 3 indicates the GenBank accession number of the homologous sequences1
Fig13 RT2PCR analysis of the novel cDNA clones
0 : Non2treated control ;
1 :Control treated with water for 4 h ;
2 :Control treated with water for 24 h ;
3 :Tobacco plant treated with ethephon for 4 h ;
4 :Tobacco plant treated with ethephon for 24 h ;
ACT: Actin gene as an internal reference1
3 　Discussion
Differential display RT2PCR ( DD RT2PCR) has
been developed as a very useful tool to detect and charac2
terize altered gene expression in eukaryotic cells1 Its oper2
ation is very quick and easy , as well as very little mRNA
is used by PCR2based method1 But this method has a very
serious defect , the high frequency of false positive re2
sults , which would greatly increase the labor of identifica2
tion and selection of the differential fragments1
The major reasons for false2positive results are low2
stringency annealing , isolation of comigrating PCR pro2
ducts (contamination) and amplification by the arbitrary
primers[17 ] 1 In order to solve this problem , we modified
the reverse2transcription (RT) primer and used longer ar2
bitrary and anchor primers (as described in methods) 1
The longer primers made us use higher annealing tempera2
ture in PCR reaction and improve the PCR stringency1
Another result of using longer anchor and arbitrary primers
was reducing the complexity of DD RT2PCR products
bands , which would greatly decrease the chance of comi2
grating PCR products1 In our experiment , 17 out of 24
cDNA fragments isolated were confirmed by dot blotting to
be true positive1 This suggested that this improved DD
RT2PCR procedure could remarkably reduce false positive
results1
As a very good generator of plant hormone ethylene ,
ethephon is widely used in agriculture1 It’s stable in room
temperature and can be decomposed easily to generate
ethylene in plant1 During the decomposing of ethephon ,
acid is also produced which was considered to affect
plant , so its application in laboratory was limited[3 ] 1
While , because it’s easy to use and was widely applifi2
cated in farm production , there was still much research
using ethephon as ethylene generator[9 ,18 ,19 ] 1
In the four novel ethephon2regulated genes isolated ,
three of them showed homology to some genes having
known function1 The DNA sequence of DF6 has homology
with a reported stolon tip protein gene ( TUB8) presented
throughout early tuberisation of potato and a ripening2
related gene ( ERT13) in tomato1 The TUB8 protein con2
tains several repeated motifs and a high proline content
72　第 1 期 YU Tao et al . :Cloning and Characterization of Novel Tobacco Genes Induced by Ethephon Using. . . 　　　

and has been suggested to play a role in the cell wall ar2
chitecture1 The ERT13 gene is ethylene2regulated , but
its function is unclear1 Tobacco related closely with potato
and tomato1 DF6 is also ethylene2regulated , it’s there2
fore possible that DF6 protein has the similar function in
both tuberisation and ripening1
The identified clone DF9 is highly similar in DNA
sequence to some auxin2induced 1032type genes in tobac2
co1 These genes were reported to express only in the root
tip , which indicated that their expression was related with
the start of cell division[20 ] 1 DF9 sequence also shows
homology to a kind of glutathione S2transferase gene
( CTU1 ) in tomato and a pathogenesis2related protein
(PR1) gene in potato1 The transcription level of CTU1
improved rapidly during programmed cell death (PCD) in
tomato cell suspensions triggered by treatment with camp2
tothecin , an inhibitor of topoisomerase 1[10 ] 1 PR1 gene is
rapidly activated in potato leaves after infected by some
pathogeny or treated with fungal elicitor[21 ] 1 It’s suggest2
ed that DF9 might belong to the 1032type genes and its
expression might be related with pathogen or PCD re2
sponse in plant1
The DF13 sequence is homology to some glycine2
rich protein genes , which were induced by tobacco mosaic
virus infection or salicylic acid treatment and were sug2
gested to play a role in controlling plant viral systemic
movement1 Therefore , it’s possible that DF13 gene
might play a role in defending against virus infection in
tobacco plant1
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