全 文 ：第 17 卷
第 4 期

Vol. 17
No . 4
草
地
学
报
ACTA AGRESTIA SINICA



2009 年
7 月

Jul.

2009
Molecular Analysis of T1 and T2 Generations of Red Clover Expressing Coat Protein
Genes of Alf alf a Mosaic Virus ( AMV) andWhite Clover Mosaic Virus (WCMV)
ZHANG Ting
t ing1 , ZHAO Gui
qin* , LIU Huan
( College of Gras sland S cien ce, Gan su Agricultu ral University, Lan zhou 730070, China)
Abstract: T hir ty
four T1 generat ion plants of transgenic r ed clov er ( Tr if olium p r etense L. ) w ere analyzed
by PCR and 12 single or double resistant t ransgenic plants w ere obtained. A rt if icial hybridizat ion w as car

r ied out betw een these positive plants of T1. Among 30 T2 progenies obtained, nine offspring expressed
bo th AMV and WCMV CP genes and nine expressed single CP gene by PCR and RT
PCR analysis. The
results o f PCR and RT
PCR indicated that the exogenous genes had been integrated into the genome of
tr ansgenic red clo ver in most of the hybrid o ffspring and could t ranscribe cor rect ly. Mo reover, the exoge

nous genes could steadily inherit fr om T 0 to T 2. T he results also demonst rated that art ificial hybridizat ion
is an ef ficient w ay o f gene pyr am iding of breeding prog ram.
Key words: tr ansgenic red clov er, molecular biolog y analysis, AMV, WCMV
表达 AMV、WCMV外壳蛋白基因红三叶
T1、T2代植株的分子生物学分析
张婷婷, 赵桂琴* ,刘
欢
(甘肃农业大学草业学院 , 甘肃 兰州
730070)
摘
要: 随着生物技术的发展,转基因技术已经越来越多的应用到植物育种领域并发挥着重要的作用, 将传统育种
技术与转基因技术相结合,既可以缩短育种时间 ,并具有良好的经济效益。为最终获得能稳定遗传且性状优良的
转基因植株,对其后代进行鉴定及筛选是非常重要的。本研究以抗苜蓿花叶病毒( AMV)转基因红三叶和抗白三
叶花叶病毒( WCM V)的转基因红三叶人工杂交后代 T1, T 2代植株为研究对象,通过 PCR及 RT
PCR 等分子生物
学手段,对 T1 代 34 株植株进行 PCR检测, 筛选出12 株阳性植株,其中兼有两种抗病基因的植株5 株, 各有一个抗
病基因的植株 7 株,在这些植株中进行杂交组合, 大部分杂交组合都结了种子, 最终得到 30 株性状优良的 T2 代植
株,对其进行 PCR及 RT
PCR检测, 结果表明 AMV 与WCMV 外壳蛋白基因能够在大部分 T2 代转基因植株中整
合并表达,并且通过人工杂交, 得到 9 株兼有两个抗病基因的杂交后代,外源基因稳定地从 T0 传至 T2。同时也表
明人工杂交是转基因植株基因累积的一种有效方法。
关键词: 转基因红三叶; 苜蓿花叶病毒;白三叶花叶病毒; 分子生物学分析
中图分类号: Q781




文献标识码: A




文章编号: 1007
0435( 2009) 04
0456
08
Introduction
Red clover ( T r if ol ium pratense L . ) is a for

age legume w idely g row n for hay, silage, pastur e
or as a g reen manure crop w ith considerable eco

nom ic importance in w orld agriculture[ 1, 2] . Because
of it s ability to f ix atmospheric nitr ogen, red clo ver
is of a high value to the environment too
[ 3]
. How

ever , the red clover of ten decline and lose it s per

sistence because o f it s poor disease to lerance and
w inter hardiness in Aust ralia, N ew Zealand,
Nor th Amer ica , and China[ 4] . The majo r v ir uses
收稿日期: 2009
03
13;修回日期: 2009
04
27
基金项目:科技支撑计划( 2008BADB3B07, 2007BAD52B06) ;甘肃省农业生物技术专项 ( GNSW0407)资助
作者简介:张婷婷( 1984
) ,女,甘肃平凉人,硕士,研究方向为牧草种质资源及育种; * 通讯作者 Author for correspon dence, E
mail : z ha

og07@ yah oo. com
第4期 ZHANG T ing
t ing et al : M olecular Ana lys is of T1 and T2 Generat ions o f Red Clover Expres sing Coa t Pro tein Genes of Al f alf a Mo saic Virus ( AMV) and White Clover M osaic Virus (WCMV)
infect ing red clo ver are Al f alf a mosaic v ir us
( AMV ) , Bean y el low mosaic vir us ( BYMV ) ,
Clover y ellow vein vi rus ( CYVV) , White clov er
mosaic vi rus ( WCMV ) , and Subter ranean clov er
stunt nanov ir us ( SCSV) [ 5] .
M ost of the t radit ional methods to prevent
plant virus infect ions are laborious, econom ically
and environmentally unsustainable[ 5] . T o over

come these problems, genet ic eng ineering technol

ogy, g enetic modification o f plants w ith plant viral
coat protein genes, w hich is an example of patho

gen
derived resistance, is being used successfully
to produce viral disease resistance
[ 6, 7]
. In this
study, the virus resistant t ransgenic red clover
plants expressing either the AMV or WCMV coat
pro tein and developed respect ively at the Plant Bio

technolog y Center, Ag riculture Victoria in Aus

t ralia w ere used[ 4] . Even though the general princi

ples of tr ansformation are known, the w ho le
pro cess is st ill far from being completely under

stood. T here is a considerable v ariat ion among in

dependent t ransgenic lines obtained under ident ical
condit ions using the same DNA const ruct [ 8] , inclu

ding featur es such as epigenet ic g ene silencing. Be

sides, the ult imate object ive of plant t ransforma

t ion is to obtain virus resistant or v ir us immune
lines of pro genies w ith good agronom ic characteris

t ics and high genet ic stability.
Accordingly , in order to obtain progenies w ith
stable inheritance, detect ion of genet ically modi

f ied plant is more impo rtant and signif icant. T he
po lymerase chain react ion ( PCR) and r everse t ran

script ion PCR ( RT
PCR) are tw o techniques w ide

ly used in molecular biolo gy. PCR is now a com

mon indispensable technique used in biolog ical re

search for a variety of applications[ 9, 10] . RT
PCR is
w idely used in expression pro filing , to determine
the expression of a gene o r to ident ify the sequence
of an RNA tr anscript . Both methods have high
sensit iv ity and eff iciency for t ransgenic detection.
Therefore, the PCR and RT
PCR were used to de

tect the integ ration and expression of the viral co at
pro tein genes in t ransgenic progenies in the study,
based on the results, the progenies w ith bo th
AMV and WCMV CP genes w ere selected and ob

tained. A rt if icial cr oss
fer tilizat ion was also carried
out betw een the t ransgenic pr ogenies in order to
combine bo th AMV and WCMV CP genes in one
plant .
T he results laid the foundat ions for further
study of stable inheritance of the viral co at pr otein
genes and eventual product ion o f double resistant
red clover plants.
Materials and methods
1
Plant materials
T ransgenic red clover plants t ransformed w ith
either the AMV or the WCMV coat pr otein gene
w ere developed from excised coty ledonary explants
of the cult ivar
Renegade
at the Plant Biotechno l

ogy Centre, Aust ralia. A binary vector containing
a chimer ic AMV4 or WCMV4 gene of interest and
a chimer ic neomycin phosphot ransferase ( npt
)
selectable marker gene w as used fo r the A gr obac

t er ium
meditated t ransformat ion[ 4] . Ar tif icial
cross
fert ilizat ion betw een the AMV and WCMV
primary t ransgenic plants w as employed to obtain
T1 seeds in Australia. The T1 red clover seeds w ere
planted in the Experimental Plot of College of Grassland
Science, GAU, and analyzed by PCR to identify trans

genic progenies. Based on the PCR results the following
crosses were made to obtain the T2 seeds: AMV
WC

MV, W
A, ( A& W)
W, ( A&W)
A, ( A&W)

( A&W) - ( Table 2) .
T he T 2 seeds w ere harv ested and stor ed fo r 3

4 months. Then the seeds w ere planted in the nu

t rit io n po t in the experimental lab for 4 months be

fore they w ere t ransplanted into the field.
2
DNA extraction and detections
T w o methods w ere used for DNA ex tract ion.
In method one ( M 1) , total DNA was ex tr acted
f rom the leaf lets of both T 1 and T2 transgenic and
non
t ransformed control red clov er plants using the
457
草
地
学
报 第 17卷
CT AB method[ 11 ] . A Plant Genomic DNA Purifi

cation Kit ( TIANGEN) w as used in Method 2 to
ex t ract T 2 generat ion plants ( M 2) accor ding to it s
manufacturer pro to co ls. The qualit ies of the DNA
samples w ere analy zed by elect rophoresis in 1%
agarose gels.
3
RNA isolation and detection
Total plant RNA was ext racted f rom T 2
tr ansgenic and non
t ransfo rmed red clover leaves
using T Rizo l ( Inv it ro gen Life T echnolo gies) . A p

prox imately 250 mg leaf t issue w as f rozen in liquid
nit rog en, g round in an RNase
and DNase
f ree
mortar and pestle, and RNA extracted as specified
by the manufactur er
s protocol. RNA was re
sus

pended in 50~ 80
L o f diethyl pyro carbonate t rea

ted w ater and 2. 5~ 4
L o f RNA Safe ( TIAN

GEN) . Water bathed at 60
for 20 m in to pro tect
the RNA samples f rom damage by RNase then
stored the samples at - 70
in the fr idge. T he
qualit ies o f the RNA obtained w ere analyzed by e

lectr ophoresis in 1% agar ose gels.
4
PCR analysis
PCR was carried out in a total vo lume of 25

L using 500 ng
1
g to tal DNA as a template and
rTaqTM po lymerase ( TaKaRa ) . Primers used for
the PCR detection of the selectable marker g ene npt
,
AMV coat protein
encoding gene, and WCMV coat
protein
encoding gene are shown in Table 1.
Table. 1
Specific primers used for PCR detection
Prim ers Sequen ce ( 5
t o 3
)
WCM V1 AAACTCGAGCAT GGACT TCACTACT T TA
WCM V2 CAGGT ACCCT GAAAT T TT AT
TAAACAGAAAGCACACAC
AMV1 CCAGATCT TCCATCAT GAGT T C
AMV2 CCAGATCT TCAATGACGATCA AGAT C
NPT
1 GAGGCT ATT CGGCT AT GACTG
NPT
2 ATCGGGAGCGGCGATACCGT A


T hermo
cycling w as performed as follow s:
95
for 5 m in, followed by 25 cycles, each cycle
comprising 1 min at 95
, 1 m in at 55
, 1 min at
72
, and an extension cycle o f 3 min at 72
. A ll
PCR analyses w er e repeated thr ee t imes.
5
RT
PCR analysis
All RT
PCR reactions w er e carried out using
the Quant OneStep RT
PCR Kit manufactured by
T IA NGEN( cat. # KR113 ) and the manufactur

er
s protocol. The specific primers used in the am

plif icat ion w ere the same as the PCR screening
showed in Table 1. RT
PCR reactions, done in a
25
L final v olume, include 2. 5
L of purif ied
RN A as template.
T hermo
cycling was performed as follow s:
50
for 30 min- ( reverse t ranscript ion) , 94
fo r
2 min, fo llow ed by 35 cycles, each cycle compri

sing 40 sec at 94
, 30 sec at 60
, 1 min at 72
,
and an ex tension cycle of 10 m in at 72
.
6
Detection of amplified products
T he amplified PCR and RT
PCR products
w ere analyzed by electrophoresis in an ethidium
brom ide stained 1% metaphor
agarose gel ( 1
1
metaphor to agar ose r at io) using sodium bo ric acid
elect rophoresis buffer. The PCR and RT
PCR
products w ere visualized under UV light using a
gel documentat ion sy stem . A 200
2000 bp mo lecu

lar DNA marker ( TIANGEN & T aKaRa) w as r un
in each gel to determ ine the fragment sizes o f the
amplified products. Posit ive and negat iv e controls
for the viruses under study w ere included in each
experiment .
Results
1
Artificial Hybridization
Art if icial hybridizat ion w as carried out be

tw een 15 transgenic T1 plants and all the cro sses
achieved 90% seeding rate. T he T 2 seeds w ere
planted in the nutr it ion po t for 4 months and then
transplanted to the f ield. T hirty st rong and
458
第4期 ZHANG T ing
t ing et al : M olecular Ana lys is of T1 and T2 Generat ions o f Red Clover Expres sing Coa t Pro tein Genes of Al f alf a Mo saic Virus ( AMV) and White Clover M osaic Virus (WCMV)
healthy T 2 plants w ere selected for molecular biol

ogy analysis w hen mature.
2
Analysis of transgene integration in
T1 and T2 progenies
2. 1
Electrophoretic analysis of total DNA from
transgenic plants
The results o f agaro se gel elect ropho resis o f
34 DNA samples o f T 1 generation, 30 DNA sam

ples of T 2 generat ion, and 1 DNA sample of con

t ro l non
t ransgenic plant obtained by either the M 1
or M 2 DNA purif ication method showed clear sin

gle high molecular w eight bands in all the samples
( F ig. 1) , both of the samples are suitable for use
in PCR.
2. 2
PCR analysis of T1 generation transgenic
plants
T he PCR was performed on the DNA from the
leaflets of 34 putat ive t ransgenic red clover plants
of T 1 generation and a non
t ransformed contro l
plant to detect the presence o f virus coat pr otein
Fig. 1
Representative DNA samples ( lanes 1
6) extracted by methods M1(A) , and M2(B)
genes and selectable marker gene ( F ig. 2, Panels
A
C) . Of the putat ive t ransgenics, f ive w ere posi

t ive for both AMV and WCMV; six w ere WCMV
posit iv e; one w as AMV posit ive; three w ere only
npt
posit ive, the rest and the non
t ransfo rmed
control plant w ere all negat iv e ( T able. 2) .
2. 3
PCR analysis of T2 Generation
The PCR was perfo rmed on the DNA from the
leaf lets o f 30 putative t ransgenic red clo ver plants
of T2 generat ion and a non
t ransformed control
plant ( F ig. 2, Panels D
F) . Of the putat ive t rans

genics, al l of which w ere nptII positive, ten w er e
AMV and WCMV posit ive, thirteen w ere WCMV
posit iv e, three w ere AMV positive, and the rest
w ere nptII positiv e. T he non
t ransfo rmed contro ls
w ere negat ive ( T able 3) .
The presence of 700
bp, 700
bp, and 1000
bp
PCR fragments for nptII, AMV and WCMV
genes, respectively, conf irmed the t ransgenic sta

tus of the plants.
From these results, f ive and ten t ransgenic
red clover plants w ith bo th AMV and WCMV viral
coat pr otein genes in T1 and T2 generat ions w ere
obtained. Seven and six teen red clo ver plants w ith
sing le viral coat protein genes in T 1 and T2 genera

t ions w ere also obtained
Table. 2
PCR screening results of T1 generation plants
Code Cul tivar npt
AMV WCM V
1. 1 Renegade + + +
2. 1 Renegade + - +
2. 2 Renegade + - -
4. 2 Renegade + + +
1. 3 Renegade + + +
2. 3 Renegade + + +
3. 3 Renegade + - +
2. 5 Renegade + + -
3. 5 Renegade + - -
4. 5 Renegade + - +
2. 9 Renegade + - -
2. 11 Renegade + - +
3. 11 Renegade + + +
5. 12 Renegade + - +
6. 12 Renegade + - +
NT Renegade - - -
459
草
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报 第 17卷
Table. 3
PCR and RT
PCR screening of T2 generation plants
code cros s combinat ion
PCR
npt
AM V WCM V
RT
PCR
npt
AMV WCMV
1 RA2. 5
RW2. 11 + + + + + +
2 RA2. 5
RW2. 11 + + + + + +
3 RA2. 5
RW5. 12 + + + + + +
4 RA2. 5
RW5. 12 + + + + + +
5 RA2. 5
RW3. 3 + - + + - +
6 RA2. 5
RW4. 5 + - + + - +
7 RA2. 5
RW3. 3 + - + + - +
8 RA2. 5
RW2. 1 + - + - - -
9 RA2. 5
RW2. 11 + - + - - -
10 RA2. 5
RW2. 11 + - + - - -
11 RW4. 5
RA2. 5 + + + + + +
12 RW4. 5
RA2. 5 + + + + + +
13 RW3. 3
RA2. 5 + + - + + -
14 RW3. 3
RA2. 5 + - + + - +
15 R( A& W) 3. 11
RA2. 11 + + + + + +
16 R( A& W) 3. 11
RW2. 11 + + + + + +
17 R( A& W) 3. 11
RA2. 11 + + + + + +
18 R( A& W) 3. 11
RW2. 11 + + - + + -
19 R( A& W) 3. 11
RA2. 11 + + - + + -
20 R( A& W) 2. 11
RW3. 11 + - + + - +
21 R( A& W) 4. 2
R( A&W) 1. 1 + - + + - +
22 RW4. 5
RA2. 5 + - + - - -
23 RW3. 3
RA2. 5 + - + - - -
24 R( A& W) 3. 11
RA2. 11 + - + - - -
25 R( A& W) 2. 11
RW3. 11 + - + - - -
26 R( A& W) 2. 11
RW3. 11 + + + - - -
27 RA2. 5
RW2. 11 + - - + - -
28 RA2. 5
RW2. 11 + - - + - -
29 RW4. 5
RA2. 5 + - - + - -
30 RA2. 5
RW2. 1 + - - - - -
NT - - - - - - -
Fig. 2
Representative PCR analyses of the different transgenes in the T1 ( lanes 1
8 in panel A, B and C) and T2 ( lanes 1
8
in panels D, E and F) transgenics showing 700 bp PCR fragment or npt
(A, D) and AMV
( B, E) and 1000 bp PCR fragment for WCMV ( C, F) CP genes
M: 200
2000 bp marker gene. NT : non
transformed contro l
460
第4期 ZHANG T ing
t ing et al : M olecular Ana lys is of T1 and T2 Generat ions o f Red Clover Expres sing Coa t Pro tein Genes of Al f alf a Mo saic Virus ( AMV) and White Clover M osaic Virus (WCMV)
3
Analysis of transgene transcription
Analy sis of RNA from the 30 transgenic red
clov er plants of T2 generat ion, in agarose gels
show ed that the 28 S, 18 S and 5 S rRNA were all
intact ( Fig 3) .
RT
PCR analyses w ere per formed to detect
AMV, WCMV and npt
t ransgene t ranscript ion
among the T 2 gener at ion t ransgenic progenies.
DNA fragments of expected sizes ( 700 bp for
AMV and nptII and 1000 bp for WCMV) w ere ob

tained in most of the T2 generat ion plants, respec

t ively ( F ig 3) . Among them, nine expressed bo th
AMV and WCMV CP transcripts; thr ee expr essed
AMV CP transcript, six expressed WCMV CP
tr asncript and three expressed the npt
gene on

ly. The rest w ere negat ive as w as the non
t rans

formed control ( Table 3) .
The results show ed that w e obtained tw enty

one t ransgenic red clov er plants o f T2 generat ion
that expressed the viral coat protein genes. A mong
them, nine of the plants expressed both o f the CP
genes, and nine of the them expressed sing le viral
CP genes.


In recent years, 120 genet ically
modified
plants w ere obtained and some impor tant tr ansgen

ic cr ops w ere commercialized [ 12 ] . During the
w ho le process of molecular plant breeding by ge

netic modif icat ion, ident ificat ion of t ransfo rmed
plants is an essent ial aspect.
In this study, the methods of mo lecular analy

sis had been successfully used for this purpose.
The PCR results in amplif icat ion of the tr ansgenes
in tw o successiv e generations o f progenies show ed
that f if teen plants w ith double viral CP genes in T 1
and T2 had been got ten, 23 plants w ith single viral
CP genes in T1 and T 2 had been also got ten. This
results indicated that the viral CP genes w er e stab

ly integrated into the red clover g enome and inheri

ted fr om T0 to T2.
At present , the technolog y of PCR analy sis
has been used to ident ified most of the t ransgenic
plants, such as occidental poplar、tomato、pepper、
grape、wheat , etc. T his method is the simplest and
most popular w ay o f t ransgenic identif icat ion.
T he RT
PCR results o f T2 generat ion plants
showed that eighteen plants w ith both the AMV
and WCMV CP genes or sing le v ir al CP genes had
been obtained, and these plants could also express
the genes w ell. The results demonstr ated the viral
CP genes could be t ranscribed by the modif ied
plants.
T he aim of artif icial hybridizat ion w as to com

bine molecular breeding and tradit ional breeding
techno logies for red clover to obtain AMV plus
WCMV double r esistant plants at last. T herefo re,
sing le and double AMV plus WCMV transgenic
plants w ere selected from T 1 generat ion and
crossed betw een single and double AMV plus WC

MV transgenic plants, and nine AMV plus WCMV
posit ive t ransgenic plants o f T2 gener at ion w ere
obtained from both str ategies. T hese results dem

onst rated that it w as po ssible to produce r ed clo ver
plants w ith double virus t ransgenes by crossing
par ents w ith single or double t ransgenes and that
both t ransgenes can be transcribed in the same
plant . M oreover, the combination of molecular
breeding and tradit ional breeding technolog ies
could enhance breeding eff icacies and improve eco

nomical ef f iciency .
Despite these over all positive results, the au

thors have also found a few abno rmal situat ions. In
the T1 generat ion, the genes of interest w ere not
integ rated into the plant genome in some of the
plants ( e. g. , plants 2. 2, 2. 9 and 3. 5 appeared to
inherit only the nptII g ene) . Similar situat ion w as
also observ ed in the T 2 generat ion progenies
( plants 27
29 ) . In addit ion, there w ere some
plants in the T 2 generat ion that could not t ran

scribe any of the exogenous genes detected by PCR
( e. g . , plants 8
10) .
T hese phenomenon could be due to tr ansgene
silencing. T he factor inf luencing gene silencing in

clude copy numbers, integr at ion sites and conf igu

rat ion of t ransgene, and act ivat ion of po st
t ran

script ional silencing, etc
[ 13]
. T ranscript ional g ene
silencing is the result o f histone modif icat ions
creat ing an environment of heter ochromatin around
461
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报 第 17卷
Fig. 3
Partial RNA samples extracted of T2 generation ( A) , RT
PCR analyses of the T2 transgenic plants
showing 700 and 1000 bp amplified for npt
(B) , AMV( C) and WCMV(D)
CP genes. M : 200
2000bp marker g ene. NT : no
tr ansfo rmed cont rol, RNA fr om a no
tr ansformed
red clo ver. 1
6: tr ansgenic red clover plants of T2 generation Discussion
a gene that makes it inaccessible to the t ranscrip

t ional machiner y
[ 14]
. Although these abno rmal ca

ses could be clar if ied by northern and Southern an

aly ses, the author
s task is to ident ify the t rans

genic pr ogenies for further cult iv ar development.
Conclusion
This is f irst report of molecular analysis on
t ransgenic r ed clover expr essing coat protein genes
of alfalfa mosaic virus and white clover mosaic vi

rus in china.
Compared w ith CT AB method and Plant Ge

nomic DNA Purif icat ion Kit, the lat ter w as more
eff icient , and the quality w as bet ter.
T he results of PCR analysis of tw o genera

t ions show s that most of the CP genes w ere inte

462
第4期 ZHANG T ing
t ing et al : M olecular Ana lys is of T1 and T2 Generat ions o f Red Clover Expres sing Coa t Pro tein Genes of Al f alf a Mo saic Virus ( AMV) and White Clover M osaic Virus (WCMV)
g rated into the red clover g enome. T he RT
PCR
results show ed that v ir al CP genes o f 21 plants
could be tr anscribed by the modified plants. T he
results also demonst rated that the ar tif icial hybrid

izat ion is an effectual w ay of g ene pyramid.
The tests o f plant resistance w ill be conducted
in the next step o f the study because the expression
of the t ransgene does not mean that all the t rans

genic plants w ill be r esistant to the targ et virus. In
addit ion, the t ransgenic plants w ere generated in
Aust ralia, and the virus coat pr otein gene used
w ere derived from Australian isolates o f the viru

ses. So it is unknown whether the t ransgenic
plants w ill protect ag ainst the virus found in Chi

na. T he hybrid of fspring s w ill be tested under field
condit ions and in the glasshouse for response to vi

r us inoculat ion.
Art if icial hybr idization and molecular analy sis
w il l be carried on w ith the t ransgenic red clover
pro genies in the study. In or der to obtain double
viral resistance tr ansgenic red clov er bred var iety.
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