农杆菌侵染植物的原理 烟草农杆菌转化
natiwanlar-free
2022年5月1日发
(作者:齐光伟个人简介)Agricultural Science&Technology,2011,12(1):62—64
Copyright⑥201 1,Information Institute of HAAS.All rights resemed Agricultural Biotechnology
Optimization of Genetic Transformation System of
Tobacco K326 Med iated by Agrobacterium
ZHAO Qin
Agronomy Depa ̄ment of Sichuan Agricultural University搜索引擎原理,Ya’an 62501 4
Abstract {O biective I The aim was lo optimize genetic lransformation system in lobacco K326 mediated by Agrobacterium 1 Method j The Iear
of fobacco aseptic seedling was faken as exp}ants lo study the optimization of Agrobacterium-mediated genetic transformation systern. Result j
The highest transformation efficiency was obtained when the explants were pre cultu red in the medium of MS+2 mg/L 6-BA+0.2 mg/L lAA
for 2 d网页挂马检测,and then infected with Agrobacterium GV3101(OD =O 6) or 5 rain.The PCR detection proved that npt ll gene had been integrated
into the regenerated tobacco p)ants [Conclusion j A highly efficient genetic transformation system of tobacco(ear mediated by Agrobacterium
was es1ab¥ished,
Key words Agrobactenum tumefaciens:Tobacco:Genetic transformation
Tobacco f Nicotiana.Solanaceae)is a kind of important then sterilized with 1O%sodium hypochlorite solution for 15
annual or perennial economic crops.It is used as model plant
min.washed with sterile water for 5 limes.and slightly dried
jn currenl field of plant genetic engineering because i{s tissue with sterile absorbenl paper.Then.they were sowed jn the
ls easy to be cultured and its transformation efficiency s medium of MS+sucrose 30 g/L+Agar 8 g/L(pH 5.8)建站培训,
high .Transgenic tobacco plant was first obtained using with 4 seeds per bottle.The seeds were cultured in 25℃con-
Agrobacterium-mediated transgenic technology by Horseh et
stanl temperature incubator for 45 d.under the conditions of
a/. .and lhen a series of significant progresses on trans- light intensity Ix 1 6OOrobots协议,photoperiod 16 h(1ight)/8 h(dark).The
genic tobacco research were obtained.For example,Glyco-
aseptic seedling was cut into smalI pieces of about 5 mm x 5
syltransferase gene had been transformed jnto tobacco via mm after four Jeaves grew.and the veins were removed.The
Agrobacterium by CEHN Qiu—ping et a1. :the expression lear pieces were pre—cultured in MS medium containing 2 mg/L
vector of potato IbNPR1 gene was constructed and success・
6一BA and 0.2 mc L IAA for 2 d.and then dipped in Agrobac—
fully transformed fnto tobacco bv CHEN Guan shui et a1.网络推广宣传,and tef um liquid.Then.the lnfected explants were cultured 1n MS
lhe resulting transgenic plant was conformed using PCR anal-
medium containing 2 mg/L 6一BA and 0.2 mg/L IAA in dark—
ysis :transgenic lobaccos carrying chitinase gene Ch¨rOm
ness for 2 d.After co.culture.5O mg/L kanamycin and 50O
Trichoderma and herbicide resistant gene Bar were estab-
mg/L carbenicillin were added into medium for screening and
Iished bv WU Wen-jun et a1.L5 3.Tobacco K326 was intro-
inducing the growth of resistant callus.culture conditions was
duced ffOm America in 1 985.its aroma quality and amount
the same as above.Subculture was peflormed once every 14
were superior to other varieties .In addition.it is most wide-
d.When the resistanl shoot of callus grew lo 2 cm.the shoot
IY cultivated jn China.Aqrobacte优化设计, m GV31 O1-mediated ge-
was transferred into the rooting medium f MS+50 mg/L ka—
netic lransformation system of the tobacco Jeaf was optimized
namycin+5o0 ma/L carbenicillin+0.2 mq/L lAA).The cal—
by lhe author目录搜索引擎,to determine the optimum conditions for the
Ius began rooting aboul 7 d Iater.When the seedling grew to
transformation of foreign functionaI gene;n tobacco
about 6 cm、the flask mouth was opened for training plantlet
fOr 2 d.And then che seedlings were transpianted into nutritive
Materials and Methods sotj which had been sterilized by high temperature.They were
Plant materiaI.strains and plasmids
cultured at 25—27℃in light.Meanwhile.plastic ffIm was
The seeds of lhe experimentaI materiaI tobacco K326 covered on them to maintain moisture. 企业网络营销方案。
were provided by Agronomy Department of Sichuan Agricul-
Comparison of infection time
tural University.Agrobacterium tumefaciens strain GV31 01 Agrobacterium GV31 01 single colony was picked and cul—
and plant binary expression vector pBI1 21 were saved by lhe
tured at 28 oC overnight}n YEB medium
.and then inoculated
Department of Biotechnology.Sichuan Agricultural University.
with the inoculum size of 1:1 00.After growing to the appropri‘
Kanamycin was impohed Sigma-packaging product,Taq DNA ate OD value.the cells were harvested by centrifuging al
polymerase was purchased fr0m Dingguo Biological Engineer— 5 000 r/min for 5 rain.and then resuspended in sterile MS Iiq—
ing Company The primers of npt II gene were:upstream uid medium so as to obtained the solution with the OD value
primer 5’-GCTA丁G ACT GGG CAC AAC AG.3’.downstream Of 0.6.Then cotyledon explant of tobacco was{nfected by the
primer 5'-ATA CCG TAA AGC ACG AGG AA-3’:they were solution.with the infection time of 1,3 5企业网站建设要求,7 and 9 min,re—
synthesized by Sangon Biotech(Shanghai)Co.商城推广方案,Ltd.. spectively.The excess solution was dried with two Iayers of
Genetic transformation of tobacco sterile filter paper.the explant was cultured}n the selective
The fulI seeds with the same size were selected.and
medium.1 5 d Iater.the callus formation was observed.
Comparison of the infection concentr ̄ion of Agrobacterium
Received:December 27,201 0 Accepted:danua ̄31.201 1
The saturated Agrobacterium culture was jnoculated with
Corresponding author.E—mail・z654321 q@1 63 com the inoculum size Of 1:1 O0 and cuItured{O the desired 0D val
ZHAO Qin.Optimization of Genetic Transformation System of Tobacco 1<,326 Mediated by Agrobacterium 63
ues of 0.2,0.4。0.6.0.8 and 1.The sterile cotyledon of to.
bacco seedling which had been pre—cultured for 2 d was infec—
ted by Agrobacte rIum solution for 6 min.and then cultured ln
dark for 2 d.before transferring Into selective medium.Af【er 1 5
d of culture.the callus fOrmation of explants was observed.
Molecular detection of tobacco carrying npt II gene
The totaI DNA of the leaves of regenerated resistant plant
and nOrmaI controI plant was extracted with C JAB method.
and then used as template for PCR detection.PCR reaction
system contained 1 ul DNA template.2 pl 10 x Taq buffer
(Mg“),0.5 ul of each primer(10 pmol/L),0.5 pl 4O mmoI/L
dNTP,0.1 pl Taq DNA polymer enzyme(5 U/p1);ddH2O
was added to the total volume of 20 p1.The PCR was started
with predenaturing at 94 oC for 5 min
,
followed by 35 cycles of
denaturing at 94 oC for 1 min
.annealing at 55℃for 1 min,
and extension at 72 oC for 1 min:the amplification was com—
pleted by holding the reaction mixture at 72 oC for 8 min to al—
Iow complete extension of PCR products.
Results and Analysis
Comparison of diferent infection time of Agrobacterium
lt could be concluded frOm Table 1 that the differentiatiOn
rate of the explants were significantly diferent under the con—
ditions of diferent infection time in Agrobacterium liquid with
the 0D value of 0.6.The diferentiation rates of explants
which were infected with Agrobacter『um for 1 and 3 min were
40%and 47%.respectively.5 min of the infection time could
obtain the highest diferentiation rate 63%.When the explants
were infected for 7 and 9 min,the diferentiation rates of ex-
plants were 56%and 53%.but Agrobacte contamination
happened at the same time.So.it could be concluded that,5
min was the most suitable infection time to obtain high rate of
callus fOrmation and reduce the contamination rate.
Table 1 The effect of jnfection time on diferentiation rate of tobacco
The data was analyzed by using DPS statistical method.The diferent
lower case letters represent significant diferences at P<O.05 leve1.
Co ̄dson of the infection concentration of Agrobacterium’
ft could be concluded frOm Table 2 that.under the condi—
tion of Infection time 5 min.the effects of diferent infection
cOncentratiOns of Agrobacte 网站建设需要什么人,n GV31 01 on the diferentiation
rates of explants varied significantly.The diferentiation rates
of the explants\nfected by Agrobacterium\iquid with the oD
values 0.2 and 0.4 respectively were 36%and 46%.The ex-
plants infected by the Agrobacterium with the OD value 0.6
showed the highest differentiatiOn rate of 63%.The diferenti-
ation rates of the explants、nfected by Agrobacte rjum with the
。D values 0.8 and 1.0 respectively were 50%and 43%.In
addition. with the increase of infection concentration,
Agrobacte rjum contamination phenomenon was observed.
Therefore.in order to obtain high rate of callus formationbaidu竞价,this
study suggests that the infection concentration of oD value
O.6 is more suitable.
Table 2 The effect of the Agrobacterium concentration on diferentia-
tion rate of tobacco
The data was analyzed by using DPS statistical method.The diferent
lower case letters represent significant diferences at P<0.05 leve1.
Molecular detection of npt II gene in tclbacco
A totaI of 25 regenerated plants were obtained in this
study.PCR detection of part of the regenerated plants was
shown in Fig.1.1t could be concluded frOm Fig.1 that the am-
plified bands of npt II gene showing the same size as expec—
ted.while no band of the negative controI non-transgenic plant
could be observed.Thus.it could be preliminarily concluded
that the resulting regenerated plants were transgenic plants.
2 3 4 M
M:DNA Maker;1—4:The regenerated plants;5:Non・trans-
genic negative contro1.
Fig.1 PCR test results of regenerated plants
Discussions
optimization of the tobacco genetic transformation sys-
tem is of great theoTetical and practical significance for obtai—
ning transgenic plants.In recent years.most researches on
transgenic tobacco were mediated bv Agrobacterium -gj.The
strength of interaction between Agrobacte c,m and plants is
the main factor to affect the induction efficiency of plants.
Some experiments show that the sensitivity of the same to—
bacco variety to diferent Agrobactenum rhizogenes differs.
the same conclusion can be obtained frOm the transfOrmation
of other plantsl1o J.SoiI Agrobacterium belongs to gram-nega—
tive bacterium .Agrobacterium commonly used in genetic
transformation experiments includs LBA4404.EHA1 O5.A4.
C58.and GV31 01 and sO On.Diferent scholars use diferent
strains in transgenic tobacco researches.For instance,
Agrobacte um EHA1 05 was used to infect aseptic seedling of
tobacco K326 bV CHEN Guan-shui L4 :Agrobacterium A4 and
ATCC1 5834 were used to mediate genetic transformation of
tobacco K326 bv SONG Zhi—hong et a1.1 12 J.Agrobacterium
LBA4404 was used to infect tobacco by WANG Wei et a1..
and the result revealed that 3—5 min of infection was the most
suitablel 13 J
.Agrobacterium C58 was used to infect tobacco
cultivar Zhongyan 99.and the result found that the highest dif—
ferentiation rate f 63%1 could be obtained under the condi—
tions of that the infection time was 5 min and the 0D value of
bacteriaI solution was 0.6:in addition.the explants were easi-
IV to be cOntaminated bV A口泰安百度公司,l06ac挎r扬中网站建设,I,m when the jnfect10n
time was 7 and 9 min,which was cOnsistent with the study Of
WANG Wei ef a ¨ 一。4 J. 1n additi0n,studies have repOrted
that a ste le fjIter paper cOve rinq On the surface Of medjum dur-
inq c0一culture medium cOuId qreatIy inhib.t the qr0Ⅵ h Of
A口,叻ac挎r『l册新泰网,and the present e×periment Obtained the same
Agricultural Science&Technology Vo1.12.No.1.201 1
result j
.
Agricultural University(吉林农业大学学报),2OO8,30(4):442—
445.
In summary.an efficient transformation system obtained
}n this study was as follows:the explant which was pre—cul-
tured for 2 d was Infected by the bacteriaI solution of oD value
[9】ZHOU SF(周树峰)google网站推广,LI QL(李秋莉),WU YQ(吴元奇),ef Im
proving tobacco oxidative stress by Agrobacterium mediated trans-
formation of choline monooxygenase gene from Suaeda liaotungen・
0f O.6:after 2 d of co—culture.jt was washed with sterile wa-
ter and cultured for 45 d on screening differentiation medium,
the transformat)on efficiency cou}d reach 63%.PCR dete ion
s『S(农杆菌介导的辽宁碱蓬胆碱单氧化酶基因CMO转化烟草提高抗
氧化胁迫的研究)[J].Chinese Agricultural Science Bulletin(中国农
学i耐艮),2010,鸽({5):44—47
f奥运软文,01 GODW】N),丁DDD G网站推广员,FD只D—L】OV B.The eff6cts D{aeetosynn—
gone and pH on Agrobacte#ummediatedtransformation vary ac-
on the regenerated transgenic p)ants proved that the resist—
ance genes had been integrated into the tobacco genome竞价排名点击,
which was significant for tobacco genetic breeding and the in—
troduction of foreign genes.
cording plant species[J].Plant CeI 1Repo ̄,1991,9(12):671一
研r5.
References
1]ZHANG N(张宁),WANG D(王蒂).High efficiency system of ac-
ceptor system of Agrobacterium-mediated transformation of tobac-
[11]CHENG YP(程焉平).Research and appliatcion 0f transgenic plants
(转基因值物的研究与应用)[J].Jilin Agricultural ciSences(吉林农
业科学学报),2001黑链代码,26(5):26—30.
[12]SONG ZH(宋志红).CUl H(崔红),LIU GS(支0国J 页)竞价产品,8f a1.Study
on factors i#luencinfl the efficiency of Ri p)asrr ̄d transforming of to-
co(农杆菌介导的烟草高效遗传转化体系研究)[J]Gansu Agricul—
tural Science and Technology(甘肃农业科技),2O04(9):11—13.
f21 H0RSEH RB百度分享互刷,FRY JE HOFFMANN NI快速网站建设,ef a A simple and gen.
bacco(Ri质粒转化烟草影响因素的研究)[J].Journal of Henan Ag—
ricultural University(河南农业大学学报)seo外链代发,2OO4百度品牌专区,38(3):259-262.
[13]WANG W(王伟),WANG YQ(王义强),XU GB(徐刚标).sludy
on the conditions of transferring Caz gene into tobacco mediated
eral method for transferring genes into plants[J].Science软文培训,1985,
227.1129—1131.
by Agrobacterium tumefaciens(根癌农杆菌介导Caz基因转化烟草
条件研究)[J]Nonwood Forest Research(经济林研究)潍坊网络营销外包,2007,
25(2):35—38. [3j CHEN QP(陈秋苹),LIU XQ(刘学),WANG CT(王春台).Re—
search of conditions on tobacco transformation mediated by f14]WANG F(王飞)网站安全检测工具,XING XS(耶新生),MA YZ(马有志),et a1.
Study on the conditions of transferring 1332 gene into tobacco me— Agrobactedum tumefaciens expressing glycosyltransferase gene
(限癌农杆蔺介导的糖苷转移酶基因转化烟草的条件研究)[J]
Chenftstry&8ioengfnee 几g(化学与生物工程)网站解决方案,20O5,22(6):9—
11
[4]CHEN GS(陈观7k)整合网络营销,ZHANG z(张铮),ZHOU YF(周以飞)关键词seo优化公司,et al
Construction of plant expression vector of Ib NPR1 gene and trans—
diated byAgrobacteriumtumefaciens(根癌农扦菌介导D32基因转
化烟草的条件研究)[J]Acta Batanica Boreali・Occidentalia Sinica
(两北植物学报)怎么建立自己的博客,20O9a5网站,29(6):1104—111O
[15]BABIC V,DATLA RS,SCOLES GJ,etaL Development of an ef
ficient Agrobacterium mediated transformation system for Brassica
formation in tobacco(甘薯IbNPR1基因表达载体的构建及转化烟
草)[J].Chinese Journal of Tropical Crops(热带作物学报)保定网络营销,2O09网路推广,
30(10):1483—1487.
carinata『J].Plant Cell Reports.1998搜狗360,17:183—188
『16]ZHANG FY,LIU ZP,BAO HZ.Genetic study on badey male ste—
[5j wU WJ(吴文俊)郑州sem,JIA XX(贾小霞),ZHANG JW(张金文),et a1.
Construction of plant expression vector containing Chi and Bar
rility[J].Agricultural Science&Technolgy,2OO9义乌seo,10(4):108— o
111.
企业营销网站、SEO优化、行业细分占位策划,营销系统开发等领域,为中小微企业和个人提供以上领域内的服务以及咨询。加微信:qq438569148 马上咨询版权声明:本文内容由互联网用户自发贡献,该文观点及内容相关仅代表作者本人。本站仅提供信息存储空间服务,不拥有所有权,不承担相关法律责任。如发现本站有涉嫌侵权/违法违规的内容请联系QQ:438569148立即清除!