农杆菌侵染植物的原理 烟草农杆菌转化

昭棠笔记 2023-01-26

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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. 

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