CN102864170A - In-vitro screening method for anti-TYLCV (tomato yellow leaf curl virus) somaclonal mutants of tobacco - Google Patents

Abstract

The invention relates to an in-vitro screening method for anti-TYLCV (tomato yellow leaf curl virus) somaclonal mutants of tobacco, belonging to the technical field of biology. The in-vitro screening method comprises the following steps: taking tobacco leaves which contain TYLCV infectious clones and are subjected to Agrobacterium-mediated transformation; massively duplicating TYLCV in in-vitro cells of tobacco to produce a virus screening pressure; performing long-time tissue culture to induce somaclonal mutants; and performing continuous screening by inoculating the mutant plants and later generation plants thereof with TYLCV-carrying tobacco whiteflies to obtain anti-TYLCV tobacco which is normal in phenotype and has no symptoms. The invention effectively overcomes the defect that a virus or a crude extract thereof can not be directly added into a culture medium and used as a selection pressure for antivirus mutant in-vitro screening, and provides a new method for the cultivation of antivirus materials.

Description

The screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus

One, technical field

The present invention relates to the screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus, belong to biological technical field, be exclusively used in the breeding of the anti-tomato yellow leaf curl virus cell engineering of crop (tobacco).

Two, background technology

The tomato yellow leaf curl virus disease is to be carried by Bemisia tabaci to propagate tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) cause, sick (the Liu Yule of a kind of geminivirus infection of the Important Economic crop production such as harm tomato, tobacco, Cai Jian and, Li Dongling, etc. a novel species of tomato yellow leaf curl china virus---geminivirus infection. Chinese science, C collects, 1998,28:148-153; Zhou X P, Xie Y, Zhang Z K. Molecular characterization of a distinct begomovirus infecting tobacco in Yunnan, China. Archives of Virology, 2001,146:1599-1606).This disease occured in Israel early than 1964, was diffused into rapidly afterwards the areas such as Europe, Asia, Africa and Oceania, had caused huge loss to tomato production.Since 2002 import China into, TYLCVD in Yunnan, the ground such as Guangdong, Guangxi, Shanghai, Zhejiang, Jiangsu, Henan, Shandong spreads, have a strong impact on local farming the production of material, even can cause total crop failure (the gorgeous plum of state, Du Yongchen, Wang Xiaoxuan, high Jianchang. the progress of tomato yellow leaf curl virus (TYLCV), Chinese agriculture science and technology Leader, 2009,11(5): 30-35).

Because TYLCV carries propagation by Bemisia tabaci, so chemical prevention is the most direct effective means, but the use along with a large amount of chemical agents, the resistance of Bemisia tabaci is more and more stronger, even drug effect is fine, also can because of a large amount of uses, people and animals and environment be impacted, therefore screen the anti-source of TYLCV, thereby the kind tool of cultivating high resistance TYLCV is of great significance.But lack effective anti-source gene in the Cultivar, only there are some germplasms to show as anti-disease, high resistance TYLCV gene all is present in (Maruthi M.N. in the nearly edge wild germplasm, Czosnek H., Vidavski F., et al. Comparison of resistance to tomato leaf curl virus (India) and tomato yellow leaf curl virus (Israel) among Lycopersicon wild species, breeding lines and hybrids. European Journal of Plant Pathology, 2003,109 (1): 1-11.).

Somaclonal variation refers to obtain regeneration plant by tissue culture procedures, phenomenon (the Larkin P J. Scowcroft W R. Somaclonal variation-a novel source of variability from cell culture for plant improvement. Theoretical and Applied Genetics of regeneration plant performance and parent's different characteristics, 1981,60:197-214.).The probability that somaclonal variation occurs in certain plants is up to 30%-40%, the aberration rate of a certain proterties between 0.2%-3% (Feng Xianhong, Li Jian, Luo Xiaogui. somaclonal variation research in the plant tissue culture. Chinese agronomy circular, 2010,26 (14): 70-73.).At present, somaclonal variation has been widely used in the germplasm innovation of the crops such as tobacco, corn, paddy rice, and obtained valuable breeding mutant, comprise resistant mutant (Carlson P S. Induction and isolation of auxotrophic mutants in somatic cell culture of Nicotiana tabacum. scince, 1970,168:487-489; Chen Qifeng, Chen Zhang, Wang Jinling. use nosotoxin to screen anti-rice blast cell plastid mutant. Acta Genetica Sinica, 1993, (4): 340-347; Zhao Xiaoming, Li Mingshan, Song Xiuying. by the anti-early blight of tissue culture screening tomato. Agricultural University Of Shanxi's journal, 1996,16 (4): 350-353).Wherein, a large amount of resistant mutants is to press screening to obtain with the toxin that pathogenic bacteria produces as selecting.And generally, plant virus only in vivo could keep its activity the host, so the thick extraction of virus or virus can not directly be added in the substratum as the vitro Screening of selecting to compress into capable plant disease-resistant virus mutants.

The TYLCV infectious clone is that TYLCV genomic dna-A tandem repetitive sequence is cloned into plant expression vector T-DNA district, by agriculture bacillus mediated T-DNA is incorporated into the transcript and expression that vegetable cell is realized the TYLCV gene, being mainly used in TYLCV infects the host and carries out transient expression research (Zhang H, Gong H R, Zhou X P. Molecular characterization and pathogenicity of tomato yellow leaf curl virus in China. Virus Ggenes, 2009,39:249-255).In order to obtain anti-TYLCV tobacco mutant, the present invention is to contain the Agrobacterium conversion tobacco of TYLCV infectious clone, be incorporated in the tobacco gene group by the dna sequence dna of agrobatcerium T-DNA with TYLCV, make TYLCV a large amount of synthetic and copy in the Vitro Plant cell, produce viral screening pressure, by the somaclonal variation that produces in the tissue culture procedures, thus the screening of realization Resistance In Tobacco TYLCV mutant.Up to the present, have no the report that utilizes the screening of infectious clone transformation of tobacco to obtain antiviral mutant.

Three, summary of the invention

Technical problem

The present invention relates to the screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus, its purpose is to overcome stripped viral or viral crude extract non-activity and can't directly adds substratum as the defective of selecting to press screening somaclone mutant, provides new method for obtaining the plant virus resistance material.

Technical scheme

The screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus (TYLCV) comprising:

1) to contain the Agrobacterium conversion tobacco blade of tomato yellow leaf curl virus (TYLCV) infectious clone;

2) tobacco leaf after the conversion brings out the somaclone mutant by long tissue culture;

3) mutant plant and offspring plant thereof the inoculation Bemisia tabaci step sizing of carrying TYLCV, obtain phenotype normal, without the anti-TYLCV tobacco of illness.

Method of the present invention, concrete steps are as follows:

1) the common tobacco leaf that will sterilize is cut into 1cm ²Size is with OD ₆₀₀The Agrobacterium that contains tomato yellow leaf curl virus infectious clone PTYj01 of=0.3-0.4 is soaked 10 min, place MS+1.0mg/L 6-BA+0.1mg/L NAA altogether on the substratum, 25 ℃ of dark culturing 2 days;

2) blade places and selects substratum (MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephamycin+50mg/L kantlex) upper, 25 ℃, the cultivation of 16/8 h periodicity of illumination, every 20 days subcultures once, and subculture 3-4 month continuously;

3) the 1-2 cm sproutings that grow on the callus are downcut, change the upper root culture of root media (1/2 MS+500mg/L cephamycin) over to;

4) the normal regrowth of phenotype carries out after PCR is accredited as the positive through the TYLCV special primer, and in the intermediate house, 50 Bemisia tabaci of carrying TYLCV of every strain inoculation are gathered in the crops the plant selfed seed that does not embody illness whole breeding time;

5) selfed seed is seeded in the greenhouse again that the Bemisia tabaci through carrying TYLCV takes food, and screens the plant that does not embody illness whole breeding time.

The TYLCV special primer is:

5'-CGCCCGTCTCGAAGGTTC-3'

5'-GCCATATACAATAACAAGGC-3'；

The PCR authentication method is:

94 ℃ of denaturation 4 min, 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1 min, totally 30 circulations, last 72 ℃ of downward-extension 10 min, getting 10 μ L PCR products is that 1% agarose gel carries out electrophoresis detection in mass percent, the result shows that the regeneration plant of the TYLCV DNA specific fragment that can amplify the 600bp size is transfer-gen plant.

Beneficial effect

The present invention has the following advantages and positively effect:

1)The present invention has overcome virus or viral crude extract can't directly add substratum as the defective of selecting to press screening somaclone mutant.

2)The present invention utilizes TYLCV infectious clone transformation of tobacco, the DNA of TYLCV is inserted in the tobacco cell that exsomatizes, make its a large amount of synthetic expression in isolated cells, producing virus selects to press, and then induce by long tissue culture and to produce anti-TYLCV tobacco mutant, offer reference for formulating anti-TYLCV plant novel material.

3)By the TYLCV resistant plant that the present invention obtains, without disease symptom, have preferably resistance whole breeding time in carrying the greenhouse that the TYLCV Bemisia tabaci takes food continuously.

4)Advantage of the present invention is: simple to operate, need not prepare toxin, and efficiency of selection is high, and the regeneration plant disease resisting effect is good.

Four, description of drawings

Fig. 1 contains the Agrobacterium conversion tobacco process of tomato yellow leaf curl virus infectious clone (kalamycin resistance)

(a) the tobacco leaf explant that transforms; (b) callus growth; (c) kalamycin resistance seedling; (d) root culture.

Fig. 2 partial regeneration plant virus distinguished sequence PCR detects

M: standard molecular weight Marker DL10000; P: plasmid positive control; WT: wild-type tobacco; 1-13: partial regeneration plant

The anti-TYLCV of Fig. 3 regeneration plant (T0) identifies

(a) inoculation 2 months rear blades of Bemisia tabaci of carrying TYLCV are the wild-type tobacco of shrunk; (b) inoculation is carried 2 months rear blades of Bemisia tabaci of TYLCV without the regeneration plant of illness; (c) after the Bemisia tabaci of TYLCV is carried in inoculation, just embody the regeneration plant of illness to later stage breeding time; (d) after the Bemisia tabaci of TYLCV is carried in inoculation, all show disease-resistant regeneration plant whole breeding time.

The anti-TYLCV of Fig. 4 tobacco plant (T1) identifies

(a) after the inoculation Bemisia tabaci, wild-type tobacco grows and is obstructed, and can't normally yield positive results; (b) after the inoculation Bemisia tabaci, the resistance tobacco plant is without illness and normally yield positive results.

Five, specific embodiments

1)Agrobacterium is EHA105(Beijing Biovector company), contain TYLCV infectious clone PTYj01 Agrobacterium (Wu Dan etc. the research of different promoters anti-tomato yellow leaf curl virus in transgene tobacco. Jiangsu agricultural journal, 2010,26 (1): 55-60) at liquid LB substratum (Boris Martinac, Matthew Buechner, Anne H.Delcour, et al. Pressure-sensitive ion channel in Escherichia coli. PNAS, 1987, April, 84:2297-2301) (additional 25 mg/L Rifampins+50 mg/L kantlex), 28 ℃, the 130rpm overnight incubation.

2)Get common tobacco ( Nicotiana tabacum) fresh blade, first by 75% alcohol disinfecting, 1 min, 0.1% HgCl ₂Soak 5 min, aseptic water washing 5 times, be cut into 1cm with sterile scissors ²Large leaflet dish is at OD ₆₀₀The Agrobacterium of=0.3-0.4 is soaked 10min, put blot blade bacterium liquid on the aseptic filter paper after, change that common substratum (MS+1.0mg/L 6-BA+0.1mg/L NAA) is upper, (Fig. 1 was a) in 2 days for 25 ℃ of dark culturing over to.The MS culture medium prescription document (C.C.Dalton that sees reference, K.lqbal, D.A.Turner. Iron phosphate precipitation in Murashige and media. Physiologia Plantarum, 1983,4 (57): 472-476).

3)After cultivating altogether 2 days, blade is placed selection substratum (MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephamycin+50mg/L kantlex) upper, 25 ℃, 16/8 h periodicity of illumination cultivation (Fig. 1 b), every 20 days subcultures once, continuously subculture 3-April, remove the lopsided seedlings such as leaf rolling during subculture.

4)The normal sprouting of 1-2 cm, the phenotype that grow on the callus is downcut, change the upper root culture (Fig. 1 c) of root media (1/2 MS+500mg/L cephamycin) over to.The seedling that also can occur part phenotype deformity in rooting process with its removal, is only transplanted phenotype normal plant (Fig. 1 d).

5)Utilize the CTAB method to extract the normal tobacco regeneration plant of phenotype leaf DNA, concrete steps are as follows: get the fresh blade of 0.1-0.5g, the liquid nitrogen grinding powdered, change 1.5 ml centrifuge tubes over to, add 600 μ l preheating CTAB lysates, vortex mixing, 65 ℃ of water-bath 1h, add 600 μ l chloroform-primary isoamyl alcohol (24:1), put upside down mixing 50 times, the centrifugal 15min of 10000rpm gets supernatant in new 1.5ml centrifuge tube, add isopyknic pre-cold isopropanol, fully mixing is placed 30min, the centrifugal 15min of 12000rpm for-20 ℃, abandon supernatant, add 1ml 75% alcohol washing DNA precipitation, abandon alcohol, air-dry, add 100 μ l TE dissolving DNAs, 4 ℃ store for future use.

6)Carry out pcr amplification with virus (TYLCV) special primer 5'-CGCCCGTCTCGAAGGTTC-3' and 5'-GCCATATACAATAACAAGGC-3' and detect the dna sequence dna whether tobacco gene group DNA contains TYLCV, the purpose clip size is about 600 bp, and the non-transgenic tobacco is contrast.PCR amplification program: 94 ℃ of denaturation 4 min, 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1 min, totally 30 circulations, last 72 ℃ of downward-extension 10 min, get 10 μ L PCR products and carry out electrophoresis detection at 1% agarose gel, the result shows, obtains the regeneration plant that 174 strains can amplify the TYLCV DNA specific fragment of 600bp size, the specific band (Fig. 2) but not transfer-gen plant does not increase.

7)In the 174 strain T0 plantlet of transplant greenhouses with the PCR tests positive, 50 of every strain inoculations carry TYLCV Bemisia tabaci (entangle quick, Zhou Xueping, Liu Shusheng. Bemisia tabaci is propagated the Study on Geminiviruses Infecting progress. insect journal, 2006,49:513-520; Wu Dan, Zhang Baolong, Yang Yuwen, Ni Wanchao, Zhao Tongmin, Zhang Yun China, Wang Rongfu. the research of different promoters anti-tomato yellow leaf curl virus in transgene tobacco. Jiangsu agricultural journal, 2010,26 (1): 55-60).

20-is after 30 days for the inoculation Bemisia tabaci, and obvious TYLCVD disease symptom has appearred in wild-type tobacco and 118 strain regrowths, and (Fig. 3 a) such as top young leaves leaf rolling thickening; The disease plant number increases along with inoculation Bemisia tabaci time lapse, and blade is obvious fold bunch shape, and be obstructed (Fig. 3 c) grows; Screening obtains inoculation with 23 of the plant that does not fall ill in whole breeding time behind the toxic smoke aleyrodid.

With 23 (Fig. 3 b, 3d) selfings of plant that inoculation is not fallen ill in whole breeding time after with the toxic smoke aleyrodid, gather in the crops its selfed seed by individual plant.

8)Individual plant results seed continues to be seeded in the greenhouse, forms 23 T2 for strain, and each strain is planted 50 strains.Bemisia tabaci through carrying TYLCV takes food and propagates TYLCV again, screens 9 strains, and its T2 does not fall ill for have an appointment 2/3rds plant of colony whole breeding time, is the mutating strain series (Fig. 4) of anti-TYLCV.

SEQUENCE LISTING

＜110〉Jiangsu Province Agriculture Science Institute

＜120〉screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus

＜130〉specification sheets

<140> 00

<141> 2012-09-10

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 18

<212> DNA

＜213〉artificial

<220>

＜221〉TYLCV special primer

<222> (1)..(18)

<223>

<400> 1

cgcccgtctc gaaggttc 18

<210> 2

<211> 20

<212> DNA

＜213〉synthetic

<220>

＜221〉TYLCV special primer

<222> (1)..(20)

<223>

<400> 2

gccatataca ataacaaggc 20

Claims (3)

1. the screening method in vitro of the somaclone mutant of Resistance In Tobacco tomato yellow leaf curl virus, its step comprises:

1) to contain the Agrobacterium conversion tobacco blade of tomato yellow leaf curl virus TYLCV infectious clone;

2) tobacco leaf after the conversion brings out the somaclone mutant by long tissue culture;

3) mutant plant and offspring plant thereof the inoculation Bemisia tabaci step sizing of carrying TYLCV, obtain phenotype normal, without the anti-TYLCV tobacco of illness.

2. method according to claim 1 is characterized in that:

1) get common tobacco ( Nicotiana tabacum) blade, after sterilization, be cut into 0.5-2cm ²Size is with OD ₆₀₀The Agrobacterium that contains tomato yellow leaf curl virus infectious clone PTYj01 of=0.3-0.4 is soaked 10 min, place MS+1.0mg/L 6-BA+0.1mg/L NAA altogether on the substratum, 25 ℃ of dark culturing 2 days;

2) blade places that MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephamycin+50mg/L kantlex is selected on the substratum, 25 ℃, 16h/8 h periodicity of illumination are cultivated, every 20 days subcultures once, and subculture 3-4 month continuously;

3) the 1-2 cm sproutings that grow on the callus are downcut, change root culture on 1/2 MS+500mg/L cephamycin root media over to;

4) the normal regrowth of phenotype carries out after PCR is accredited as the positive through the TYLCV special primer, and in the intermediate house, 50 Bemisia tabaci of carrying TYLCV of every strain inoculation do not embody the plant results selfed seed of illness whole breeding time;

5) selfed seed is seeded in the greenhouse again that the Bemisia tabaci through carrying TYLCV takes food, and selects not embody whole breeding time the plant of illness.

3. method according to claim 2 is characterized in that:

The TYLCV special primer is:

5'-CGCCCGTCTCGAAGGTTC-3'

5'-GCCATATACAATAACAAGGC-3'；

The PCR authentication method is:

94 ℃ of denaturation 4 min, 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1 min, totally 30 circulations, last 72 ℃ of downward-extension 10 min, getting 10 μ L PCR products is that 1% agarose gel carries out electrophoresis detection in mass percent, the result shows that the regeneration plant of the TYLCV DNA specific fragment that can amplify the 600bp size is transfer-gen plant.

Images