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

In Vitro Screening Method of Tobacco Somatic Clonal Mutants Resistant to Tomato Yellow Leaf Curl Virus

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1. Technical field

The invention relates to an in vitro screening method for somatic clone mutants of tobacco resistant to tomato yellow leaf curl virus, belongs to the field of biotechnology, and is specially used for cell engineering breeding of crops (tobacco) resistant to tomato yellow leaf curl virus.

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2. Background technology

Tomato yellow leaf curl virus disease is caused by the transmission of tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) by Bemisia tabaci, and is a twin virus disease that endangers the production of important economic crops such as tomato and tobacco (Liu Yule, Cai Jianhe, Li Dongling, et al. Tomato yellow leaf curl virus in China—a new species of geminivirus. Chinese Science, Series C, 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). The disease first occurred in Israel in 1964, and then spread rapidly to Europe, Asia, Africa, Oceania and other regions, causing huge losses to tomato production. Since it was introduced into my country in 2002, TYLCVD has spread in Yunnan, Guangdong, Guangxi, Shanghai, Zhejiang, Jiangsu, Henan, Shandong and other places, seriously affecting the production of local crops, and even leading to crop failure (Guo Yanmei, Du Yongchen, Wang Xiaoxuan, Gao Jianchang. Research progress of tomato yellow leaf curl virus (TYLCV), China Agricultural Science and Technology Herald, 2009, 11(5): 30-35).

Since TYLCV is carried and transmitted by whitefly, chemical control is the most direct and effective method. However, with the use of a large number of chemical agents, the resistance of whitefly is getting stronger and stronger. A large number of uses will affect humans, animals and the environment. Therefore, it is of great significance to screen the source of TYLCV resistance and cultivate varieties with high resistance to TYLCV. However, there is a lack of effective resistance genes in cultivated varieties, and only some germplasms are resistant to disease, and the high resistance TYLCV genes are all present in related wild germplasms (Maruthi M.N., 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-a novel source of variability from cell culture for plant improvement. Theoretical and Applied Genetics, 1981, 60:197-214.). The probability of somaclonal variation in some plants is as high as 30%-40%, and the mutation rate of a certain trait is between 0.2%-3% (Feng Xianhong, Li Jian, Luo Xiaogui. Somatic Cells in Plant Tissue Culture Research on Clonal Variation. China Agricultural Science Bulletin, 2010, 26(14):70-73.). At present, somaclonal variation has been widely used in the germplasm innovation of crops such as tobacco, corn, and rice, and has obtained valuable breeding mutants, including disease-resistant mutants (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. Screening blast-resistant mutants of rice blast plasmocytes using virugenic toxins. Acta Genetica Sinica, 1993, ( 4 ): 340- 347; Zhao Xiaoming, Li Mingshan, Song Xiuying. Screening tomato against early blight through tissue culture. Journal of Shanxi Agricultural University, 1996, 16 (4): 350-353). Among them, a large number of disease-resistant mutants are screened using toxins produced by pathogenic bacteria as selection pressure. Under normal circumstances, plant viruses can only maintain their activity in the living host, so viruses or crude virus extracts cannot be directly added to the medium as a selection pressure for in vitro screening of plant anti-virus mutants.

TYLCV invasive cloning is to clone the TYLCV genomic DNA-A tandem repeat sequence into the T-DNA region of the plant expression vector, and integrate the T-DNA into plant cells through Agrobacterium-mediated transcription and expression of the TYLCV gene, which is mainly used for TYLCV infected hosts for transient expression studies (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 TYLCV-resistant tobacco mutants, the present invention transforms tobacco with Agrobacterium containing TYLCV-infectious clones, and integrates the DNA sequence of TYLCV into the tobacco genome through Agrobacterium T-DNA, so that TYLCV can be synthesized in large quantities in isolated plant cells And replication, to generate virus selection pressure, with the help of somatic cell clone variation produced in the tissue culture process, so as to realize the screening of tobacco resistance TYLCV mutants. So far, there has been no report on the use of infectious clones to transform tobacco to screen for virus-resistant mutants.

3. Contents of the invention

technical problem

The invention relates to an in vitro screening method for somatic cell clone mutants of tobacco resistant to tomato yellow leaf curl virus. The defects of cell clone mutants provide a new method for obtaining plant antiviral materials.

Technical solutions

The in vitro screening method of the somatic clone mutants of tobacco resistance to tomato yellow leaf curl virus (TYLCV), comprising:

1) Transform tobacco leaves with Agrobacterium containing tomato yellow leaf curl virus (TYLCV) infectious clone;

2) Transformed tobacco leaves induce somaclonal mutants through long-term tissue culture;

3) Mutant plants and their progeny were inoculated with TYLCV-carrying Bemisia tabaci for continuous screening to obtain TYLCV-resistant tobacco with normal phenotype and no symptoms.

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

1) Cut the sterilized leaves of common tobacco into 1cm ² size, soak them in Agrobacterium with OD ₆₀₀ =0.3-0.4 containing tomato yellow leaf curl virus infective clone PTYj01 for 10 min, and place them in MS+1.0mg/L 6 -BA+0.1mg/L NAA co-culture medium, cultured in the dark at 25°C for 2 days;

2) Leaves were placed on selective medium (MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephalosporin+50mg/L kanamycin), 25°C, 16/8 h light Periodic culture, subculture once every 20 days, continuous subculture for 3-4 months;

3) Cut off the 1-2 cm shoots grown on the callus, and transfer them to the rooting medium (1/2 MS +500mg/L cephalosporin) for rooting culture;

4) After the regenerated seedlings with normal phenotype were identified as positive by PCR with TYLCV-specific primers, they were transplanted into the greenhouse, and each plant was inoculated with 50 Bemisia tabaci carrying TYLCV, and the self-bred seeds of plants that did not show symptoms throughout the growth period were harvested;

5) The selfed seeds were sown in the greenhouse and fed by Bemisia tabaci carrying TYLCV, and the plants that did not show symptoms throughout the growth period were screened.

TYLCV-specific primers are:

5'-CGCCCGTCTCGAAGGTTC-3'

5'-GCCATATACAATAACAAGGC-3';

The PCR identification method is:

Pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min, a total of 30 cycles, and finally extension at 72°C for 10 min, take 10 μL of PCR products on 1% agarose gel Carry out electrophoresis detection, the result shows that the regenerated plant that can amplify the TYLCV DNA specific fragment of 600bp size is transgenic plant.

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Beneficial effect

The present invention has following advantage and positive effect:

1) The present invention overcomes the defect that viruses or virus crude extracts cannot be directly added to the culture medium as a selection pressure to screen somaclonal mutants.

2) The present invention utilizes TYLCV invasive clones to transform tobacco, inserts the DNA of TYLCV into isolated tobacco cells, makes it synthesized and expressed in large quantities in isolated cells, generates virus selection pressure, and then induces the production by long-term tissue culture Tobacco mutants resistant to TYLCV provide reference for creating new plant materials resistant to TYLCV.

3) The TYLCV-resistant plants obtained by the present invention have no disease symptoms throughout the growth period in a greenhouse environment where Bemisia tabaci carry TYLCV and continuously feed on them, and have good resistance.

4) The invention has the advantages of simple operation, no need to prepare toxin, high selection efficiency, and good disease resistance effect of regenerated plants.

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4. Description of drawings

Figure 1 Transformation of tobacco with Agrobacterium containing tomato yellow leaf curl virus infectious clone (kanamycin resistance)

(a) Transformed tobacco leaf explants; (b) callus growth; (c) kanamycin-resistant shoots; (d) rooting culture.

Figure 2 PCR detection of virus-specific sequences in some regenerated plants

M: standard molecular weight Marker DL10000; P: plasmid positive control; WT: wild-type tobacco; 1-13: partially regenerated plants

Figure 3 Identification of regenerated plants (T0) resistant to TYLCV

(a) Wild-type tobacco with shrunken leaves 2 months after inoculation with B. tabaci carrying TYLCV; (b) Regenerated plants with no symptoms 2 months after inoculation with B. tabaci carrying TYLCV; (c) Inoculation with B. tabaci carrying After the TYLCV-carrying whitefly, the regenerated plants showed the disease in the late growth period; (d) After inoculation with the TYLCV-carrying whitefly, the regenerated plants showed disease resistance throughout the growth period.

Figure 4 Tobacco plant (T1) identification of resistance to TYLCV

(a) After inoculation with Bemisia tabaci, the growth and development of wild-type tobacco was blocked, and normal flowering and fruiting were not possible; (b) After inoculation with Bemisia tabaci, the resistant tobacco plants were asymptomatic and normal flowering and fruiting.

5. Specific implementation plan

1) Agrobacterium is EHA105 (Beijing Biovector Company), Agrobacterium containing TYLCV-infectious clone PTYj01 (Wu Dan et al. Research on resistance to tomato yellow leaf curl virus in transgenic tobacco with different promoters. Journal of Jiangsu Agricultural Science, 2010, 26(1):55-60) in liquid LB medium (Boris Martinac, Matthew Buechner, Anne H.Delcour, et al. Pressure-sensitive ion channel in Escherichia coli. PNAS, 1987, April, 84:2297-2301) (Add 25 mg/L rifampicin + 50 mg/L kanamycin) and culture overnight at 28°C and 130rpm.

2) Fresh leaves of common tobacco ( Nicotiana tabacum ) were taken, sterilized with 75% alcohol for 1 min, soaked in 0.1% HgCl ₂ for 5 min, rinsed with sterile water for 5 times, cut into 1 cm ² leaf disks with sterile scissors, and put them at OD ₆₀₀ = 0.3-0.4 Agrobacterium soaked for 10min, put it on sterile filter paper to blot the leaf bacterial solution, then transferred to the co-culture medium (MS+1.0mg/L 6-BA+0.1mg/L NAA) and kept in the dark at 25℃ Cultured for 2 days (Fig. 1a). For the recipe of MS medium, see references (CCDalton, K.lqbal, DATurner. Iron phosphate precipitation in Murashige and media. Physiologia Plantarum, 1983, 4(57):472-476).

3) After 2 days of co-cultivation, the leaves were placed on the selection medium (MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephalosporin+50mg/L kanamycin) at 25°C , 16/8 h photoperiod culture (Figure 1b), subculture once every 20 days, continuous subculture for 3-4 months, and remove deformed seedlings such as curled leaves during subculture.

4) Excise the 1-2 cm shoots from the callus with normal phenotype, and transfer them to the rooting medium (1/2 MS + 500 mg/L cephalosporin) for rooting culture (Figure 1c). During the rooting process, some seedlings with deformed phenotypes also appeared, which were removed, and only plants with normal phenotypes were transplanted (Fig. 1d).

5) Use the CTAB method to extract DNA from leaves of regenerated tobacco plants with normal phenotypes. The specific steps are as follows: take 0.1-0.5 g of fresh leaves, grind them into powder with liquid nitrogen, transfer them to a 1.5 ml centrifuge tube, add 600 μl of preheated CTAB lysate, and vortex Rotate and mix well, bathe in 65°C water for 1 hour, add 600 μl chloroform-isoamyl alcohol (24:1), invert 50 times to mix, centrifuge at 10,000 rpm for 15 minutes, take the supernatant into a new 1.5ml centrifuge tube, add an equal volume of pre-cooled isopropyl alcohol Alcohol, mix well, place at -20°C for 30min, centrifuge at 12000rpm for 15min, discard the supernatant, add 1ml of 75% alcohol to wash the DNA precipitate, discard the alcohol, air dry, add 100μl TE to dissolve the DNA, and store at 4°C for later use.

6) Use virus (TYLCV) specific primers 5'-CGCCCGTCTCGAAGGTTC-3' and 5'-GCCATATACAATAACAAGGC-3' to perform PCR amplification to detect whether the tobacco genomic DNA contains the DNA sequence of TYLCV. The target fragment size is about 600 bp, non-transgenic tobacco For control. PCR amplification program: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min, a total of 30 cycles, and a final extension at 72°C for 10 min, take 10 μL of PCR product in 1% agarose Electrophoresis was performed on the gel, and the results showed that 174 regenerated plants that could amplify a 600bp TYLCV DNA-specific fragment were obtained, while the non-transgenic plants did not amplify the specific band (Figure 2).

7) Transplant 174 T0 plants that were positive in the PCR test into the greenhouse, and each plant was inoculated with 50 Bemisia tabaci carrying TYLCV (Jiao Min, Zhou Xueping, Liu Shusheng. Progress in Research on Geminivirus Transmission by Bemisia tabaci. Acta Entomology, 2006, 49:513-520; Wu Dan, Zhang Baolong, Yang Yuwen, Ni Wanchao, Zhao Tongmin, Zhang Yunhua, Wang Rongfu. Research on the resistance of different promoters to tomato yellow leaf curl virus in transgenic tobacco. Journal of Jiangsu Agricultural Science, 2010, 26(1): 55-60).

20-30 days after inoculation with Bemisia tabaci, wild-type tobacco and 118 regenerated seedlings showed obvious symptoms of TYLCVD, such as curling and thickening of new leaves at the top (Fig. 3a); The leaves were obviously wrinkled and clustered, and the growth and development were hindered (Fig. 3c); 23 plants that were inoculated with the poisonous Bemisia tabaci and did not develop disease throughout the growth period were screened.

23 plants (Fig. 3b, 3d) that were free from disease during the entire growth period after inoculation with the poisonous Bemisia tabaci were selfed, and the inbred species were harvested as a single plant.

8) Harvesting of individual plants Seeds were continued to be sown in the greenhouse to form 23 T2 generation lines, and 50 plants were planted for each line. After eating and spreading TYLCV by Bemisia tabaci carrying TYLCV, 9 strains were screened, and about two-thirds of the plants in the T2 generation population did not develop disease throughout the growth period, which was the mutant strain resistant to TYLCV (Fig. 4).

SEQUENCE LISTING

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<110> Jiangsu Academy of Agricultural Sciences

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<120> In Vitro Screening Method of Tobacco Somaclonal Mutants Resistant to Tomato Yellow Leaf Curl Virus

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<130> Manual

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<140> 00

<141> 2012-09-10

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<160> 2

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<170> PatentIn version 3.1

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<210> 1

<211> 18

<212> DNA

<213> Artificial

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<220>

<221> TYLCV-specific primers

<222> (1)..(18)

<223>

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<400> 1

cgcccgtctc gaaggttc 18

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<210> 2

<211> 20

<212> DNA

<213> Synthetic

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<220>

<221> TYLCV-specific primers

<222> (1)..(20)

<223>

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<400> 2

gccatataca ataacaaggc 20

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Claims (3)

1. The in vitro screening method for somatic clone mutants of tobacco resistance to tomato yellow leaf curl virus, the steps comprising: 1) Transform tobacco leaves with Agrobacterium containing tomato yellow leaf curl virus TYLCV invasive clone; 2) Transformed tobacco leaves induce somaclonal mutants through long-term tissue culture; 3) Mutant plants and their progeny were inoculated with TYLCV-carrying Bemisia tabaci for continuous screening to obtain TYLCV-resistant tobacco with normal phenotype and no symptoms. 2. The method according to claim 1, characterized in that: 1) Take the leaves of common tobacco ( Nicotiana tabacum ), cut them into ^0.5-2cm2 size after sterilizing, soak them in the Agrobacterium containing tomato yellow leaf curl virus infectious clone PTYj01 with OD ₆₀₀ =0.3-0.4 for 10 min, put them in Culture in the dark at 25°C for 2 days on MS+1.0mg/L 6-BA+0.1mg/L NAA co-culture medium; 2) Leaves were placed on MS+1.0mg/L 6-BA+0.1mg/L NAA+500mg/L cephalosporin+50mg/L kanamycin selection medium, cultured at 25°C, 16h/8h photoperiod , subculture once every 20 days, continuous subculture for 3-4 months; 3) Excise the 1-2 cm shoots grown on the callus, and transfer to 1/2 MS + 500mg/L cephalosporin rooting medium for rooting culture; 4) After the regenerated seedlings with normal phenotype were identified as positive by PCR with TYLCV-specific primers, they were transplanted into the greenhouse, and each plant was inoculated with 50 Bemisia tabaci carrying TYLCV, and self-bred seeds were harvested from plants that did not show symptoms throughout the growth period; 5) The selfed seeds were sown in the greenhouse and then fed by Bemisia tabaci carrying TYLCV, and the plants that did not show symptoms throughout the growth period were selected. 3. The method according to claim 2, characterized in that: TYLCV-specific primers are: 5'-CGCCCGTCTCGAAGGTTC-3' 5'-GCCATATACAATAACAAGGC-3'; The PCR identification method is: Pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min, a total of 30 cycles, and finally extension at 72°C for 10 min, take 10 μL of PCR products on 1% agarose gel Carry out electrophoresis detection, the result shows that the regenerated plant that can amplify the TYLCV DNA specific fragment of 600bp size is transgenic plant.

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