KR100358755B1 - Transgenic tomato resistant to virus and process for preparation thereof - Google Patents

Abstract

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a virus resistant tomato and a method of manufacturing the same, and specifically, to agro-logo-inducing tomato and CP gene transformed with a coat protein (CP) gene of tobacco mosaic virus (TMV). The present invention relates to a method for producing a virus-resistant tomato consisting of a process of co-culture of bacterium and tomato tissue, induction of callus, plant regeneration, purifying and pot transplantation.

The virus resistant tomato of the present invention can be usefully used as a parent tomato because the introduced gene has been stably maintained up to six generations.

In addition, the production method according to the present invention can greatly contribute to the development of useful plants in the future by drastically shortening the development period of a variety that takes more than 10-15 years using a transformation method as a conventional breeding method.

Description

Virus resistant tomato and process for preparing the same

The present invention relates to virus resistant tomatoes and methods for their preparation.

More specifically, the present invention relates to a virus-resistant tomato transformed with a coat protein (CP) gene of tobacco mosaic virus (Tobacco mosaicvirus, TMV) and a method for producing the same, wherein the virus-resistant tomato is up to 6 generations. As the transgene is kept stable due to the development, it can be usefully used as a parent tomato.

Transformation studies on virus resistance have been reported since Abel et al. (Abel, Science 232: 738-742pp, 1986) first reported the development of tobacco incorporating the coat protein gene of tobacco mosaic virus (TMV). It has been strongly suggested that various viral resistant plants can be developed. Accordingly, potato virus X (PVX) resistant potato (Cuozzo et al., Embo J., 75: 1273-1280pp, 1998), potato virus Y (potato virus Y, PVY) resistant potato (Lawson et al., Biotechnology, 8 : 127-134pp, 1990), alfalfa mosaic virus (AIMV) resistant tobacco and tomato (Tumer et al., Life Science 140: 351-356pp, 1987), cucumber mosaic virus (CMV) resistant tobacco ( Various transgenic plants have been produced and reported, including Harrison et al., Nature 328: 799-802pp, 1987).

In Korea, Lee et al. (Mol. Cells, 4: 67-71, 1994) transformed tomato mosaic RNA of cucumber mosaic virus (CMV) into tomatoes to obtain resistant individuals. Cho et al., Mol. Cells, 2: 329-334, 1992) also reported obtaining tomato individuals resistant to larvae (coleopteran larvae).

Tomato mosaic virus produced in our country is known as cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), tomato mosaic virus (Tomato Mosaic) (ToMV). It is also infected with seeds, the symptom of which is a mosaic symptom on the leaves, and when further developed, the leaves become malformed or thinner. It has also been reported that necrosis of shoots and necrotic spots appear on stems and fruits.

On the other hand, in the case of tomatoes, a number of tobacco mosaic virus resistant varieties have been developed by conventional breeding methods, but since the breeding period is long, the development of varieties is not easy. There is no situation.

In general, coat proteins have been reported to inhibit or tolerate the invasion of the virus by interference when the gene is introduced into the transgenic plant. In addition, the envelope protein gene is not only resistant to the target gene transformed, but is known to be resistant to several strains at the same time.

Accordingly, the present inventors have prepared a carrier containing the envelope protein gene of tobacco mosaic virus (TMV) and transformed Agrobacterium ( A.tumefaciens ) in order to develop a virus resistant tomato by the transformation method using the envelope protein. The virus-resistant tomato was prepared by co-culturing and co-cultured tomato tissues, and then the outer protein was stably expressed, thereby confirming that the virus resistance was completed.

It is an object of the present invention to provide a transformed tomato having a viral resistance and a method for producing the same.

1 is a schematic diagram showing a process of preparing a virus resistant tomato using the Agrobacterium vector containing the envelope protein (TMV-T CP) gene of tobacco mosaic virus,

FIG. 2 sequentially shows the regeneration process of transformed tomatoes from co-culture with Agrobacterium tumefaciens to transplantation into a pot.

A: co-culture with Agrobacterium;

B: induction of callus and shoot in selective medium;

C: transplanted in medium;

D: expression of GUS gene in roots;

E: purifying regenerative plants;

F: Port Transplantation of Transformed Plant

Figure 3 shows the chromosomal DNA from the transformed tomato showing the TMV CP gene insertion in Southern blot,

M: size marker (lambda DNA digested with restriction enzyme Hind III / Eco RI);

Ag: Agrobacterium; 1: control group;

2, 3: T1 transformed plant; 4, 7: T4 transformed plants;

5, 6, 8: untransformed plants;

Figure 4 shows the Northern blot that the transcription of the inserted TMV CP gene by separating the RNA from the transformed tomato,

A-a, B-a: coat protein genes and PCR products;

A-b, B-g, h: T1 (B-g) and T5 (A-b, B-h);

5 is a graph showing the inoculation of the purified virus to each line of the transformed plant and confirmed virus resistance by Eliza method,

Figure 6 shows the degree of viral resistance of the transformed tomato compared to the control,

Figure 7 schematically shows the process of selecting a virus resistant tomato individual of the present invention.

In order to achieve the above object, the present invention provides a virus resistant tomato transformed with the coat protein gene of Tobacco mosaic virus (TMV).

The virus resistant tomato of the present invention has advanced five generations or more, and includes virus resistant tomato strains T16-11-5-3 and T44-2-6-14-28.

In addition, the present invention is a virus consisting of a process of co-culturing the Agrobacterium and tomato tissues introduced with the foreign genes to induce callus and shoot, re-differentiate the plant from the purified and transplanted to the port Provided are methods for preparing resistant tomatoes.

In the above process, it is preferable to use a hypocotyl having a seedling of 6-11 days and a size of 0.4-0.6 cm as tomato tissue. In addition, it is preferable to pre-culture before the process of redifferentiation, and it is preferable that the co-culture time is 4-6 minutes. In addition, it is desirable to allow 6-8 days in advance when transplanted into the pot.

Hereinafter, the present invention will be described in detail.

The present invention provides a virus resistant tomato prepared by the method of transformation.

Specifically, the present invention provides a virus resistant tomato transformed using the envelope protein gene of Tobacco mosaic virus (TMV), which has the characteristics that the resistance trait is stabilized as generation progresses.

The viral resistant tomato includes viral resistant tomatoes T16-11-5-3, T44-2-6-14-28, and the like, which have advanced five generations or more.

The present invention also provides a method for producing a virus resistant tomato by the transformation process.

The production method of the present invention is a process of co-culture of Agrobacterium and tomato tissues into which foreign genes are introduced to induce callus and shoot, and then re-differentiate and then purify plants and transplant them into pots. It is composed.

The present invention improves the transformation efficiency of tomatoes by examining various factors affecting tomato regeneration in the above process. Such factors include the presence or absence of preculture, coexisting incubation time of tomato tissue and Agrobacterium, and composition of an appropriate regeneration medium.

In order to obtain Agrobacterium into which a foreign gene is introduced, the present invention first isolates tobacco mosaic virus from tomatoes and synthesizes cDNA from it to prepare a vector having the full-length coat protein (CP) gene inserted therein. Next, E. coli is transformed with the vector, and the transformant is mixed with Agrobacterium to select a transformed Agrobacterium clone using a medium containing antibiotics (see FIG. 1).

Next, Agrobacterium into which the coat protein gene is introduced is co-cultured with tomato tissues to induce callus and the like.

At this time, the tomato tissue is preferably 6 to 11 days old and the axis of the axis is 0.4-0.6 cm, it is more preferable to use 5 to 10 days of seedlings and 0.5 cm in size. Moreover, it is preferable that the coexistence incubation time is 4-6 minutes, and it is more preferable that it is 5 minutes.

Next, the plants are regenerated using the callus and the like, and the regenerated plants are transplanted into pots. At this time, it is preferable to carry out preculture before regeneration process, and it is preferable to purify about 6-8 days before transplanting into a pot.

In addition, the present invention, in order to obtain a tomato having a stable virus resistance, by culturing the transformed tomato continuously to advance the generation of the transformed tomato and selects individuals having a stable virus resistance therefrom (see Fig. 7).

The present invention identifies the individual to which the TMV CP gene is inserted from the transgenic plant by presence and expression of GUS (β-glucronidase gene) and NPTII gene and viral resistance. Specifically, the subsequent inheritance of the target gene is confirmed by Southern blot and the like, and the stable expression of the gene is examined by the northern blot.

In addition, the present invention induces the resistant trait is fixed by the development of more than five generations of individuals resistant to virus among the individual generation of each generation. In the above process, the present invention confirmed that more than 90% of the transformed tomatoes exhibited resistant traits when they were advanced to six generations.

In addition, the present invention examines whether the horticultural trait of the virus resistant tomato can be used for parental copy compared with the brightening tomato.

Specifically, the growth type, cotyledon, leaf color, leaf shape, leaf complexion degree, leaf length, leaf width, cultivation, herbaceous, first-flowered fruiting arthroplasty, flower water, fruiting fruit, fruiting, fruiting, fruiting, Check for hypertype, color, and sugar.

In addition, the present invention is inoculated to the transformed tomato at various concentrations to determine the degree of virus resistance (see Figure 6).

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only examples of the present invention, and the scope of the present invention is not limited by the examples.

Example 1 Cloning of Envelope Protein Gene of Tobacco Mosaic Virus

RNA was isolated and purified from tobacco mosaic virus (TMV-T) isolated from tomatoes, and cDNA was synthesized therefrom to select a vector containing the full-length TMV-T envelope protein (full-length TMV-T CP) gene. . In this case, the vector pBI 121 (Promega) having a β-glucuronidase (GUS) gene and a kanamycin resistance gene between the CaMV 35S promoter and the NOS terminator was used. Used. Transfected with the vector so produced by culturing in a medium a mixture of transformants having pRK2013 / HB101 E. coli strain and the Agrobacterium strain LBA 4404 in E. coli HB101 strain containing kanamycin, rifampicin (rifampicin) and streptomycin (streptomycin) conversion Clones were selected (see FIG. 1).

Example 2 Coexistence Culture of Agrobacterium and Tomato Tissue Inserted with Coat Protein Gene and Inorganic Transformant

To re-differentiate the tomato dentate tissue, it was recommended to use a hypocotyl with a size of about 7-10 days and 0.5 cm in size. Before transferring to the selection medium, the dentate tissue was re-differentiated (MS salt, 3% sucrose, 0.8% agar, 1). 1 day preculture with x B5 vitamin, 0.02 mg / l IAA, 3 mg / l zeatin) enhanced the vitality and regeneration rate of the explants. The best coexistence time of tomato tissue and Agrobacterium ( A. tumefaciens) was 5 minutes (see FIG. Selection medium (primary, secondary) was 100 mg / in basal medium (MS salt, 3% sucrose, 0.8% agar, 1 × B5 vitamin, 0.02 mg / l IAA, 3 mg / l zeatine) Addition of 1 kanamycin and 500 mg / l carbenicillin was preferred, and the addition of the same amount of antibiotics to rooting medium greatly enhanced callus organic and regeneration rates (see FIG. 2C). When transferring from the cabin to the pot, it was effective to purify in the cabin for about a week and transfer to the greenhouse (see FIG.

Example 3 Subsequent Selection of Transformants

To confirm the insertion of the NPTII gene, a pair of primers NPTII-F (5'-CTG AAT GAA GTG CAG GAC GAG GAG G-3 ') and NPT- corresponding to the 5' and 3 'terminal sequences of the NPTII gene PCR primers were prepared to amplify a band of about 500 bp using Ⅱ-R (5'-ACC AAC GCT ATG TCC TGA TAG C-3 '), and to confirm the insertion of the envelope protein (CP) gene. A pair of primers corresponding to the 5 'and 3' terminal sequences of the protein gene (480 bp) were prepared (TMV-T: 5'-ATG TCT TAC TCA ATC ACT TCT CC-3 ', TMV-R: 5' -CAG GTG CAG AGG TCC AGA CC-3 ') A primer was prepared to amplify a band of about 480 bp in size. The PCR reaction was repeated at different annealing temperatures of 48 ° C., 52 ° C. and 56 ° C., resulting in PCR products of approximately 480 bp in size equal to the desired size from genomic DNA isolated from the transformants under all conditions. It could be confirmed.

In addition, it was analyzed by Southern blot to investigate the stable expression and isolation of the target gene in the future. Genomic DNA was extracted after inoculating the leaves of about 8 to 9 cm of the transformants and storing them at −70 ° C. The transgenic tomato was used as a probe (TM 2, FIG. 3). The reaction was confirmed only in 3, 4, and 7). In particular, it was confirmed that both transgenic tomatoes were inserted with two pairs of TMV CP genes, and the transgenic tomatoes obtained in the present invention were confirmed to stably inherit TMV CP genes to later T4 plants.

Gene expression was analyzed by Northern blot for lines identified by the Southern blot analysis. At this time, total RNA (total RNA) was isolated from the leaves stored, and the TMV CP gene was used as a probe. As shown in FIG. 4, the CP gene was expressed as a hybridization band in the transformed tomato (lanes A-b, B-g, and B-h), and it was confirmed that the CP gene was stably expressed in tissues (see FIG. 4).

As a result of the subsequent test, the selection rate was 24.4% for T0 plants, 57.9% for T1 plants, 19.3% for T2 plants, 12.9% for T3 plants, and 44% for T4 plants. Four strains (T16-11-5-3, T18-8-2-2-7, T 44-2-6-13-27 and T44-2-6-14-28) were selected from the following T4 plants. 2 lines (T16-11-5-3 and T44-2-6-14-28) at T5 were selected by Northern blot analysis (see FIG. 7). On the other hand, the seeds harvested from the two selected strains were partially stored in the seed storage (temperature 1-2 ℃ humidity 40%), and some were grown for 10 weeks per strain in the pot state for seed growth.

Example 4 Investigation of horticultural characteristics of transformed tomato

In order to investigate the horticultural characteristics of transgenic tomato, plant growth type, cotyledon color, leaf color, leaf shape, grafting degree of leaf, leaf length, leaf width, cultivation, grass length, first flower condensation, flower plant, fruit tree, fruit tree, Hypercapacity, overweight, hyperplasia, hypercolor, and sugar were confirmed. As a result, there was no significant variation according to generation progress, and abnormal stems or albinos occurred in some lines derived from T16, T36, and T44 plants, but they were inherited only to later generation T4 plants. In the early generation, individuals with lobe and hypermorphic variability appeared, but it was thought to be due to somaclonal variation rather than transformation. In addition, there was no difference between the transformant and the non-convertant in the analysis of the inorganic component of the fruit.

Example 5 Investigation of Virus Resistance of Transformed Tomato

To determine the degree of virus resistance, the purified purified virus was inoculated with TMV at concentrations of 1 mg / ml, 100 µg / ml, 10 µg / ml and 1 µg / ml to 3 strains of T5 plants, respectively. The increase and decrease of virus after days and 15 days was examined. As a result, the transformed tomato exhibiting resistance showed greater resistance compared to fluorescence after 10 days of inoculation (see FIG. 5). In addition, among the same transgenic lines, the resistance tendency showed the same type as the control group.

In addition, as shown in Figure 6, the transformed tomatoes were individuals exhibiting resistance to TMV-T, but in addition to TMV-T, which TMV strain (strain) was examined. As a result, it was assumed that the TMV-OM strain is also resistant. However, in recent years, globally regulated stability of GMOs (transformers) must be followed by packaging assays and stability assessments to find a response.

As described above, the virus resistant tomato of the present invention has been advanced for more than five generations, so that the introduction gene is kept stable, so that it can be usefully used as a parent tomato, and its resistance to various tobacco mosaic virus strains is further utilized. It is expected. In addition, the production method of the present invention can be widely used to develop useful plants in the future by drastically shortening the development period of a variety that takes more than 10-15 years as a conventional breeding method using a transformation method.

Viral resistant tomato and a method for producing the same according to the present invention have been described above with reference to the drawings and examples, but this is only for describing the most preferred embodiments of the present invention, and the present invention is not limited thereto. In addition, anyone of ordinary skill in the art will be able to make various modifications and imitations by the description of the present specification, but it will be apparent that this is also outside the scope of the present invention.

Claims (6)

(a) separating TMV-T envelope protein (TMV-T CP) RNA from tobacco mosaic virus (TMV-T) isolated from tomatoes and synthesizing cDNA therefrom; (b) preparing a recombinant expression vector containing the envelope protein encoding gene cDNA; (c) introducing the recombinant expression vector into Agrobacterium tumefaciens and co-cultured with tomato tissues to select a transformed clone in a selection medium; And (d) inducing callus and chute from the selected transgenic clones and regenerating plants from the selected transgenic clones. delete According to claim 1, wherein the tomato tissue has a seedling of about 7 to 10 days and a size of 0.4 ~ 0.6cm embryonic axis, re-differentiated medium (MS salt, 3% sucrose, 0.8% agar, 1 before transfer to selection medium) XB5 vitamin, 0.02 mg / L IAA, 3 mg / L zeatine) for 1 day, co-culture with Agrobacterium tumefaciens for 4-6 minutes, and 6-8 days when transplanted into pot Characterized in that the method. A virus resistant tomato prepared by the method of claim 1. 5. The virus resistant tomato according to claim 4, wherein the virus resistant tomato is a strain that has been advanced over 5 generations. delete