KR102791470B1 - Chinese cabbage varieties resistant to Turnip mosaic virus by eIF(iso)4E gene editing and Method for breeding of the same - Google Patents

Abstract

The present invention relates to a cabbage plant having increased resistance to turnip mosaic virus by eIF(iso)4E gene correction and a method for producing the same. The expression vector of the present invention and the cabbage plant genetically corrected through the same have TuMV resistance using gene correction technology, and it is expected that the development of cabbage varieties will be promoted by securing such mutants.

Description

{Chinese cabbage varieties resistant to Turnip mosaic virus by eIF(iso)4E gene editing and method for breeding of the same}

The present invention relates to a cabbage plant having increased resistance to turnip mosaic virus by eIF(iso)4E gene correction and a method for producing the same.

In Korea, cabbage production amounted to 634.6 billion won in 2006, making cabbage one of the four most cultivated vegetables after red pepper. Since cabbage is mainly produced intensively, it is very vulnerable to various pests and diseases, especially viruses. Viruses that damage cabbage include Turnip mosaic virus, Cucumber mosaic virus, and Ribgrass mosaic virus, and Turnip mosaic virus, which belongs to the Potyvirus genus, is particularly serious. In Korea, it is one of the main culprits that causes yield loss in cabbage farming in the Gangwon highlands and reduces marketability due to poor quality.

The reality is that there is still no proper control method for most viral diseases, and the best farmers can do is to use clean seeds or parent plants and prevent physical contact or the introduction of insects that the virus uses. However, the most certain way to control viruses is to use resistant varieties.

Plants have dominant resistance, recessive resistance, and non-host resistance as virus resistance, but research on recessive resistance is still insufficient. Recessive resistance is a resistance that utilizes the host factor of the host, and compared to dominant resistance determined by the R gene, it fundamentally blocks the virus infection mechanism, so it has the advantage of being able to develop varieties that are resistant to a longer period of time and a wider variety of viruses.

Many studies have already reported that the infectivity of TuMV (Turnip mosaic virus) in the cabbage family is determined by the interaction between the transcription initiation factors of the plant, eIF4E and eIF(iso)4E, and the characteristic protein of potyvirus, VPg (Virus genome linked protein). Recently, it has been found that specific amino acid mutations in the eIF(iso)4E of the cabbage family can induce the recessive resistance of the virus by destroying the interaction with TuMV VPg. Identifying these specific amino acids that regulate the virus recessive resistance mechanism will enable the selection and breeding of more stable and efficient resistant cabbages in the future.

KR 10-2007-0055889 A

One object of the present invention is to provide a recombinant expression vector comprising at least one nucleotide sequence of SEQ ID NOs: 1 to 3 encoding a gRNA that hybridizes to an eIF(iso)4E gene derived from cabbage; a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to the nucleotide sequence.

Another object of the present invention is to provide a genetically modified cabbage plant resistant to Turnip mosaic virus obtained through selfing from cabbage into which the recombinant expression vector has been introduced.

Another object of the present invention is to provide a method for producing a gene-edited cabbage plant resistant to Turnip mosaic virus, comprising the steps of: introducing an expression vector into the hypocotyl of cabbage; culturing the hypocotyl of cabbage into which the expression vector has been introduced to obtain a cabbage transformed plant ( _{T 0 )} ; and obtaining a gene-edited cabbage plant (T ₁ ), which is a progeny cabbage gene-edited plant obtained through selfing of the cabbage transformed plant (T 0 ), and is resistant to Turnip mosaic virus.

One aspect of the present invention provides a recombinant expression vector comprising: at least one nucleotide sequence of SEQ ID NOs: 1 to 3 encoding a gRNA that hybridizes to an eIF(iso)4E gene derived from cabbage; a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to the nucleotide sequence.

In this specification, a method is provided for producing a genetically modified cabbage plant having resistance to Turnip mosaic virus by mutating the eIF(iso)4E gene derived from cabbage using the recombinant expression vector.

The 'cabbage-derived eIF(iso)4E gene' of the present invention represents an isoform of eIF4E, and eIF(iso)4E exhibits a function similar to eIF4E. It is known that eIF4E and eIF(iso)4E proteins of Arabidopsis thaliana are 44% to 49% identical at the amino acid level.

The term "gRNA (guide RNA)" used in the present invention is RNA specific for target DNA, and the guide RNA can form a complex with Cas9hc:NLS.

Any one of the gRNA sequences of the above sequence numbers 1 to 3 hybridizes to the eIF(iso)4E gene derived from cabbage, and after hybridization, through the action of Cas9hc:NLS, the gRNA induces a mutation in the DNA encoding the eIF(iso)4E gene derived from cabbage.

Sequence name sequences Sequence number gRNA1 CCCCAGGCGGCGGCGCCTTGTTTGG Sequence number 1 gRNA2 CACTCAGGATCTTCCCACTTAGG Sequence number 2 gRNA3 GAACGAAGCCCTTGCGGCGGCGG Sequence number 3

In one specific embodiment of the present invention, the expression vector may include sequence numbers 1 to 3 encoding gRNA. The expression vector includes sequence numbers 1 to 3 encoding gRNA, thereby mutating the eIF(iso)4E gene derived from cabbage, specifically, mutating the eIF(iso)4E gene sequence of sequence number 4 to any one of sequence numbers 5 to 10, thereby increasing resistance to Turnip mosaic virus in cabbage.

The term "Cas9 protein" used in the present invention is an essential protein element in the CRISPR/Cas9 system, which can form a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), and act as an active endonuclease or nickase. The Cas9 protein may be derived from the genus Staphylococcus , the genus Streptococcus , the genus Neisseria , the genus Pasteurella , the genus Francisella , and the genus Campylobacter , but is not limited thereto, and as a specific example, the Cag9 protein may be Cas9hc:NLS.

The term "operably linked" as used in the present invention means a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be at least one selected from the group consisting of a replication origin, a promoter, and a transcription terminator. The transcription terminator sequence may be a polyadenylation sequence (pA), and the replication origin may be, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin.

The term "promoter" as used in the present invention means a region of DNA upstream from a structural gene, and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

A promoter according to one specific example of the present invention is one of the transcription regulatory sequences that regulates the initiation of transcription of a specific gene, and may be a polynucleotide fragment having a length of about 100 bp to about 2500 bp. The promoter may be used without limitation as long as it can regulate the initiation of transcription in a cell, for example, a eukaryotic cell (e.g., a plant cell, or an animal cell (e.g., a mammalian cell such as a human or a mouse, etc.)). For example, the promoter may be a cytomegalovirus promoter (e.g., human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL λ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (RSV) promoter, metallothionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 gene promoter, human It may be selected from the group consisting of, but is not limited to, human lymphotoxin gene promoter and human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter.

The term "expression" as used herein refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression can involve splicing of the mRNA in a eukaryotic cell.

A recombinant expression vector according to one specific embodiment of the present invention may be selected from the group consisting of a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. Vectors that can be used as recombinant expression vectors can be produced based on, but are not limited to, plasmids used in the art (e.g., pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phage (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (e.g., adeno-associated virus (AAV) vectors, etc.).

The term "recombinant expression vector" as used in the present invention means a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid sequences essential for expressing the coding sequence operably linked in a specific host organism. Promoters, enhancers, termination signals, and polyadenylation signals available in eukaryotic cells are known.

The recombinant expression vector of the present invention may further comprise one or more selectable markers. The markers are nucleic acid sequences having properties that can be selected, typically by chemical methods, and include any genes that can distinguish transfected cells from non-transfected cells. For example, the markers may be, but are not limited to, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, antibiotic resistance genes such as ampicillin, kanamycin, G418, Bleomycin, hygromycin or chloramphenicol.

The recombinant expression vector of the present invention can be produced using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

Another aspect of the present invention provides a genetically modified cabbage plant resistant to Turnip mosaic virus obtained through selfing from cabbage into which the recombinant expression vector has been introduced.

The gene-edited cabbage plant resistant to the above Turnip mosaic virus may be a cabbage transgenic plant ( _T0 ) obtained by introducing the above-described recombinant expression vector into cabbage, specifically, the hypocotyl of cabbage, and then a progeny cabbage gene-edited plant ( _T1 ) obtained through selfing.

The method for introducing the above expression vector into the hypocotyl of cabbage can be introduced through a known method, and can be performed by selecting an appropriate standard technique depending on the host cell. Such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, and specifically, the expression vector can be introduced into the hypocotyl through a microorganism, Agrobacterium (strain GV3101).

Through the introduction and expression of the above recombinant expression vector, a mutation in the eIF(iso)4E gene can be induced, thereby increasing resistance to Turnip mosaic virus.

As a specific example of the present invention, the cabbage variety may include a mutation in the eIF(iso)4E gene, and specifically, the eIF(iso)4E gene sequence of SEQ ID NO: 4 may be mutated to any one of SEQ ID NOs: 5 to 9.

Sequence name sequence Sequence number eIF(iso)4E WT gene sequence AGAAGTCTTGGACGGTGTCGAAGGTATAGGCTTTGCGAAGGGAGGCTCCCCAGGCGGCGCCTTGCTTTGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCTCTCTGTTGCCGGTACTTCCGCCGCCGCAAGGGCTTCGTTCACATCCTCTGTCGCCATCGCACTCGATGGGTCAA Sequence number 4 6-19 GGTTGGCATGCGAGGGAGGCTCCCAGGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGCCCGCAAGGGCTTCGTTCACATCCTCTGTCGCCATCGCACTCGATCGGTCAAA Sequence number 5 6-41 GGATGCTGCGAGGAGGCTCCCAGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGCCCGCAAGGGCTTCGTTCACATCCTCTGTCGCCATCGCACTCGATCGGTCAAA Sequence number 6 6-42 GTGGTGCTGCGAGGAGGCTCCCAGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGCCCGCAAGGGCTTCGTTCACTTCCTCTGTCGCCATCGCACTCGATCGTTCAAAA Sequence number 7 6-46 ATGTATGCTGCGAGGGAGGCTCCCAGGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGCCCGCAGGGGCTTCTTTCACTTCCTCGGTCCCCTTCCCATTCAATCGGCCAAAA Sequence number 8 7-44 TTATCCTTACGAGGAGGCTCCCAGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGCCGGGGCGGCTTTCACATCCTCCGTCGCCATCATCCTCCATCAGTCAAAAAAA Sequence number 9 7-49 GGAGCTTGCGAGGGAGGCTCCCAGGGCGGCGCCTTGCTTTGGTTTGGATTGGTTATCGAACCAGAAACTCCACTTTCTTTCGAGCTTGTCAGCAGGCTGCTTCTCCGTCGTCTCTGTTGCCGGTACTTCCGCCGACCGCAAGGGCTTCGTTCACATCCTCTGTCGCCATCGCACTCGATCGGTCAAAA Sequence number 10

The cabbage into which the above recombinant expression vector has been introduced can be any known cabbage, but as a specific example of the present invention, the cabbage can be Seoul cabbage (Dongwon Agricultural Products).

In another aspect of the present invention, a method for producing a gene-edited cabbage plant resistant to Turnip mosaic virus is provided, comprising the steps of: introducing the expression vector into the hypocotyl of cabbage; culturing the hypocotyl of cabbage into which the expression vector has been introduced to obtain a cabbage transformed plant (T ₀ ); and obtaining a gene-edited cabbage plant resistant to Turnip mosaic virus, which is a progeny cabbage transformed plant (T ₁ ) obtained through selfing _of the cabbage transformed plant (T 0 ).

The step of introducing into the hypocotyl of the above-mentioned plant is a step of introducing the above-mentioned expression vector into the hypocotyl of the cabbage, and the method for introduction is as described above.

The hypocotyl refers to the part between the root and cotyledon in a germinating young plant, and when the hypocotyl is cultured under appropriate culture conditions, a sprout can be formed.

The step of obtaining the above-mentioned cabbage transformed plant (T ₀ ) is a step of culturing the hypocotyl of the cabbage into which the expression vector has been introduced, and as a specific method in the present invention, the cabbage transformed plant (T ₀ ) can be obtained by culturing the hypocotyl of the cabbage in the dark together with a strain containing the expression vector and then culturing it in the light.

The step of obtaining a gene-edited cabbage plant resistant to the above Turnip mosaic virus can be accomplished by obtaining a progeny gene-edited cabbage plant (T ₁ ) through selfing of the above cabbage transformed plant (T ₀ ). The selfing method can use a known method.

The expression vector of the present invention and the cabbage variety genetically corrected using the same are expected to enable the development of cabbage varieties by securing such mutants having TuMV resistance using genetic correction technology.

Figure 1 is a diagram showing the transformation process of cabbage.
Figure 2 shows a schematic diagram of the pECO101_eIF(iso)4E_gRNA1,2,3 vector.
Figure 3 is a diagram showing the TumV inoculation method.
Figure 4 is a diagram showing a transformed cabbage plant.
Figure 5 shows the results of PCR analysis for transgene testing of cabbage transgenic plants.
Figure 6 shows the results of confirming mutations within the target locus.
Figure 7 shows the results of the Seoul cabbage 6 T ₁ line PCR assay.
Figure 8 shows the results of the Seoul cabbage 7 T ₁ line PCR assay.
Figure 9 is a diagram showing the results of direct sequencing analysis of Seoul cabbage 6, 7 T ₁ line.
Figure 10 shows the results of ELISA analysis of Seoul cabbage _T1 plants for TuMV resistance.

Below, one or more specific examples are described in more detail by way of examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

Experimental Example 1: Experimental Materials and Methods

Experimental Example 1-1. Experimental Materials

About 3,500 cabbages (Seoul cabbage, Dongwon Agricultural Seed) for transformation were prepared, and the pECO101_eIF(iso)4E_gRNA1,2,3 vector was prepared as the transgene (Fig. 2). In addition, primers for PCR assay were prepared as shown in Table 1. Materials for transformation 1/2 Murashige & Skoog selection and redifferentiation medium, Hygromycine, and Agrobacterium (strain GV3101) for gene introduction were prepared.

Primer name Primer sequence (5'- 3') Size (bp) Transgene
specific primer AAGAAGAGAAGCAGGCCC 600 AAACCCGCCGCAAGGGCTTCGTTC

Experimental Example 1-2. Transformation Process

A medium was prepared with the composition shown in Table 4.

Badge Types MS sucrose BA
(4 mg/ml) NAA
(1 mg/ml) AgNO3
(4 mg/ml) Cefotaxime
(250 mg/ml) Hygromycin
(50 mg/ml) Plant agar pH In-flight sowing (1/2 MS) 2.2 g 20 g - - - - - 5 g 5.8 Co-culture (CM) 4.4 g 20 g 1 ml 1 ml - - - 9 g 5.2 Selection (SⅠ) 4.4 g 20 g 1 ml 1 ml 1 ml 1 ml 80 μL 9.5 g 5.2 RIM 4.4 g 20 g - - - - - 6 g 5.8

For seed disinfection and sowing, seeds were soaked in a 70% ethanol solution for 1 minute and then washed 2-3 times with SDW. Then, they were disinfected in a 25% bleach solution for 20 minutes (shaking), washed 5-6 times with SDW, and left for about 1 hour with SDW. They were sown on a 1/2MS, 2% sucrose, and 0.5% plant agar medium. They were cultured in the dark at 25℃ for 3 days. For the selection of inoculated cutting bodies and pre-culture, the germinated plantlets were transferred to the light culture and exposed to light for 3-4 hours (hardening process). Then, the part of the hypocotyl where chlorophyll is not visible was cut into 0.7-1 cm long, planted horizontally on the pre-culture medium (MS+BA 4.0 mg/L+NAA 1.0 mg/mL), and then cultured in the dark at 23℃ for 2 days. For inoculation and selection of strain culture, strains were cultured (OD: 0.6-0.8) the day before the scheduled inoculation date. On the day of inoculation, the strains obtained by centrifugation at 4000 rpm for 20 minutes were diluted with MS (MS + glucose 36 g / L, pH 5.2) and shaken for 1 hour (23 ° C). Pre-cultured hypocotyls were inoculated with the strain (10 minutes) and then cultured in the dark at 23 ° C for 2 days. After transferring to the selection medium and performing light culture (SI: MS + BA 4.0 mg / L, AgNO3 4.0 m / L, cefotaxime 250 mg / L, hygromacin 4.0 mg / L), the medium was replaced while observing the condition at intervals of 2-3 weeks. When complete sprouts were formed, they were transplanted to the rooting medium (MS + sucrose 20 g / L) (Fig. 1).

Experimental Example 1-3. TuMV inoculation method

Prepare healthy plants to be inoculated, and spray Carborundum on the leaves to be inoculated. Then, inoculate the prepared virus source onto the healthy plants, and wash the leaves after inoculation with water and observe the symptoms (Fig. 3).

Experimental Example 2: Experimental Results

Experimental Example 2-1. Obtaining Transgenic Cabbage Plants

Cabbage transgenic plants were selected using hygromycin 4 mg/ml antibiotic medium (Fig. 4). As a result, 53 transgenic plants were obtained.

Experimental Example 2-2. Confirmation of target gene insertion in cabbage T ₀ transgenic plants

PCR assay was performed on the obtained transgenic plants. Primers containing the gRNA target site were prepared (Table 1) and PCR assay was performed using Takare exTaq (Takara Shuzo Co., Ltd., Shiga, Japan). As a result of PCR assay on cabbage transgenic plants, insertion of the target gene was confirmed in 33 out of 53 plants (Fig. 5).

Experimental Example 2-3. Confirmation of mutation in target locus of cabbage T ₀ transgenic plant

The T ₀ transgenic plants with confirmed gene insertion were subjected to deep sequencing to identify mutations within the target locus. As a result, In/Del within the target locus were identified, and transgenic plants with large deletions were also identified (Fig. 6). Transgenic plants with confirmed mutations within the target locus were used to obtain T ₁ progeny seeds through selfing of the T ₀ transgenic plants. PCR analysis was performed on three of the ten T ₁ lines, Seoul cabbage 6 and 7 T ₁ lines.

Experimental Example 2-4. Confirmation of insertion of target gene in cabbage _T1 gene-edited plant

In the case of the Seoul cabbage 6 T ₁ line, it was confirmed that the eIF (iso) 4E target gene was inserted in 26 out of a total of 46 individuals (Fig. 7). In the case of the Seoul cabbage 7 T ₁ line, it was confirmed that the eIF (iso) 4E target gene was inserted in 6 out of a total of 49 individuals (Fig. 8).

Experimental Example 2-5. Confirmation of mutation in target locus of transgenic cabbage _T1 plants

Direct sequencing analysis was performed on T ₁ plants, excluding those with poor growth status among those in which the target gene insertion was confirmed. As a result, in the case of the Seoul cabbage 6 T ₁ line, it was confirmed that mutations occurred in the target locus in 19 out of 26 plants, and in the case of the Seoul cabbage 7 T ₁ line, it was confirmed that mutations occurred in all 5 out of 5 plants in the target locus. In particular, a large InDel was confirmed in the case of the 7-44 plant (Fig. 9).

Experimental Example 2-6. Virus resistance test of transgenic cabbage _T1 plants

Two weeks after TuMV inoculation, the phenotypes of the transgenic plants of cabbage lines 6 and 7 T ₁ were examined by visual observation. The disease evaluation was classified into four stages as follows (0, immune with no detectable symptom; R, Resistance, infection limited to inoculated leaves but no systemic infection; +, susceptibility, systemic mosaic infection; +N, susceptibility, systemic infection with necrosis). Resistance (0 or R) and susceptibility (N or +N) were compared with the results of ELISA analysis. In addition, a value lower than 1 in the ELISA result was classified as resistance (R), and a value higher than 1 was classified as susceptible (S).

And the TuMV phenotypic and ELISA results for transgenic plant lines 6 and 7 T1 were compared. As a result, plants 6-19, 6-41, 6-42, 6-46, 7-44, and 7-49 showed resistance in both phenotypic and ELISA results (Tables 5 and 6) (Fig. 10) .

Line Phenotype ELISA Resistance (R) Susceptible (S) Resistance (R) Susceptible (S) #6 17 9 4 22 #7 5 0 2 3

Line/plants PhenotypeZ ELISA Line/plants PhenotypeZ ELISA 6-2 R 3.020 7-1 0 3.050 6-3 R 3.030 7-2 R 1.810 6-4 R 3.050 7-37 R 3.020 6-5 R 3.010 7-44 0 0.093 6-7 + 3.080 7-49 0 0.092 6-9 + 3.040 6-11 0 2.750 Pc-1 +N 3.050 6-12 + 3.000 Pc-2 +N 3.030 6-14 R 3.000 Pc-3 +N 3.050 6-19 0 0.108 Pc-4 +N 3.040 6-20 0 3.000 6-24 0 3.000 Nc-1 0 0.089 6-25 + 3.000 Nc-2 0 0.095 6-26 + 3.000 Nc-3 0 0.075 6-32 R 3.000 Nc-4 0 0.080 6-34 0 3.050 6-35 + 2.290 6-36 +N 3.020 6-37 +N 3.040 6-38 + 3.050 6-39 0 1.510 6-41 0 0.220 6-42 0 0.640 6-44 R 3.080 6-45 0 1.250 6-46 R 0.090

^Z 0, Immune with no detectable symptom (no symptoms); R, resistance, infection limited to inoculated leaves but no systemic infection (limited infection at the site of inoculation but no symptoms, hypersensitivity (HR)); +, susceptibility, systemic mosaic infection (mosaic phenomenon); +N, susceptibility, systemic infection with necrosis (necrosis)

* PC: POTY positive control, NC: POTY negative control

Experimental Example 2-7. Confirmation of TuMV-resistant cabbage

Direct sequencing of six isolates that showed resistance to TuMV based on visual phenotypic assay and ELISA results was performed. As a result, large and small InDels were confirmed in all six isolates.

The above results can be summarized as follows:

In this study, we selected an sgRNA that can simultaneously target the eIF(iso)4E gene, an isomer of eukaryotic translation initiation factor 4E (eIF4E), as the target gene, and constructed a plasmid vector using pECO101.

The constructed plasmid vector was transformed into Agrobacterium (GV3101) for plant transformation, and transformation was performed using this.

Approximately 3,500 cabbage seeds were sown, and more than 10,000 hypocotyls were obtained. These were co-cultured with Agrobacterium (GV3101) to perform Agrotransformation. Cabbage transformants for pECO101_eIF(iso)4E_gRNA1,2,3 were obtained through hygromycin selection and redifferentiation.

The presence or absence of transgenic gene insertion was confirmed by performing a PCR assay using a fragment base sequence (primer) of the transgene gene, and deep-sequencing analysis was performed on plants in which gene insertion was confirmed to confirm mutations within the target locus.

Transgenic cabbage plants with mutations in the target locus were passed through generations to obtain _T1 plants.

The _T1 generation was subjected to PCR assay to confirm whether the target gene was inserted, and plants in which the target gene was confirmed to have been inserted were subjected to direct-sequencing analysis to confirm mutations within the target locus.

Additionally, individuals with the target gene inserted through PCR assay were inoculated with TuMV, and visual identification and ELISA analysis were performed.

Finally, it was confirmed that the developed TuMV-resistant cabbage can be used as a breeding material based on gene editing technology.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

<110> REPUBLIC OF KOREA(RURAL DEVELOPMENT ADMINISTRATION) <120> Chinese cabbage varieties resistant to Turnip mosaic virus by eIF(iso)4E gene editing and Method for breeding of the same <130>RDA-BPN210024 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223>gRNA1 <400> 1 ccccaggcgg cggcgccttg tttgg 25 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> gRNA2 <400> 2 cactcaggat cttcccactt agg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223>gRNA3 <400> 3 gaacgaagcc cttgcggcgg cgg 23 <210> 4 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E WT sequence <400> 4 agaagtcttg gacggtgtcg aaggtatagg ctttgcgaag ggaggctccc caggcggcgc 60 cttgctttgt ttggattggt tatcgaacca gaaactccac tttctttcga gcttgtcagc 120 aggctgcttc tccgtctctc tgttgccggt acttccgccg ccgcaagggc ttcgttcaca 180 tcctctgtcg ccatcgcact cgatggtcaa 210 <210> 5 <211> 188 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 6-19 sequence <400> 5 ggttggcatg cgagggaggc tcccaggcgg cgccttgctt tggtttggat tggttatcga 60 accagaaact ccactttctt tcgagcttgt cagcaggctg cttctccgtc gtctctgttg 120 ccggtacttc cgccgcccgc aagggcttcg ttcacatcct ctgtcgccat cgcactcgat 180 cggtcaaa 188 <210> 6 <211> 185 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 6-41 sequence <400> 6 ggatgctgcg aggaggctcc caggcggcgc cttgctttgg tttggattgg ttatcgaacc 60 agaaactcca ctttctttcg agcttgtcag caggctgctt ctccgtcgtc tctgttgccg 120 gtacttccgc cgcccgcaag ggcttcgttc acatcctctg tcgccatcgc actcgatcgg 180 tcaaa 185 <210> 7 <211> 187 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 6-42 sequence <400> 7 gtggtgctgc gaggaggctc ccaggcggcg ccttgctttg gtttggattg gttatcgaac 60 cagaaactcc actttctttc gagcttgtca gcaggctgct tctccgtcgt ctctgttgcc 120 ggtacttccg ccgccccgcaa gggcttcgtt cacttcctct gtcgccatcg cactcgatcg 180 ttcaaaa 187 <210> 8 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 6-46 sequence <400> 8 atgtatgctg cgagggaggc tcccaggcgg cgccttgctt tggtttggat tggttatcga 60 accagaaact ccactttctt tcgagcttgt cagcaggctg cttctccgtc gtctctgttg 120 ccggtacttc cgccgcccgc aggggcttct ttcacttcct cggtcccctt cccattcaat 180 cggccaaaa 189 <210> 9 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 7-44 sequence <400> 9 ttatccttac gaggaggctc ccaggcggcg ccttgctttg gtttggattg gttatcgaac 60 cagaaactcc actttctttc gagcttgtca gcaggctgct tctccgtcgt ctctgttgcc 120 ggtacttccg ccgccggggc ggctttcaca tcctccgtcg ccatcatcct ccatcagtca 180 aaaaaa 186 <210> 10 <211> 187 <212> DNA <213> Artificial Sequence <220> <223> eIF(iso)4E 7-49 sequence <400> 10 ggagcttgcg agggaggctc ccaggcggcg ccttgctttg gtttggattg gttatcgaac 60 cagaaactcc actttctttc gagcttgtca gcaggctgct tctccgtcgt ctctgttgcc 120 ggtacttccg ccgaccgcaa gggcttcgtt cacatcctct gtcgccatcg cactcgatcg 180 gtcaaaa 187

Claims (6)

delete delete A genetically modified cabbage plant resistant to Turnip mosaic virus obtained through selfing from a cabbage into which a recombinant expression vector has been introduced,
The above recombinant expression vector comprises at least one nucleotide sequence selected from SEQ ID NOS: 1 to 3 encoding a gRNA that hybridizes to the eIF(iso)4E gene derived from cabbage;
A nucleotide sequence encoding the Cas9 protein; and
A recombinant expression vector comprising a promoter operably linked to the above nucleotide sequence,
The above genetically corrected cabbage plant is a genetically corrected cabbage plant in which the sequence of the eIF(iso)4E gene of sequence number 4 is mutated to any one of sequence numbers 6 to 9.
delete In the third paragraph,
The cabbage into which the above recombinant expression vector has been introduced is a genetically modified cabbage plant called Seoul cabbage.
delete