KR20110026545A - Cabbage resistant to diamondback moth transformed with cryiac gene and production method thereof - Google Patents

Abstract

PURPOSE: A Brassica oleracea var. Capitata with Plutella xylostella resistance transformed by CryIAc gene is provided to save labor and agricultural chemical. CONSTITUTION: A Brassica oleracea var. Capitata with Plutella xylostella resistance is obtained by transforming CryIAc gene having a base sequence of sequence number 1 with a recombinant plant vector. The recombinant vector is pNW2300/CryIAc vector.

Description

Cabbage resistant to diamondback moth transformed with CryIAc gene and production method

The present invention relates to cabbage moth-resistant cabbage transformed with the CryIAc gene and a method for preparing the same, more specifically, a cabbage plant transformed with a recombinant plant expression vector comprising the CryIAc gene and resistant to cabbage moth; The present invention relates to a method for producing a cabbage plant having a resistance to cabbage moths comprising the step of transforming cabbage plant cells with a recombinant plant expression vector comprising the CryIAc gene to overexpress the CryIAc gene.

This problem is overcome because long-term use and abuse of chemical synthetic pesticides, which have contributed greatly to the productivity of crops, not only causes problems such as environmental pollution and ecosystem destruction, but also causes pathogens and insects to resist pesticides. In order to do so, the development of pollution-free or low pollution pesticides is urgently required.

Bacillus Picchu ringi N-Sys (Bacillus thuringiensis ) Bacteria produce specific toxins that kill certain insects and have long been used as biopesticides, which are much safer than common pesticides. This toxin is from B. thuringiensis It is expressed from the Bt gene (creating the CryI type protein as the CryI type gene) and is particularly activated in the midgut of Lepidoptera insects. The toxin binds to receptors in the midgut's epithelial tissue, penetrates into the tissue, dissolves the tissue, and eventually kills the insect. Most lepidoptera insects have receptors that bind CryIAa, CryIAb, and CryIAc toxins.

Chinese cabbage moth ( Plutella) xylostella ) is a representative lepidoptera insect that is severely damaging cruciferous crops, and by eating the leaves of the larvae, it not only reduces the commercial value of the crop but also hinders the growth of the crop.

Korean Patent Registration No. 0475674 discloses a method for reproducing using the cabbage of the cabbage and a method for producing a cabbage transformed with a useful foreign gene.

The present invention has been made in accordance with the above requirements, the present invention is transformed by inserting the toxin expressed from the Bt gene into the cabbage of the world's largest market value of cruciferous crops transformed to grow while being protected from the cabbage moth We want to build a system that can

In order to solve the above problems, the present invention is transformed with a recombinant plant expression vector containing the CryIAc gene to provide a cabbage plant having resistance to cabbage moth.

The present invention is by transforming the cabbage plant cell with a recombinant plant expression vector containing the CryIAc gene provides a method of manufacturing a cabbage plant having resistance to the cabbage moth, comprising over-expressing the CryIAc gene.

According to the present invention, by introducing the CryIAc gene into a cabbage with a high marketing value, the development of cabbage resistant to cabbage moth, which is a major pest of cabbage and crops, by using pesticides and labor reduction effect in the control of Chinese cabbage moth Can be obtained.

In order to achieve the object of the present invention, the present invention is transformed with a recombinant plant expression vector comprising the Cry1Ac gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 to provide a cabbage plant having a resistance to cabbage moth.

Bt's native endotoxin protein gene CryIAc encodes 1,178 amino acids at 3,537 bp, and about 1,653 bp of the C-terminal region is involved in the crystallization of endotoxin protein and is not related to toxicity. N-terminal 1,854bp of the site was modified and synthesized. On the other hand, when the gene was modified, the codon was changed to change the codon of the insecticidal-related site of the CryIAc gene similarly to the cabbage crop, but not to change the amino acid sequence (SEQ ID NO: 1).

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the CryIAc gene has a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The recombinant plant expression vector may be the pNW2300 / CryIAc vector described in FIG. 1, but is not limited thereto.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the fetuin DNA according to the invention into a plant host are viruses such as those that can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses and the like. Vector, for example an incomplete plant virus vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes include, but are not limited to.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminators of the octopine gene of bacterium tumefaciens, but are not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides seeds of the transformed cabbage plant.

The present invention also relates to a cabbage plant having a resistance to cabbage moths comprising the step of transforming cabbage plant cells with a recombinant plant expression vector comprising a Cry1Ac gene consisting of the nucleotide sequence represented by SEQ ID NO: 1, overexpressing the Cry1Ac gene. It provides a method for producing.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by infiltration of plants or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell. Preferably, the plant is cabbage.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: CryIAc  Preparation of Plant Expression Vectors That Express Genes

A plant expression vector pNW2300 / Cry1Ac (FIG. 1) for transforming cabbage was prepared.

After removing the hygromycin resistance gene from the pCAMBIA1390 vector using hygromycin as the selection marker, the CryIAc gene sequence described in SEQ ID NO: 1 was prepared by forward binding to the CaMV 35S promoter. At this time, the CryIAc gene was inserted into the blunt end at the Kpn I and Nco I restriction enzyme sites. The restriction enzyme sites were combined with the blunt ends of Sma I and Kpn I and Nco I to introduce these into pNW2300 for selection using kanamycin. PCR was performed with the prepared primers to amplify the CryIAc gene to ensure normal cloning. At this time, PCR was denatured at 94 ° C. for 5 minutes as a precycling reaction, and then repeated 35 times in the order of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C., and then The PCR reaction was terminated by reacting at 72 ° C. for 5 minutes.

Plasmid pNW2300 / Cry1Ac of each vector prepared as described above was used as Agrobacterium tumefaciens EHA105 ( Agrobacterium tumefaciens EHA105) and used for the transformation of plants.

Example  2: Agro Bacteria  Used CryIAc  Cabbage transformation of gene

Cabbage cabbage inbred line (ad-bentam) ( Brassica oleracea var . capitata ) axle was used. Cleaved excreta were precultured for 2 days in MS medium containing 2 mg / L BA and 1 mg / L NAA.

The precultured embryos were placed in a conical tube containing 20 ml of MS liquid medium, 2 ml of Agrobacterium solution was added and shaken at 200 rpm for 20 minutes to allow bacteria to grow in the embryo. Application was made. The MS liquid medium was then discarded and excess MS solution was removed with sterile filter paper. Acobacterium-infected cabbage embryos are approximately 50-100 in MS cocultivation medium (MS basal medium, 2 mg / L BA, 1 mg / L NAA, 100 μM acetosilling source) coated with sterile 9 cm filter paper. The dogs were placed and incubated under dark conditions for 2 days.

Cultured cocultured embryos were cultured in a selection medium (2 mg / L AgNO ₃ , 200 mg / L Lilacillin, 15 mg / L Kanamycin-containing preculture medium) at 25 ° C. under light conditions in order to select cells with gene conversion. Subsequently, every four weeks, subcultures form callus. Once the leaves are formed from callus, a well-grown leaf (regrowth of more than 1 cm) is selected from one callus, which is then renal medium (MS + 3% sucrose + 2 mg / L BA + 0.1 mg / L NAA + Elongate in 2 mg / L AgNO ₃ and 200 mg / L Lilacillin + 15 mg / L Kanamycin) and organically root in root organic medium (1/2 MS basal medium, 200 mg / L Lilacillin and 15 mg / L Kanamycin) I was. Green plants with roots were planted in a mixture of vermiculite, perlite, and peat moss (2: 1: 1), purified for one week, and then transferred to soil pots and grown in greenhouses.

Transgenic cabbages transformed with the synthetic CryIAc gene were cold treated for 30-40 days in a low temperature room at 4 ° C., and then transferred to a greenhouse to induce pollen, self-pollinated, and seed seed was obtained.

2 is a step-by-step diagram of the development of the transformed cabbage plant. As can be seen in FIG. 2, the transformed cabbage plants were shown to grow into normal plants.

As a result of analyzing the transformation rate of the transformed cabbage plants, the shoots showing PCR positive reaction showed 1.5% (Table 1).

Table 1. Cabbage transformation efficiency

Number of explants Number of shoots (%) PCR positive shoots (%) 3,500 1,130 (32.3) 52 (1.5)

Example  3: transgenic cabbage PCR  analysis

In order to confirm whether the CryIAc gene was introduced into the plant after transformation, a polymerase chain reaction (PCR) was performed. Specifically, after chromosomal DNA was isolated from the leaf sections of the selected news, forward primer 5'-ATG ACG CAC AAT CCC ACT AT-3 '(SEQ ID NO: 2: 35S promoter site) and reverse primer 5'-TGT GGC TCT CTT CCC GAA CT-3 '(SEQ ID NO. 3: CryIAc site) was used for the PCR reaction. At this time, PCR was denatured at 94 ° C. for 5 minutes as a precycling reaction, and then repeated 35 times in the order of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C., and then The PCR reaction was terminated by reacting at 72 ° C. for 5 minutes. Figure 3 is a diagram showing the results of PCR analysis of the transformed cabbage plant. As can be seen in Figure 3, the transgenic cabbage plant was able to observe a 0.8kb PCR product.

Example  4: Southern of transformed cabbage plants Blot  analysis

Southern blot analysis was performed to confirm whether the CryIAc gene was introduced into the plant after transformation. Specifically, chromosomal DNA was isolated from the leaf sections of the selected newsletter, then cleaved with DraI and HindIII, the cleavage product was subjected to agarose gel electrophoresis, and the CryIAc gene was used as a probe to perform a routine Southern blot analysis procedure. Southern blot analysis was performed.

4 shows Southern blot analysis of transformed cabbage plants. As can be seen in Figure 4, C2, C3, C10, C20, C24, C30 in the case of DraI cleavage in a transgenic cabbage plant appeared as a single copy, C3, C20, C24, C30 is single for HindIII cleavage I could confirm that it is a copy.

Example  5: Infection test of transformed cabbage plants

Cabbage T ₁ for cabbage moth assay A total of 210 points were planted in a greenhouse. One month after the meal, cabbage root moth larvae 1-2 and 3-4 years old were injected into the cabbage. The larvae had been active for about two months, eating up cabbage leaves and inhibiting the growth of cabbage.

6 shows the results of resistance to cabbage myth moth between transgenic cabbage and non-transformed cabbage. As can be seen in Figure 6, in the case of synthetic CryIAc transgenic cabbage cabbage leaf moth was not damaged at all, while it can be seen that the leaves of non-transformed cabbage is much damaged.

Therefore, it can be seen that the cabbage transformed with the CryIAc gene shows very strong resistance to the cabbage moth.

1 is a schematic diagram of a pNW2300 / CryIAc vector.

2 is a step-by-step diagram of the development of the transformed cabbage plant. A: selection medium, B: shoot induction, C: shoot extension, D: root induction, E: Jiffy purified, F: soil purified

Figure 3 is a diagram showing the results of PCR analysis of the transformed cabbage plant.

M: Molecular marker; 1-26: Transformed (T ₀ ); N: Non-transformed; P: Bacterial cells harboring CryIAc

4 shows Southern blot analysis of transformed cabbage plants.

Cont: non-transformed; C2 ~ C39: transformed

5 shows T _o cabbage and T ₁ seeds of transgenic cabbage.

A: T _o cabbage after spring treatment, B: T ₁ Cabbage seeds

6 shows the results of resistance to cabbage myth moth between transgenic cabbage and non-transformed cabbage.

<110> NONG WOO BIO CO., LTD <120> Cabbage resistant to diamondback moth transformed with CryIAc          gene and production method <130> PN09166 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1865 <212> DNA <213> Bacillus thuringiensis <400> 1 ccatggacaa caacccaaac atcaacgaat gcattccata caactgcttg agtaacccag 60 aagttgaagt acttggtgga gaacgcattg aaaccggtta cactcccatc gacatctcct 120 tgtccttgac acagtttctg ctcagcgagt tcgtgccagg agctgggttc gttctcggac 180 tagttgacat catctggggt atctttggtc catctcaatg ggatgcattc ctggtgcaaa 240 ttgagcagtt gatcaaccag aggatcgaag agttcgccag gaaccaggcc atctctcgtt 300 tggaaggatt gagcaatctc taccaaatct atgcagagag cttcagagag tgggaagccg 360 atcctactaa cccagctctc cgcgaggaaa tgcgtattca attcaacgac atgaacagcg 420 ccttgaccac agctatccca ttgttcgcag tccagaacta ccaagttcct ctcttgtccg 480 tgtacgttca agcagctaat cttcacctca gcgtgcttcg agacgttagc gtgtttgggc 540 aaagatgggg attcgatgct gcaaccatca atagccgtta caacgacctt actaggctga 600 ttggaaacta caccgactac gctgttcgtt ggtacaacac tggcttggag cgtgtctggg 660 gtcctgattc tagagattgg gtgagataca accagttcag gagagaattg accctcacag 720 ttttggacat tgtggctctc ttcccgaact atgactccag acgttaccct atccgtacag 780 tgtcccaact taccagagaa atctacacta acccagttct tgagaacttc gacggtagct 840 tccgtggttc tgcccagggt atcgaaagat ccatcaggag cccacacttg atggacatct 900 tgaacagcat aactatctac accgatgctc acagaggata ctattactgg tctggacacc 960 agatcatggc ctctccagtt ggattctccg gacctgagtt tacctttcct ctctatggaa 1020 ctatgggaaa cgccgctcca caacaacgta tcgttgctca actaggacag ggtgtctaca 1080 gaaccttgtc ttccaccttg tacagaagac ccttcaatat cggtatcaac aaccagcaac 1140 tttccgttct tgacggaaca gagttcgcct atggaacctc ttctaacttg ccatccgctg 1200 tttacagaaa gagcggaacc gttgattcct tggacgaaat cccaccacag aacaacaatg 1260 tgccacccag gcaaggattc tcccacaggc ttagccacgt gtccatgttc cgttccggat 1320 tcagcaacag ttccgtgagc atcatcagag ctcctatgtt ctcttggatt caccgttctg 1380 ccgagttcaa caacatcatc gcatctgata gtattactca aatccctgcc gtgaagggaa 1440 acttcctttt caatggaagc gtaatcagcg gaccaggatt cactggcgga gatcttgtga 1500 gacttaacag ctctggcaac aacattcaga atagaggcta catcgaagtt cctatccact 1560 tcccatccac atctactaga tacagagtta gggttagata cgcctctgtg accccaatcc 1620 accttaacgt gaactggggc aattcatcta tcttctccaa caccgttcca gctactgcta 1680 cctcactcga taatcttcaa tccagcgatt ttggttactt cgaaagtgcc aacgcattca 1740 cttcttcatt gggcaacatc gtgggtgtta ggaatttcag cggtactgca ggagtgatca 1800 ttgacagatt cgagttcatt cctgttactg ccactcttga ggctgagtac aatctttaag 1860 gtacc 1865 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 35S promoter primer <400> 2 atgacgcaca atcccactat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CryIAc primer <400> 3 tgtggctctc ttcccgaact 20  

Claims (4)

A cabbage plant transformed with a recombinant plant expression vector comprising a CryIAc gene consisting of the nucleotide sequence represented by SEQ ID NO: 1, and having a resistance to cabbage myth moths. The cabbage plant of claim 1, wherein the recombinant plant expression vector is a pNW2300 / CryIAc vector described in FIG. 1. Seed of cabbage plant according to claim 1. A method of producing a cabbage plant having resistance to cabbage moths comprising the step of transforming cabbage plant cells with a recombinant plant expression vector comprising a CryIAc gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 and overexpressing the CryIAc gene.

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