KR100899993B1 - Low Nicotine Transformed Tobacco Plant and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a low nicotine tobacco transformed by inhibiting the expression of putrescine-N-methyltransferase (PMT) gene in tobacco plants using RNA inhibition technology and a method of producing the same Specifically, amplifying a target gene using a primer containing a recognition site of restriction enzymes BamHI and XbaI or BamHI and SacI for RNA inhibition, and inserting a spacer gene between the amplified target genes The present invention relates to a method for producing low nicotine transformed tobacco using RNA inhibition technology that inhibits the expression of genes, and a low nicotine tobacco plant produced thereby.

RNAi, Low Nicotine Tobacco, Transgenic Plant

Description

A low nicotine transgenic tabacco and a method of producing thereof

1 is a result of restriction enzyme reaction of PMT BS and BX target genes cloned in pGEM-T easy vector ^TM (Lane M: 1 kbp ladder, Lane 1: PMT BS intact, Lane 2: PMT BX intact, Lane 3: PMT BX EcoRI cut, Lane 4: PMT BX BamHI cut, Lane 5: PMT BS EcoRI cut, Lane 6: PMT BS BamHI cut).

Figure 2 inserts the identified sized gene fragment into the pGEM-T easy vector ^TM and into the vector The resulting plasmid was confirmed (lane M: molecular size marker, lane 1, 2, 3 PCR products).

FIG. 3 shows the results of electrophoresis after PCR using primers containing restriction enzymes Xba I, Sac I and Bam HI recognition sites in SEQ ID NOs 1,2 and 3 (lane M: molecular weight marker, lanes 1-4 : PCR product SB, lane 5-6: PCR product XB).

Figure 4 shows the results of electrophoresis by cutting with the restriction enzyme Eco RI after cleaving with restriction enzyme Xba I and then extracting self-igation pXBpmt DNA to select pXBpmt (pXBpmtΔ Eco RI) from which the restriction enzyme Eco RI recognition site was removed. (M: molecular weight marker, *: clones, lanes 2, 4, 5, 6, and 10 were selected from which the Eco RI recognition site in the vector was removed).

Fig. 5 shows base sequences of PMT genes (Group1, Group2) of Burley's tobacco and PMT genes (PMT1, PMT2, PMT3, PMT4) of Zanti's tobacco. Blue is the sequence used to construct the primer for conjugation cloning.

6 is a schematic diagram of a result of recombination of the pBluescript II SK (-) vector from which the Bam HI recognition site was removed and the reverse repetition DNA fragment with Sac I and Xba I and recombination through an ligation reaction.

Figure 7 shows the results of electrophoresis performed after introducing a spacer gene of about 3.0kbp size between the target PMT gene reversely recombined (Lane M; molecular size marker, Lane 1: restriction enzyme digested, Lane 2: intact)

Figure 8 is a spacer gene of various sizes amplified for insertion between the reversely recombined target PMT gene ( Figure 8a , Lane M: 1kbp ladder, Lane 1-10: spacer gene, Figure 8b , after the spacer gene is inserted Schematic of RNA hairpin structure predicted to produce).

9 is a result confirming that the recombinant gene was introduced into the plant transformation vector pBI121.

10 is 10A is a tobacco tissue cultured after introduction of the recombinant gene, and FIG. 10B is a tobacco transformant derived from above ground and root. FIG. 10C is a transformed tobacco plant growing in a greenhouse. ).

11 is a photograph confirming that a recombinant gene has been introduced into a transformed tobacco plant (Lane M; 100 bp ladder marker, Lane 1-3; transgenic plant number 1-3, Lane C1-C2; Control plant number 1-2, : positive signal).

12 shows target genes for preparing nucleotides for RNAi of the PMT gene. Cccaaaaa to agtatgatcgagt ( SEQ ID NO: 11 ) and aaatgctt to agtatgatcgagt ( SEQ ID NO: 12 ), which are yellow in the common part of the PMT gene ( SEQ ID NO: 4 ), are introduced repeatedly into the vector as target genes, with the spacer gene in between. Is inserted.

Tobacco has been enjoyed by many people as a favorite of humans, but recently, with increasing interest in health, anti-smoking campaigns are spreading around the world, and the demand for tobacco is low nicotine and low tar while maintaining the flavor of tobacco. Accordingly, tobacco manufacturers need breakthrough technology to improve the quality and price competitiveness of tobacco leaf material.

Alkaloids in plants, including tobacco nicotine, have been studied for their biosynthesis due to their pharmacological activity (De Luca 1993, Hashimoto and Yamada 1992, Hashimoto and Yamata 1993), and interest in the mass production of alkaloids has increased. (Kinnersley and Dougall 1980, Saitoh et al. 1985, Tiburcio et al. 1985a, 1985b). Recently, researches on controlling the biosynthesis process of alkaloids have also been reported (Fumihiko et al., 1999).

Alkaloids in tobacco have been detected in the development of analytical technology, such as nicotine, nornicotine and anabasine, and 10 kinds of alkaloids, and production of nicotine, scopolamine and bernerine. Several genes associated with have been cloned. In addition, Fumihiko et al. (1999) have been working to modify the alkaloid biosynthesis process in tobacco using the putrescine-N-methyltransferase (PMT) gene published by Hibi et al.

Nicotine, the representative alkaloid of tobacco, is put into N-methylputrescine by putrescine biosynthesized from ornithine and arginine at the roots, such as tropane alkaloid. Converted and oxidized by diamine oxidase to 4-methylaminobutanol to 1-methyl-Δ1-pyrrolinium cation and nicotinic acid ( biosynthesis via nicotinic acid). Among them, putrescine-N-methyltransferase is an important enzyme in the biosynthesis process of nicotine and can be called a rate limiting enzyme in the biosynthesis process of nicotine. Hibi (1994) et al. Have successfully cloned PMT genes by comparing low nicotine mutants with normal tobacco. See also Riechers, D.E. And Timko, M.P. et al. (1999) have described the tobacco species Nicotiana tabacum cv. The cloned PMT gene was reported from Xanthi and the nucleotide sequence was reported. In addition, the PMT gene was analyzed for several species of the genus Nicotiana such as N. tomentosiformis, N. otophora, and N. attenuata. However, PMT genes have been found to differ in sequence among several species in the genus Nicotiana, and four different PMT genes have been reported from cultivar Xanthi, including PMT1, PMT2, PMT3, and PMT 4, even in N. tabacum. Only one type of PMT is reported. The inventors have developed a cigarette in which nicotine biosynthesis is inhibited by inhibiting the expression of the gene of putrescine N-methyltransferase (PMT), an enzyme of alkaloid biosynthesis in tobacco. Was completed.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

An object of the present invention is to provide a method for producing low nicotine transgenic tobacco plants comprising the step of inhibiting the expression of PMT gene using RNA inhibition technology.

Another object of the present invention to provide a low nicotine tobacco plant produced by the method for producing a transformed tobacco plant.

For this purpose, in one embodiment of the present invention, the step of inhibiting the expression of putrescine-N-methyltransferase (PMT) gene using RNA interference (interference) technology It provides a low nicotine type transgenic tobacco plant production method comprising a. Specifically, the present invention, RNA designed to amplify a target gene using a primer containing a recognition site of restriction enzymes BamHI and XbaI or BamHI and SacI for RNA inhibition, and insert a spacer gene between the amplified target gene It includes using an inhibition vector.

Recently, the RNA interference (RNA interference, RNAi) method has been spotlighted as a method for inhibiting gene expression more efficiently than the method by antisense nucleic acid as a gene expression suppression technology.

An antisense nucleic acid refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein.

In contrast, "RNAi" is naturally present in plants and animals and specifically "silenced" certain genes. It helps regulate gene expression and protects the host from the dangers of "jumping genes," which can spread to the genome by replicating viral infections. This process can be experimentally induced by inserting artificially-made genetic sequences into cells, thus becoming a method of deliberately silencing the target gene. This method is now widely used as one of the basic genetic tools and is considered one of the promising future therapies. Injection of certain double-stranded RNAs can silence corresponding genes, and this can happen with only a few molecules injected.

Double helix RNA is recognized by Dicer and cut into small double helix fragments. These fragments then bind to the RNA-induced silencing complex (RISC), which unwinds one strand of the double helix and becomes a complex with a tiny strip of RNA. This process is single stranded, so starting with a single strand of RNA does not have the same effect because it does not activate Dicer or RISC. The resulting complex binds to naturally occurring mRNA, cuts and breaks down its strands, silencing its parent gene.

The double chain that can induce RNA inhibition by cleaving mRNA of a target gene is called siRNA (short interfering RNA). siRNA consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases. siRNA terminal structures can be either blunt or cohesive. The cohesive end structure is possible in both the structure which protrudes at the 3 terminal side, and the structure which protrudes at the 5 terminal side, and the number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, siRNA is a low-molecular RNA (e.g., natural RNA molecules such as tRNA, rRNA, viral RNA, or artificial RNA) in the protruding portion of one end thereof in a range capable of maintaining the expression inhibitory effect of the target gene. RNA molecules). The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions. The method of preparing siRNA is a method of directly synthesizing siRNA in vitro and then introducing it into a cell through a transfection process, and siRNA expression vector or PCR-derived siRNA expression cassette prepared to express siRNA in a cell. There is a method of transformation or infection in.

In the present invention, the term "specific" or "specific" refers to the ability to inhibit only the target gene without affecting other genes in the cell, and is specific for PMT in the present invention. In a specific embodiment of the present invention, the Burley Tobacco PMT gene and the PMT gene of Zanti Tobacco, which have already been cloned and reported, were used.

The siRNA of the present invention can specifically reduce the mRNA of PMT in tobacco plants, and the sequence and length are not particularly limited. Compositions comprising such gene specific siRNAs may include additional substances that inhibit apoptosis and may also include agents that promote the intracellular influx of siRNAs. In preparations for promoting intracellular influx of siRNAs, agents that promote nucleic acid inflow can generally be used. For example, liposomes may be used or combined with one of the lipophilic carriers of many sterols, including cholesterol, cholate and deoxycholic acid. Poly-L-lysine, spermine, polysilazane, polyethylenimine, polydihydroimidazolenium, polyallylamine Cationic polymers such as chitosan), succinylated PLL, succinylated PEI, polyglutamic acid, and polyaspartic acid. anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, hyaluronic acid, etc. It can also be used.

 In using the RNA inhibition method for suppressing the expression of PMT genes of tobacco plants, since they may be affected by different PMT types of several tobacco objects, specific embodiments of the present invention may be easily used for various tobacco objects. In search for the common nucleotide sequence (SEQ ID NO: 4) of the PMT gene of several tobacco objects, and based on this to prepare a primer for RNAi to add a restriction enzyme recognition site to the specific nucleotide sequence shown in SEQ ID NO: 1,2, and 3 It was. These primers were used to amplify fragments of the consensus nucleotide sequence portion of the PMT gene, and introduced into the vector so that these amplified fragments could be repeatedly recombined in opposite directions.

In the present invention, a spacer was introduced in order to keep the recombinant gene repetitively reversed in vivo. A "spacer" is a gene that exists between two or more cistrons consisting of <starting codons> -expression sites (codons)-<ending codons>, also called intergenic DNA or intercistronic sequences. Spacers do not encode specific proteins and serve to distinguish between codons. In the present invention, the genes were manipulated by recombining spacer-adaptable sites, and spacers of various sizes were prepared. In a specific embodiment of the present invention, spacers of about 100bp to 3kb in size were prepared and introduced between the amplified PMT target gene segments. The RNAi nucleic acid for the recombinant PMT gene was recombined into a plant transformation vector and introduced into E. coli DH5α for propagation of the gene. In addition, the recombinant vector propagated was transformed into the Agrobacterium tumefaciens LBA4404 strain, and the transformed plant was completed using the transformed Agrobacterium strain.

In another aspect, the present invention provides a low nicotine transgenic tobacco plant produced using RNA inhibition technology. In addition, the tobacco plant is preferably Burley species (Wisconsin 38). In order to obtain a transgenic plant using genetic engineering methods, a method of effectively grafting a foreign gene into a target cell, obtaining a suitable target sample, and a regeneration technique of obtaining a complete plant from the transformed cell are essential. There are two methods of effective plant cell transformation (gene transplantation). The first method is to transfer foreign gene DNA to plant cells using a biological vector, which is mainly used for the soil plant pathogen Agrobacterium tumefaciens (Agrobacterium tumefaciens). The second method is a method of projecting DNA-coated metal microparticles by directly delivering foreign gene DNA into plant cells (Hansen and Wright, Trends Plant Sci. 4; 226-231, 1999). .

The most commonly used plant transformation technology today is the plant pathogen Agrobacterium, which is a part of their Ti (tumor-inducing) or Ri (root-inducing) plasmid DNA. (T-DNA) has the ability to metastasize and transplant into infected plant cells. Therefore, when the target gene is recombined into the T-DNA and co-cultured with the bacteria and the plants having the same, the transformation of plant cells becomes possible. However, the critical limitation of this technique lies in the compatibility, including the host range of Agrobacterium and the target cell of transformation, ultimately affecting the successful regeneration of the transformed cells. Therefore, many economically useful crops have not yet been transformed by this method (Hansen and Wright, Trends Plant Sci. 4; 226-231, 1999).

In an effort to develop a method capable of transplanting genes regardless of plant species or genotype, several techniques for delivering DNA directly to plant cells have been studied. These include electroporation and microinjection. Microinjection, PEG- or liposome-mediated DNA inhalation, silicon carbide whiskers, and microparticle projection. Among them, microparticle projection technology, known as biolistics or microprojectiles, is to coat small carrier metal particles with recombinant DNA, and then physically project these particles into plant cells (Klein). et al., Nature 327; 70-73, 1987: Sanford, Plant Physiol. 79; 206-209, 1990). This method has several advantages. First, the removal of the cell wall is not necessary to introduce DNA. Second, DNA can be easily introduced into cells of meristems and differentiated tissues. Third, no special biological carrier manipulation is required. Finally, the transfer of DNA into the cell does not depend on the binding or mutual recognition of the transgenic target cell and the biological vector. Examples of transformation with these particle projection methods include corn (Fromm et al., 1990 Biotechnology 8; 833-839, 1990), rice (Christou et al., Biotechnology 9; 957-962, 1991), barley (Wan and Lemaux, Plant Physiol. 104; 37-48, 1994), wheat (Vasil et al., 1992 Biotechnology 10; 667-674, 1992), soybean (McCabe et al., Biotechnology 6; 923-926, 1988), candy It has been proven in important crops such as sorghum (Bower and Birch, Plant J. 2; 409-416, 1992). The advantage of this method is that shoots that are re-differentiated in transformed cells can be generated directly from primary and secondary meristems, reducing treatment of plant hormones and minimizing somatic mutations.

In a specific embodiment of the present invention, genes were introduced into Agrobacterium tumefaciens LBA4404 by freeze-thaw method (An et al. 1988). The transgenic Agrobacterium obtained by culturing after the gene was introduced was grown in LB (kanamycin) medium and extracted again by plasmid. After back transformation with Escherichia coli, the plasmid was extracted again, followed by restriction enzyme treatment. It was confirmed through.

In the present invention, E. coli DH5α was used as a strain for propagation of genes, and Agrobacterium tumefacience LBA4404 was used as a strain for plant transformation. As a medium for the growth of strains, LB medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl) was used, and antibiotics ampicillin, cefataxime and kanamycin were used as necessary. It was added and used. As other restriction enzymes, ligase, Taq polymerase, etc., products of InvitrogenTM, TakaraTM, LocheTM, etc. were used.

Transgenic tobacco tissue for the production of transformed plants of the present invention induced germination in a medium supplemented with hormone BAP (6-Benzylaminopurine), and the development of roots was induced by the plant hormone IAA (Indole 3-acetic acid). Of these, 16 individuals were grown as complete plants and recombinant genes were introduced: T ₀ and T ₁ generations of redifferentiated plants had 70% or more nicotine synthesis compared to normal plants before transformation. In the second generation of transgenic plants, plants with 94% inhibition of nicotine synthesis were selected.

Seed of the strain R-6 in the plant whose nicotine synthesis was suppressed was transferred to the Nicotiana Bank of Korea, Korea Research Institute of Bioscience and Biotechnology on February 23, 2007. Deposited with tabacum denico R-6 (Accession No. KCTC11072BP).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  One. RNA  Interference( interference Target Gene Manufacturing

In order to introduce RNAi technology, it is necessary to prepare nucleic acid sequences in which fragments of the target PMT gene are cloned in a reverse direction. In order to avoid being influenced by PMT type, the common part of Burley's PMT gene and Zant's PMT 1,2,3, and 4 genes (SEQ ID NO: 4) were searched for cloning by PCR based on the same. Primers were selected (see below under SEQ ID NO: 4 and SEQ ID NOs: 1-3). Restriction enzyme recognition sites such as SacI, XbaI, and BamHI were inserted at both ends of SEQ ID NOs: 1,2,3 to facilitate cloning by repeating the target gene in the reverse direction. PCR was performed using the primer and the common part as a template. To amplify the target gene. Gene fragments whose size was confirmed by electrophoresis were inserted into pGEM-T easy vector ^TM and confirmed by insertion with restriction enzymes, followed by analysis of nucleotide sequences to identify genes ( FIGS. 1 and 2 ). A spacer gene in SEQ ID NO: 1 and 3 PMT target gene amplified by using the part of the primer between (SEQ ID NO: 11) and PMT target gene is amplified using SEQ ID NOS: 2 and 3, as part of the primers (SEQ ID NO: 12) Insertion was made to prepare recombinant nucleotides for RNAi. The target genes may be represented by a sequence from cccaaaaa to agtatgatcgagt ( SEQ ID NO: 11 ) and aaatgctt to agtatgatcgagt ( SEQ ID NO: 12 ), which are indicated in yellow as shown in FIG. Is introduced.

a forward primer1 for target PMT gene PCR ( SEQ ID NO: 1 )

cccaaaaaag gttttgatca tcg

a forward primer2 for target PMT gene PCR ( SEQ ID NO: 2 )

aatgcttcgt tatccttcaa tcg

a reverse primer for target PMT gene PCR ( SEQ ID NO: 3 )

atctttcgcc agaagtatga tcgag

<PMT gene searched for common partial cloning ( SEQ ID NO: 4 )>

tccgactcta ttaagcctgg ctggttttca gagtttagcg cattatggcc aggtgaagca ttctcactta aggttgagaa gttactattc caggggaagt ctgattacca agatgtcatg ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacatgaa gagtcat aggttttgat catcg gcgga ggaattggtt ttacattatt cga aatgctt cgttatcctt caatcg aaaa aattgacatt gttgagatcg atgacgtggt agttgatgta tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag gcagtagcca aagcccttag gccaggagga gttgtatgca cacaggctga aagcatttgg cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgatcgg ttatatgctc tgctctactg aagggccaga agttgacttc aagaatccag taaatccaat tgacaaagag acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa gcagcattca ttttacc atc tttcgccaga agtatgatcg ag tc

Sequences containing restriction enzyme recognition sites such as SacI (GAGGCT / C), XbaI (T / CTAGA), and BamHI (G / GATCC) at both ends of the underlined parts (SEQ ID NOs: 1, 2, 3) were used as primers.

Example 2. Cloning and Sequencing of Related Genes

PCR products were ligation to the PCR cloning vector pDrive ^™ of Invitrogen ^™ , and extracted DNA was digested with EcoRI and compared by electrophoresis on a 1% agarose gel to confirm the cloned insert ( FIG. 4 ) . ). Other ligations, restriction enzyme reactions and electrophoresis were performed according to the "Melocular cloning laboratory manual" by Sambrook et al. And the product manual of the manufacturer. The cloned gene was reacted with BigDyeTM cycle sequencing kit, and then sequenced on ABITM's autosequencer 377TM. The results were analyzed using Genebank's database and ClustalW. ( FIG. 5 )

Example  3. RNAi  Development of Recombinant Vectors

Since the recombination of the target gene in reverse direction is essential for effective action of RNAi, fragments of the PMT gene, which is the target gene prepared in Example 1, are introduced into the pBluescriptIISK (-) vector to be reversed with each other using restriction enzymes. (See FIG. 6 ). A spacer gene having a size of about 3 kbp was inserted into the spacer insertion site formed between the target genes in a vector in which the gene constructed as shown in FIG. 6 was introduced in the reverse direction ( FIG. 7 ).

The spacer gene was expected to affect RNAi activity according to the spacer size, and the following sequence (SEQ ID NO: 5) was added as a repeating unit in succession to synthesize 10 kinds of spacer genes of various sizes, Example 1 The target gene prepared in was confirmed after recombination into pBluescript II SK + introduced in the reverse direction ( FIG. 8A ).

5'-TACGAGGATC CTTTGAGGTA ATTAATATTC TAATACACAT GCTTTAATTT

3'-ATGCTCCTAG GAAACTCCAT TAATTATAAG ATTATGTGTA CGAAATTAAA

AAAGTGATAC TTTTAATTTA CTTTTAGTTT ATTGCATGTG CACGTACAGT

TTTCACTATG AAAATTAAAT GAAAATCAAA TAACGTACAC GTGCATGTCA

CAGCAACTTA TGGATCCTAC GA-3 '

GTCGTTGAAT ACCTAGGATG CT-5 '( SEQ ID NO: 5 )

As shown in FIG. 8B, RNAi nucleotides in which a spacer gene is recombined between target genes are expected to exhibit hairpin structures having different sizes of hairpin loops depending on the size of the spacer gene ( FIG. 8B ). The RNAi nucleotides thus prepared were framed and introduced into pB1121, which is a plant transformation vector, and confirmed them ( FIG. 9 ).

Example  4. Plant introduction of related genes

The transformation vector pB1121 prepared in Example 3 was introduced into Agrobacterium tumefaciens LBA4404 by freeze-thaw method (An et al. 1988). The transformed Agrobacterium was grown in LB (kanamycin) medium and extracted again from the plasmid. The transformed plasmid was transformed back to E. coli DH5α (back transformation). Introduction of related genes was confirmed.

Example 5 Development of Tobacco Transformant Using RNAi Technology

The leaf tissue of the tobacco plant to which the RNAi gene is to be introduced was sterilized with 70% alcohol and 1% sodium hypochloride, and cut to a size of 0.5 X 0.5 (cm X cm), which was obtained in MS medium (Murashige & Skoog, 1962). The cells were transformed by incubating with transformed agrobacteria according to Example 5. The transformed leaflets were incubated for 4 weeks in germination induction medium, MS medium containing 1.0 mg / l benzylaminopurine and 100 mg / l kanamycin for 4 weeks, to induce germination and callus. It was. This was again transferred to root induction medium, which is an MS medium containing 0.1 mg / l Indole 3-acetic acid, 300 mg / l cefotaxim, and 100 mg / l kanamycin, to induce roots ( FIG. 10A ) . ). The cultured transgenic tobacco tissue induced germination in medium supplemented with hormone BAP (6-Benzylaminopurine), and induced root development with plant hormone IAA (Indole 3-acetic acid) ( FIG. 10B ). Individuals were grown as complete plants and grown in greenhouses ( FIG. 10C ).

Example  6. Assay of Transgenic Plants

In order to confirm that the RNAi recombinant vector was introduced into the transgenic plant grown in the greenhouse, DNA was extracted from the transgenic plant, PCR was performed on the extracted DNA as a template, and as a result, the recombinant gene was normally introduced ( FIG. 11 ).

DNA was extracted from the transgenic plants using Qiagen ^TM DNA isolation kit, and RT-PCR reaction was performed using Qiagen ^TM One Shot RT-PCR cloning kit. PCR was carried out using Takara ^TM's ExTaq ^TM from PerkinElmer ^TM's GeneAmp 9600 ^TM, RT-PCR reaction and then at 94 ℃ heated for 15 minutes in the reaction solution, a total of 20ul, 1 minute 30 seconds at 94 ℃ denature, 72 Annealing at 1 ° C. for 1 minute at 55 ° C. was performed for 35 cycles of extension. After reacting at 72 ℃ for 15 minutes and stored at 4 ℃. The reaction of the second PCR carried out to confirm the product obtained through RT-PCR was heated for 15 minutes at 94 ℃, then denature for 1 minute 30 seconds at 94 ℃, anealing for 1 minute at 72 ℃, extension reaction for 1 minute at 55 ℃ Reacted at 35 cycles. The primers used for RT-PCR and PCR were used as the primers (SEQ ID NOS: 6 to 10) to search for the nucleotide sequences of PMT genes known from various species of Nicotiana from the Gene Bank. The resulting reaction was confirmed by electrophoresis on 0.8% to 1.2% agarose gel ( FIG. 3 ).

1F (first forward): 5'- ggaagtcatatctaccaacac-3 ', ( SEQ ID NO 6 )

2R (second reverse): 5'-gccataatgcgctaaactctg-3 ', ( SEQ ID NO: 7 )

3F (third forward): 5'-tttgcaggtgaagcattctcac -3 ', ( SEQ ID NO: 8 )

4R (forth reverse): 5'- gactcgatcatacttctggcg -3 ', ( SEQ ID NO: 9 )

5R (fifth reverse): 5'- atcaactaccacgtcatcg -3 ', ( SEQ ID NO: 10 )

Example 7 Nicotine Synthesis of Transgenic Plants

Leaves of the transformed tobacco plants were harvested at each growth period, dried at 60 ° C. for 2 days, and the amount of nicotine synthesized was quantitatively analyzed using GC-FID. After a review of synthetic nicotine level of the first generation transgenic lines 8 Plant Regeneration from T ₀ generation transgenic plant it is given in Table 1.

Table 1. Transgenic Tobacco Plants (T ₀ Investigation of nicotine content after wetting (generation 1 regeneration, ug / g)

System name division  After soaking R-1 Transformed Plant 2622 (73% decrease) Normal plant 9836 R-2 Transformed Plant 1493 (72% decrease) Normal plant 5301 R-3 Transformed Plant 2019 (51% decrease) Normal plant 4099 R-4 Transformed Plant 1794 (60% decrease) Normal plant 4472 R-5 Transformed Plant 1686 (60% decrease) Normal plant 4170 R-6 Transformed Plant 645 (64% decrease) Normal plant 1815 R-7 Transformed Plant 660 (77% decrease) Normal plant 2871 R-8 Transformed Plant 634 (71% decrease) Normal plant 2190

Inhibition of nicotine synthesis was inhibited by 51% to 77% compared to the control group, which differed from plant to plant.

Seven generations of newly regenerated transgenic plants were collected from each seedling, before and after wetting to examine nicotine synthesis inhibition ( Table 2 ).

In the present invention, the seed of the strain R-6 in the plant whose nicotine synthesis was inhibited was deposited as Nicotiana tabacum denico R-6 in the Korea Biotechnology Research Institute Gene Bank on February 23, 2007 (Accession No. KCTC11072BP).

Table 2. Transgenic Tobacco Plants (T ₀ Generations)  Investigation of Nicotine Content by Growth Period ( 2nd year  subdivision, ug / g)

System name division Seedlings Weeping 27 days after soaking R-9 Transformed Plant 2090 (29% decrease) 5353 (37% decrease) 20752 (43% decrease) Normal plant 2957 8480 36248 R-10 Transformed Plant 1715 (18% decrease) 3330 (60% decrease) 23567 (48% decrease) Normal plant 2081 8269 45028 R-11 Transformed Plant 875 (54% decrease) 3242 (68% decrease) 6564 ( 87% decrease ) Normal plant 1922 10044 50233 R-12 Transformed Plant 1148 (82% increase) 8716 (1% increase) 32980 (36% decrease) Normal plant 631 8612 51254 R-13 Transformed Plant 606 (26% decrease) 4965 (54% decrease) 6597 (5% decrease) Normal 821 10702 6959 R-14 Transformed Plant 953 (44% decrease) 1751 (84% decrease) 3869 (45% decrease) Normal plant 1715 10811 6972 R-15 Transformed Plant - - 7235 (34% decrease) Normal plant - - 10939

The degree of inhibition of nicotine synthesis was varied in seedlings and plants before wetting. R-11 transgenic plants were 68% inhibited before wetting and 87% decreased after wetting. The transformed plants were inhibited in nicotine synthesis compared to the non-transformed normal plants. The degree of inhibition was varied and up to 87% of the suppressed plants were selected. In order to investigate the degree of nicotine synthesis inhibition of the second generation plants of these transgenic plants, each plant was harvested.

Example  8. Investigation of Nicotine Content in Second Generation Plants Transgenic

Harvested transgenic plants, T ₁ in greenhouses and fields, respectively. The extent of nicotine synthesis inhibition was examined in each generation ( Table 3 , Table 4 ). The second generation plants in the transgenic plants were inhibited nicotine synthesis irrespective of the growth time, and the synthesis was inhibited in various ways even after wetting, the maximum nicotine synthesis period, and the result was similar to the first generation of transgenic plants. Plants that inhibit nicotine synthesis up to 94% were selected from the R-8 strain.

Table 3. Transgenic second-generation plants grown in greenhouses (R _One Investigation of Average Nicotine Content by Growth Period (ug / g)

System name Seedlings Weeping After soaking R-1 2666 (46% decrease) 2134 (53% decrease, minimum 84% decrease) 2417 (66% decrease, minimum 84.3% decrease) R-2 3603 (27% decrease) 2183 (52% decrease, minimum 79% decrease) 2779 (61% decrease, minimum 85.1% decrease) R-3 5467 (10% increase) 3490 (23% decrease, minimum 61% decrease) 7967 (11% decrease, minimum 78.5% decrease) R-4 6840 (37% increase) 4126 (9% decrease, minimum 62% decrease) 6460 (10% decrease, minimum 59.5% decrease) R-5 6362 (28% increase) 5230 (15% increase, minimum 59% decrease) 7924 (10% increase, minimum 46.8% decrease) R-5-1 5496 (10% increase) 5188 (14% increase, minimum 39% decrease) 9507 (up 32%, down 23%) R-6 2791 (44% decrease) 2731 (40% decrease, minimum 82% decrease) 3352 (53% decrease, minimum 83% decrease) R-7 3965 (20% decrease) 2902 (36% decrease, minimum 83% decrease) 3751 (48% decrease, minimum 87% decrease) R-8 1776 (64% decrease) 853 (82% decrease, minimum 88% decrease) 1130.7 (84% decrease, minimum 94% decrease) Normal plant 4969 4551 7182

Table 4. Transformation Grown in Packaging 2nd generation plant ( R1 Average by growth period

  Nicotine content investigation ug / g)

System name Weeping After soaking R-1 3310 (61% decrease, minimum 85%) 16468 (53% decrease, minimum 81% decrease) R-2 3573 (58% decrease, minimum 82%) 18779 (46% decrease, minimum 82% decrease) R-3 8890 (6% decrease, minimum 27%) 38672 (10% decrease, minimum 30% decrease) R-4 10.023 (19% increase, minimum 18%) 32636 (6% decrease, minimum 46% decrease) R-5 10,495 (25% increase, lowest 22%) 29624 (15% decrease, minimum 60% decrease) R-5-1 10,195 (21% increase, minimum 26%) 31795 (9% decrease, minimum 42% decrease) R-6 5105 (39% decrease, minimum 84%) 15411 (56% decrease, minimum 85% decrease) R-7 6718 (60% decrease, lowest 69%) 22046 (37% decrease, minimum 82% decrease) R-8 2487 (70% decrease, minimum 89%) 7560 (78% decrease, minimum 94 % decrease ) Normal plant 8426 34880

In the present invention, the low nicotine tobacco that suppresses the expression of the PMT gene in tobacco plants by using RNA inhibition technology was raised, which is high in use value as a raw material for producing high-quality tobacco with low nicotine content.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Korea Research Institute of Bioscience and biotechnology <120> A Low Nicotine Transgenic Tabacco and A method of prodicing          about <130> PA9612-985KR <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a forward primer 1 for target PMT gene PCR <400> 1 cccaaaaaag gttttgatca tcg 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a forward primer2 for target PMT gene PCR <400> 2 aatgcttcgt tatccttcaa tcg 23 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> a reverse primer for target PMT gene PCR <400> 3 atctttcgcc agaagtatga tcgag 25 <210> 4 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> PMT gene for common gene cloning <400> 4 tccgactcta ttaagcctgg ctggttttca gagtttagcg cattatggcc aggtgaagca 60 ttctcactta aggttgagaa gttactattc caggggaagt ctgattacca agatgtcatg 120 ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacataca 180 gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg ttccatccca 240 aacccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt cgaaatgctt 300 cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt agttgatgta 360 tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt 420 ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata 480 gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag 540 gcagtagcca aagcccttag gccaggagga gttgtatgca cacaggctga aagcatttgg 600 cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct 660 gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgatcgg ttatatgctc 720 tgctctactg aagggccaga agttgacttc aagaatccag taaatccaat tgacaaagag 780 acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa 840 gcagcattca ttttaccatc tttcgccaga agtatgatcg agtc 884 <210> 5 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> spacer gene <400> 5 tacgaggatc ctttgaggta attaatattc taatacacat gctttaattt aaagtgatac 60 ttttaattta cttttagttt attgcatgtg cacgtacagt cagcaactta tggatcctac 120 ga 122 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> first forward primer for RT-PCR and PCR <400> 6 ggaagtcata tctaccaaca c 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> second reverse primer for RT-PCR and PCR <400> 7 gccataatgc gctaaactct g 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> third forward primer for RT-PCR and PCR <400> 8 tttgcaggtg aagcattctc ac 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forth reverse primer for RT-PCR and PCR <400> 9 gactcgatca tacttctggc g 21 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> fifth reverse primer for RT-PCR and PCR <400> 10 atcaactacc acgtcatcg 19 <210> 11 <211> 641 <212> DNA <213> Artificial Sequence <220> <223> target gene 1 for RNAi <400> 11 cccaaaaaag gttttgatca tcggcggagg aattggtttt acattattcg aaatgcttcg 60 ttatccttca atcgaaaaaa ttgacattgt tgagatcgat gacgtggtag ttgatgtatc 120 cagaaaattt ttcccttatc tggcagctaa ttttaacgat cctcgtgtaa ccctagttct 180 cggagatgga gctgcatttg taaaggctgc acaagcggga tattatgatg ctattatagt 240 ggactcttct gatcccattg gtccagcaaa agatttgttt gagaggccat tctttgaggc 300 agtagccaaa gcccttaggc caggaggagt tgtatgcaca caggctgaaa gcatttggct 360 tcatatgcat attattaagc aaatcattgc taactgtcgt caagtcttta agggttctgt 420 caactatgct tggacaaccg ttccaacata tcccaccggt gtgatcggtt atatgctctg 480 ctctactgaa gggccagaag ttgacttcaa gaatccagta aatccaattg acaaagagac 540 aactcaagtc aagtccaaat taggacctct caagttctac aactctgata ttcacaaagc 600 agcattcatt ttaccatctt tcgccagaag tatgatcgag t 641 <210> 12 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> target gene 2 for RNAi <400> 12 aaatgcttcg ttatccttca atcgaaaaaa ttgacattgt tgagatcgat gacgtggtag 60 ttgatgtatc cagaaaattt ttcccttatc tggcagctaa ttttaacgat cctcgtgtaa 120 ccctagttct cggagatgga gctgcatttg taaaggctgc acaagcggga tattatgatg 180 ctattatagt ggactcttct gatcccattg gtccagcaaa agatttgttt gagaggccat 240 tctttgaggc agtagccaaa gcccttaggc caggaggagt tgtatgcaca caggctgaaa 300 gcatttggct tcatatgcat attattaagc aaatcattgc taactgtcgt caagtcttta 360 agggttctgt caactatgct tggacaaccg ttccaacata tcccaccggt gtgatcggtt 420 atatgctctg ctctactgaa gggccagaag ttgacttcaa gaatccagta aatccaattg 480 acaaagagac aactcaagtc aagtccaaat taggacctct caagttctac aactctgata 540 ttcacaaagc agcattcatt ttaccatctt tcgccagaag tatgatcgag t 591  

Claims (8)

Recombinant nucleotides for RNAi produced by inserting the spacer gene of SEQ ID NO: 5 between the PMT gene fragments of SEQ ID NO: 11 and SEQ ID NO: 12 among the PMT gene sequences of the tobacco plants were transformed into tobacco plants and putrescine-N- A method for producing a low nicotine transgenic tobacco plant comprising the step of inhibiting the expression of a methyl transferase (putrescine-N-methyltransferase (PMT) gene). delete delete delete The method of claim 1, wherein the transformation is carried out by transforming the Agribacterium strain into which the recombinant nucleotide for RNAi is introduced into a pale plant. Low nicotine transformed tobacco plants ( Nitiana tabacum ), in which the expression of the PMT gene produced by the method of claim 1 or 5 is suppressed to decrease nicotine content. The low nicotine transformed tobacco plant ( Nicotiana tabacum ) according to claim 6, wherein the tobacco plant is Burley species. Seed of Nicotiana tabacum denico R-6, a low nicotine transgenic tobacco plant produced by the method of claim 1 or 5 (Accession No. KCTC11072BP).

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