KR100639303B1 - Plant seed or the anther-specific expression promoter derived from Perilla Japonica and seed or the anther-specific recombination vector comprising the same - Google Patents

Abstract

The present invention provides a transgenic plant comprising a seed specific expression promoter gene (PfFAD-3) derived from perilla and a plant seed or anther specific expression vector containing the same. The present invention has developed a promoter capable of high expression in sesame seeds. Through this, it can be used for the development of high-expression promoter of other oils and fats, and provides regeneration and transformation technology of sesame seeds with high reproducibility and regeneration rate. It can be usefully used to develop transgenic plants that functionally modify the production material of seeds and anthers.

Perilla, PfFAD-3, promoter, expression vector, GUS, binary vector

Description

Plant seed or anther-specific expression promoter derived from Perilla Japonica and seed or the anther-specific recombination vector comprising the same}

Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of the promoter region and the transcriptional start of the PfFAD-3 genomic gene isolated from perilla of the present invention.

Figure 2 is a schematic diagram showing a binary vector of the present invention prepared by inserting the promoter region of PfFAD-3 of the present invention into pBI101 containing the GUS gene.

Figure 3 is a photograph showing the deletion structure and electrophoresis of the deletion structure sequentially deleted by 140bp in the promoter region of the PfFAD-3 genomic gene of the present invention.

4 is a photograph showing the expression level of GUS in Arabidopsis in which pPfFAD-3 binary vector and pCaMV355 vector are inserted, respectively.

FIG. 5 illustrates the introduction of nine binary vectors and pBI121 binary vectors, prepared by deleting pPfFAD-3 binary vector and pPfFAD-3, into Agrobacteria, and then to Arabinopsis. After transformation, the photographs show that GUS is expressed in the seed of Arabidopsis inserted with pPfFAD-3.

FIG. 6 is a photograph showing that GUS is expressed in a flower of Arabidopsis inserted with pPfFAD-3 in FIG. 5.

The present invention provides a transgenic plant comprising a seed specific expression promoter gene (PfFAD-3) derived from perilla and a plant seed or anther specific expression vector containing the same.

 In order for the biotechnological transformation of plant fatty acids to be successful, it has been demonstrated that expression and regulatory regions (promoters and terminators) are essential for the expression of these genes, as well as the presence of related genes (Science 1995, 268: 681-686). . In addition, ω-3 fatty acid synthesis gene was first introduced into the plant transformation technology, Hamada et al Transgenic Res. 1996, 5: 115-121) in tobacco and the transgenic transcripts of tobacco transformed by this gene significantly increased, but antisense reported the opposite. The gene has been shown to alter the composition of plant fatty acids. Martin et al. (Eur. J. Biochem. 1999, 262: 283-290) describe the transformation of potato from anti-sense ω-3 fatty acid genes isolated from potato leaves. The study found that the amount of transcripts in the mice was much lower. It is also known that oilseed crops incorporating this technique are rapeseed and soybeans, primarily fatty acids with shorter carbon or shorter carbon lengths, such as caprics, to minimize rancidity during processing. Techniques for increasing the content of acid have been developed (Lipids 1996, 31: 557-569; Curr. Opin. Plant Biol. 1999, 2: 123-127; Curr. Opin. Biotech. 1999, 10: 175-180 Nature Biotech. 1999, 17: 593-597).

 On the other hand, about 80% of the fatty acids in sesame seeds are concentrated into oleic acid and linoleic acid, and since linolenic acid (ω-3 fatty acid), which has important functions, is hardly synthesized, the content of linolenic acid is extremely low, but in vivo Omega-3 fatty acid (linolenic acid), an essential fatty acid that is not biosynthesized, is known to inhibit the synthesis of eicosenoic acid that causes adult diseases such as hypertension and allergic diseases, and has bioregulatory functions such as improving learning ability and prolonging sleep. With economic growth, the average life expectancy is increasing, making quality more important than quantity.

However, the study of the promoter of the plant ω-3 gene is still only basic research (Plant Mol. Biol. 1995, 29: 599-609). In addition, there is almost no synthesis of plant vectors capable of transforming sesame seeds, and there is no information on a promoter specifically expressed in sesame seeds. Therefore, the research related to the transformation technology of sesame is very poor, and the development of the above technique is required for the practical use of the functional sesame applied the biotechnology technology of sesame seeds.

The present invention has been made to solve the above problems, an object of the present invention is to provide a nucleotide sequence of the expression promoter gene (PfFAD-3) specific to seed or anther derived from perilla.

Another object of the present invention is to provide a recombinant vector containing an expression promoter gene (PfFAD-3) specific for seed or anther derived from perilla.

Still another object of the present invention is to provide a plant transformed with a plant seed or anther specific recombinant vector.

The present invention relates to a seed or anther specific expression promoter gene of a plant represented by the nucleotide sequence of SEQ ID NO: 1, more specifically, 501 to 954 or 1086 to 1360 of SEQ ID NO: 1 of the present invention is a tissue specific fragment. A seed or anther specific expression promoter of a plant characterized by. The present invention also relates to a recombinant vector containing the promoter gene, and more particularly, to a transgenic plant including the recombinant vector together with a recombinant vector, wherein the promoter gene is included in a binary vector. will be.

In order to achieve the object of the present invention, the present invention provides seed and anther specific promoter genes of the plant of SEQ ID NO: 1.

In the nucleotide sequence of SEQ ID NO: 1, there was an ABRE motif and CE 1 which are elements that respond to ABA. In addition, TATC box and P box, which are elements that respond to gibberellin, existed. PfFAD-3 specifically expresses seed and anther specifically, so that it specifically expresses RY repeat motif and endosperm that express seed specificly and GCN4 motif and Skin 1 motif (motif) is included (see Example 4).

In order to achieve another object of the present invention, the present invention provides a recombinant vector containing the seed or anther specific promoter gene of the plant.

Preferably, the plant seed or anther specific recombinant vector of the present invention is characterized in that it comprises the PfFAD-3 promoter gene of the present invention, that is, the nucleotide sequence of SEQ ID NO: 1. In addition, the promoter of the present invention is capable of transcriptional coupling with foreign genes contained in the binary vector.In the present invention, for example, the promoter of the present invention is inserted into a pBI101 vector containing the GUS gene, which is a reporter gene, to perform a binary vector. Example 2) was used to transform plants.

In order to achieve another object of the present invention, the present invention provides a plant cell or plant transformed with the plant seed or anther specific recombinant vector of the present invention.

Preferably, when the seed and anther specific recombinant vector of the plant is a binary vector, the plant may be transformed according to a dip-floral method. The plant seed or anther specific recombinant vector of the present invention can transform any plant regardless of the type of plant, but in the present invention, only transformation of Arabidopsis and sesame was provided as an example (see Examples 5 and 9). .

Hereinafter will be described the specific configuration and effect of the present invention through the examples. However, the following examples are only examples of the present invention, and the scope of the present invention is not limited to the following examples.

Example 1 GenomeWalker PCR

      Genomewalker PCR was performed using the Universal GenomeWalker ™ Kit (ClONTECH). PCR-based GenomeWalker DNA Walking methods have been used to obtain unknown genomic DNA sequences from adjacent regions from the prepared cDNA or DNA sequences.

Step 1: Isolate Whole Genomic DNA

       300 mg of leaf tissues of young perilla (Oleum perilla perilla) are made of fine powder with liquid nitrogen and then 500 μl of CTAB buffer (0.25 M NaCl, 0.2 M Tris-Cl pH 8.0, 2.5% PVP, 50 mM EDTA, 0.1%). β-mercaptoethanol (β-mercapto ethanol) was added and left for 10 minutes at 68 ° C. Then, an equal amount of chloroform: IAA (24: 1 / v: v) was added thereto at a speed of 13,000 rpm. After centrifugation, only supernatant was collected and the same amount of phenol: chloroform: IAA (25: 24: 1 / v: v: v) was added thereto for 10 minutes at 13,000 rpm. 0.6 times isopropanol (isopropanol) was added to the volume of the supernatant and allowed to stand at -20 ° C for 1 hour, followed by centrifugation for 20 minutes at a speed of 13,000 rpm. After washing, 50 μl of distilled water was added to dissolve the RNA, and RNA was removed using RNase (10 mg / ml).

Stage 2: Genomic DNA digestion

Into 25 μl of Genomic DNA (0.1 μg / μl), add 8 μl of restriction enzymes such as Pvu II, Dra I, Eco RV, and Stu I provided in the kit, and place the 10 × restriction enzyme buffer corresponding to each restriction enzyme ( buffer) 10 µl was added, and 57 µl of distilled water was added thereto to adjust the total volume to 100 µl. It was then vortexed briefly and reacted at 37 ° C. for 2 hours. After vortexing for 5-10 seconds at a low speed, it was reacted at 37 ° C for 18 hours. After incubation, 5 μl of each tube was taken and subjected to electrophoresis on 0.5% agarose gel to confirm complete degradation.

Third step: DNA purification

Each An equal amount (95 μl) of phenol was added to genomic DNA cut with enzyme, vortexed for 5-10 seconds, and only the supernatant was separated. Again, an equal amount (95 μl) of chloroform was added thereto and vortexed for 5-10 seconds. Only the supernatant was separated and vortexed for 5-10 seconds with 95% ethanol, 0.1 times 3 M sodium acetate (pH 4.5) and 20 μg of glycogen, twice the total volume. It was centrifuged for 10 minutes at the speed of 13,000 rpm. The supernatant was discarded and the pellet washed with 80% ethanol, kept cold on ice. The supernatant was discarded by centrifuging for 5 minutes at a speed of 13,000 rpm and the pellet dried. 20 μl TE (10 mM Tris-Cl pH 7.5, 1 mM EDTA) was dissolved in the solution, and then vortexed for 5-10 seconds. 1 μl of each tube was used for electrophoresis on 0.5% agarose gel to ensure complete degradation.

Step 4: GenomeWalker adapter ligation

Treated with enzyme and then the genome walker adapter (GenomeWalker adaptor) (25 μM) 1.9 ㎕ with T4 DNA ligase (ligase) (1 unit / ㎕) 5 × ligation buffer (ligation buffer) 1.6 ㎕ corresponding insert the 0.5 ㎕ After the reaction, the reaction was carried out at 16 ° C. for 12 hours. The reaction was stopped by leaving it at 70 ° C. for 5 minutes and then vortexed for 5-10 seconds by adding 72 μL of TE (10 mM Tris-Cl pH 7.5, 1 mM EDTA) to each tube. It was then used as a template for PCR reaction while stored at -20 ℃.

Step 5: GenomeWalker PCR

PCR was performed under the following conditions.

     <First PCR>

     Using 1 μl of GenomeWalker library DNA (0.1 μg / μl) as a template, 1 μl of adapter primer (AP1, 10 pmole / μl) provided in the kit and gene specific primer 1 (GSP1, 10) pmole / μl) 1μl were mixed together. Add 3 μl of 25 mM MgCl2 and 2 μl of 10 mM dNTP mixture, add 1 unit of Taq polymerase (promega) and the corresponding 10 × PCR buffer, and adjust the total volume to 50 μl with distilled water. It was. Then, the reaction was performed 25 times at 94 ° C., 7 times for 3 minutes at 65 ° C., 25 seconds at 94 ° C., and 32 times at 62 ° C. for 3 minutes, followed by an additional 7 minutes at 62 ° C. after the last cycle. The sequences of AP1 and GSP1 primers were 5'-GTAATACGAC TCACTATAGG GC-3 'and 5'-GCGGCGACGT CCCAAACGAC GTAGCTCAA-3', respectively.

     <Second PCR>

     Dilute the GenomeWalker first PCR product to 1/50 and use 1 μl of this as a template, and add 1 μl of adapter primer 2 (AP2, 10 pmole / μl) provided in the kit. 1 μl of gene specific primer 2 (GSP2, 10 pmole / μl) was mixed together. 3 μl of 25 mM MgCl 2 and 2 μl of 10 mM dNTP mixture were added, 1 unit of Taq polymerase (promega) and the corresponding 10 × PCR buffer were added, and distilled water was added to adjust the total volume to 50 μl. . Then, 25 seconds at 94 ° C, 5 times for 3 minutes at 65 ° C, 25 seconds at 94 ° C, 20 times at 3 ° C for 62 minutes, and then at 62 ° C for 7 minutes after the last cycle. The sequences of AP2 and GSP2 primers were 5′-ACTATAGGGC ACGCGTGGT-3 ′ and 5′-AACCTCTCCA TCAGCGCCAC TCTTCGAGA-3 ′, respectively.

     5 μl of each of the PCR products was used to determine size by hanging on a 1.5% agarose gel. The band visible on the gel was purely separated using a gel extraction kit described below, and then cloned into a pCR 2.1 kb TOPO vector.

Example 2: Pure separation of specific DNA fragments

     Pure separation of specific DNA fragments was carried out using a gel extraction kit (Q · BIOGENE, Korea). The agarose gel of the site containing the DNA fragments was cut and weighed. The cut sections were placed in a 1.5 ml tube, and 100 µl of GENECLEAN® Turbo solution per 0.1 g of gel was added and left at 55 ° C. for 5 minutes to dissolve the gel. After the gel was completely dissolved, it was transferred to a unit consisting of a GENECLEAN® Turbo Catridge and a cap-less catch tube. After centrifugation at 13,000 rpm for 5 seconds, the solution collected in the catch tube was discarded, and 500 µl of GENECLEAN® Turbo Wash collected in GENECLEAN® Turbo Catridge was added and centrifuged for 5 seconds. The solution collected in the catch tube was discarded, and 500 µl of GENECLEAN® Turbo Wash collected in the GENECLEAN® Turbo Catridge was added and centrifuged for 5 seconds. Centrifuge at 13,000 rpm for 2 minutes to remove remaining ethanol filtrate. GENECLEAN® Turbo Catridge was placed in a new 1.5 ml tube, and then 20 µl of distilled water was added thereto for 5 minutes. Thereafter, centrifugation was performed for 1 minute at 13,000 rpm to purely separate the desired DNA fragments.

Example 3 PCR 2.1 kb TOPO Vector Cloning

     The band seen on the gel was purified using the gel extraction kit (gel estraction kit) described above and then cloned into pCR 2.1kb Topo vector (Invitrogen). 4 μl of the PCR product was added with 1 μl of Topo vector and 1 μl of sterile water, followed by incubation at room temperature for 5 minutes. 2 μl of the reacted product was placed in a prepared one shot competent cell and placed on ice for 30 minutes. Heat shock was applied at 42 ° C. for 30 seconds, and then the tube was immediately transferred to ice. 500 μl of LB media was added thereto, followed by incubation at 37 ° C. for 1 hour. 150 μl of the culture solution was applied to a plate containing X-gal (50 mg / ml) and ampicillin (50 mg / ml), and incubated at 37 ° C. for at least 12 hours. White colony, a transformed ligation, was selected. Base sequences were generated for clones obtained through genome walking.

Example 4 DNA Sequence Analysis and Preparation

     Base sequences were determined using automatic sequencing (Automatic sequence).

In order to isolate the promoter of FAD3 from perilla by the above-described GenomeWalker PCR, four libraries were prepared by separating genomic DNA as described above to clone the promoter of PfFAD-3 from perilla. PCR was performed on each library using a gene specific primer (GSP) based on an adapter primer and a PfFAD3 gene. As a result, a genome clone of 1598bp was obtained from the PvuII library. (genomic clone) was obtained. The genomic clone contained a sequence to be transcribed into PfFAD-3 RNA in a nucleotide sequence. Thus, a promoter of 1485 bp was obtained (see FIG. 1).

Meanwhile, in order to examine the function of the promoter, the cis-element of the PfFAD3 promoter is used. Analysis by PlantCARE revealed the ABRE motif and CE 1, which are elements that respond to ABA. In addition, TATC box and P box, which are elements that respond to gibberellin, existed. As PrFAD-3 expresses seed-specifically, there was RY repat motif (motif) to express seed-specifically. ) Was found (see Table 1).

Table 1

Cis-element (cis-element) estimated in the 5′-flangking region of the PfFAD-3 gene

category Motif Consensus sequence Explanation Endosperm specific elements and ABA-inducible elements ABRE motif ACGTG ABA Sensitive Element CE-1 TGCCACCGC Element related to ABRE, related to sensitivity to ABA GCEN4_motif TTAGTCA Endosperm specific expression RY repeat motif CATGCATT Seed-specific response element skn-1_motif GTCAT Required for Endosperm Expression Gibberellin-inducible elements TATA-box TATCNCA Gibberellin-responsiveness p-box CCTTTTN Gibberellin Responsive Element

Example 5 Promoter-GUS Fusion Fusion Mapping

Step 1: Promote Detachment

     In order to isolate the promoter of PuvII-PrFAD-3, a sense and antisense promoter was prepared by including restriction enzyme sequences of HindIII and Sac I on 5 'and 3' sides of the promoter, respectively. PuvII-PrFAD3.3 (100 ng / μl) was used as a template, primer (25 pmole / μl), 3 μl of 25 mM MgCl 2 and 1 μl of 10 mM dNTP mixture were added, followed by 1 unit of Taq polymerase ( promega) and the corresponding 10 × PCR buffer were added and distilled water was added to adjust the total volume to 50 μl. 25 times were repeated for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C. Finally, the reaction was continued for 10 minutes at 72 ° C. Sections formed after PCR under the above conditions were separated purely using the VIOGENE gel extraction kit described above. The obtained product was subcloned into pGEM vector (Promega).

Step 2: Preparation of the pBI101 Vector

     pBI101 vectors were isolated using Alkaline lysis method. The pBI101 vector was inoculated into 10 ml LB medium containing kanamycin (50 mg / ml) and shaken at 37 ° C. at a rate of 220 rpm for at least 16 hours. The cells were transferred to a 1.5 ml eppendorf tube and centrifuged several times to recover the cells, and then suspended in 100 µl of Sol I (50 mM glucose, 10 mM EDTA, 25 mM TRis-Cl pH 8.0, mg / ml lysozyme) solution. After 5 minutes at room temperature. To this, add 200 μl of Sol II (0.2 N NaOH, 1% SDS) solution stored on ice, leave for 5 minutes on ice, and then 150 μl of Sol III (60 mL 50 M potassium acetate pH 4.7 11.5 mL glacial actic). acid, 28.5 mL H20) solution was added, and left for 5 minutes. It was treated with phenol: chloroform: isoamylalcohol (25: 24: 1 / v: v: v). After centrifugation under the same conditions, 2 times of ethanol was added to the supernatant, and left at -20 ° C for 30 minutes, followed by centrifugation to recover DNA precipitate. The recovered precipitate was dissolved by adding TE buffer (10 mM Tris-Cl, 1 mM EDTA pH 8.0) or sterile tertiary distilled water, and used as a sample.

Step 3: pBI101 vector-pGEM-FAD3 promoter ligation

     Restriction enzymes of HindIII and Sac I were respectively treated, and agarrose gels were used to elution (Q · VIOGENE, DNA.RNA estraction kit) to dissolve the fragments in a small amount of TE buffer. T4 DNA ligase (1 unit / μl) was added and the corresponding 5 × Ligation buffer was added, followed by reaction at 16 ° C. for 12 hours. The reaction was stopped by treatment at 70 ° C. for 5 minutes, followed by vortexing for 5-10 seconds by adding 72 μl of TE (10 mM tris-Cl pH 7.5, 1 mM EDTA) to each tube. Store at 20 ° C.

A cleavage map coupled with the GUS reporter gene was prepared to identify the gene expression control specificity of the PrFAD-3 promoter as described above (see FIG. 2). And it was introduced into Agrobacteria in order to transform the plant by the method described below.

Stage 4: Agrobacteria Transformation

     1 μg of plasmid DNA was added to competent cells dissolved in ice, mixed, and incubated for 5 minutes on ice, 5 minutes on liquid nitrogen, and 5 minutes at room temperature. After diluting with 1 ml of fresh YMB medium, the solution was incubated at 28 ° C. for 2-4 hours. Then, 200 μl was applied to the YMB plate containing kanamycin (kanamycin) (50 mg / ml) and incubated at 28 ° C. for 2 days.

Example 6: Preparation of deletion series of seed specific expression gene PfFAD-3 promoter

The seed specific gene expression promoter is used to analyze cis-elements involved in the expression of genes specifically for seed, based on the nucleotide sequence of the promoter, about 140 bp from the 5 'end of the promoter. A total of nine primers were made to decrease. PCR was performed on the DNA of perilla as a template to obtain a total of nine deletion promoters.

About 140 bp on the basis of the promoter sequence to analyze the cis-element involved in the expression of the gene using the 1485 bp PfFAD-3 promoter secured through the above method Reduced pPfFAD3 deletion series: pPfFAD3 -1375, pPfFAD3 -1235, pPfFAD3 -1095, pPfFAD3 -955, pPfFAD3 -785, pPfFAD3 -647 , pPfFAD3 -510, pPfFAf3 PCR -3653 , p236 Paid. The PfFAD-3 promoter was constructed by PCR to reduce deletion structure according to cis-element and confirmed it on the gel.

Example 7 Cloning into Binary Vector Containing Reporter Gene GUS

Nine deletion promoters obtained by PCR were cloned into the pBI101 binary vector containing the GUS gene as a reporter gene.

Example 8 Agrobacteria Transformation

Binary vectors containing nine kinds of PfFAD-3 deletion promoters were introduced into the Agrobacteria GV3101 strain by the freeze / thaw method.

Example 9 Arabidopsis Arabidopsis Plant transformation and transformant analysis

Transformed Agrobacteria were transformed into Arabidopsis plants according to the dip-floral method. Arabidopsis transformants resistant to the antibiotic kanamycin (kanamycin) were collected by tissue and seed development time, stained with X-glucuronic acid, and analyzed for the activity of the GUS gene using MUG as a substrate. In addition, Arabidopsis transformants responded to plant hormone treatment and photoperiod changes to analyze the effects of plant hormone ABA on seed-specific gene expression and the effects of photoperiods on seed tissues of transgenic plants. The activity of GUS was analyzed by treatment with ABA or various photoperiods.

pBfFAD-3 binary vectors (vectors) and pPfFAD-3 deletion series (nine binary vectors, pBI121 binary vectors) were introduced into Agrobacteria and transformed into Arabidopsis . GUS was expressed in seeds and flowers of Arabidopsis in which pPfFAD-3 was inserted (see FIG. 3). Based on GUS expression in seeds and flowers, GUS expression was observed in seeds and flowers of the deletion series (see FIGS. 4 and 5). As the promoter length became shorter, it was confirmed that GUS expression disappeared from seeds and flowers. In the case of seed, the disappearance of GUS expression between 501 and 954 of SEQ ID NO: 1 was observed, which is the PfFAD- in the seed by P-box, a cis-element present between them. The expression of 3 can be said to be regulated. In the case of flowers, the expression of GUS disappeared between 1086 and 1360 of SEQ ID NO. .

Eventually, based on the above results, the promoter of pPfFAD-3 shows tissue-specific regulation of plants, and in particular, it can be seen that the expression is very high in seeds and pollen. In particular, it can be seen that 501 to 954 parts of the pPfFAD-3 sequence are involved in the expression of PfFAD-3 in the seed and 1086 to 1360 parts are involved in the expression of PfFAD-3 in anther.

As described in the above embodiments, the seed specific expression promoter gene (PfFAD-3) derived from the perilla and the plant seed or anther specific expression vector containing the same according to the present invention will develop a promoter capable of high expression in sesame seeds. It can be used for the development of high-expression promoters of oil and fat plants, and it is much more efficient than the methods published so far through regeneration and transformation technology of sesame seeds with high reproducibility and regeneration rate. do. Furthermore, it is a very useful invention for the functional food industry because it can be usefully used to produce seed or anther specific useful substances of plants or to develop transformed plants functionally modifying existing seeds and anther's producing substances.

Attach sequence list electronic file  

Claims (5)

Seed or anther specific expression promoter gene of a plant represented by the nucleotide sequence of SEQ ID NO: 1. The seed or anther specific expression promoter of a plant according to claim 1, wherein 501 to 954 or 1086 to 1360 of SEQ ID NO: 1 are tissue specific fragments. A recombinant vector containing the promoter gene of claim 1. The recombinant vector of claim 3, wherein the promoter gene is included in a binary vector. Arabidopsis transformed with the recombinant vector of claim 3.

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