KR100561190B1 - Novel vector system using promoters of gene involving resveratrol biosynthesis - Google Patents

Abstract

The present invention relates to a new vector system using a promoter of stilbene synthase gene, a gene involved in resveratrol biosynthesis, and the vector system of the present invention using a promoter of stilbene synthase gene whose activity is increased by various environmental signals It provides an excellent effect that can optimally express the desired gene more effectively than the plant expression vector system. In addition, the vector system of the present invention enables a search for an optimal environmental factor that highly enhances the content of resveratrol in major domestic grape varieties, and proposes a method for mass production of resveratrol having anticancer and antimicrobial activity. It is a very useful invention because it can produce an environmental stress resistant transgenic plant.

Grape, resveratrol, stilbene synthase gene, promoter, GUS

Description

Novel vector system using promoters of gene involving resveratrol biosynthesis

Figure 1 shows the PCR product produced by walking (walking) with GenomeWalker libraries of giant grapes. Here, M represents a DNA size marker, lane 1 represents a Dra I library, lane 2 represents an EcoRV library, lane 3 represents an EcoRV library, and lane 4 represents a PvuII library.

Figure 2 is a result of comparing the nucleotide sequence of the four kinds of giant grape stilbene synthase gene promoter. Where Pro 1 is the stilbene synthase gene promoter amplified by the PvuII library, Pro 2 is the promoter amplified by the Dra I library, Pro 3 is the promoter amplified by the EcoRV library, and Pro 4 is the EcoRV library. The amplified promoter is shown.

Figure 3 shows the nucleotide sequence of the promoter 1 and cis elements (cis elements). The promoter includes a TATA box, GATA motif, HSE, LTR, WUN, MBS, I box and GT 1 elements. Where WUN is the wound-response element, GT-1 is the light response element, AE box is the part of the module for light response, LTR is the cis-acting element involved in low temperature reactivity, I box is part of the light response element, ACE Is a cis-acting element involved in light reactivity, GAG-motif is part of the light response element, HSE is a cis-acting element involved in heat stress reactivity, MBS is a MYB bond involved in drought-inducibility The site, As-1, is a cis-acting regulatory element involved in root specific expression, a prolamin box refers to a cis-acting regulatory element bound to GCN4.

Figure 4 shows the physical map of the stilbene synthase gene promoter-GUS fusion constructs.

5 is a result of analyzing the GUS expression in seedlings of wild type and transgenic tobacco treated with UV, AlCl ₃ , yeast extract. Figure 5a is a wild type control, 5b is a seedling of untreated transgenic tobacco, 5c is a seedling of transgenic tobacco treated with UV (304nm) 15 minutes, 5d is a seedling of transgenic tobacco treated with 75mM AlCl ₃ for 24 hours, 5e is Seedlings of transgenic tobaccos treated with 100 mM AlCl ₃ for 24 hours, 5f shows seedlings of transgenic tobaccos treated with 0.1% yeast extract for 24 hours, and 5g resulted from seedlings of transgenic tobaccos treated with 0.5% yeast extract for 24 hours to be.

Figure 6 shows a deletion construct of stilbene synthase gene promoter 1. Where 6a is the structure of the temporal vector containing the GUS gene under the 5 'flanking region of the deleted promoter, and the left side in 6b represents the putative cis component of the deleted promoter region, and the lane M in the right figure 100 bp DNA ladder and lanes 2-12 represent deletion constructs.

7 shows the results of analyzing histochemical GUS activity of Arabidopsis plants transformed with the deletion construct of Promoter 1. FIG. Here, the first row is -513 promoter, the second row is -406 promoter, the third row is -308 promoter, and the fourth row is -258 promoter. From the left, the first row is the control group, the second row is UV 15 minutes treatment, the third row is 0.1% yeast extract treatment, the fourth row is 0.5% yeast extract treatment, the fifth row is 75mM AlCl ₃ treatment, and the sixth row is 100mM AlCl ₃ treatment. The treatment tool is shown.

8 is a result of fluorescence analysis of GUS activity of the deletion list of promoter 1 treated with UV, yeast extract, AlCl ₃ . Here, 8a is treated with UV for 15 minutes, 8b is treated with 0.1% and 0.5% yeast extract, and 8c is treated with 75mM and 100mM AlCl ₃ .

9 is a result of fluorescence analysis of GUS activity of promoters 1, 2, 3, 4 treated with various stresses. Where c is the control, 0.1 is 0.1% yeast extract, 0.5 is 0.5% yeast extract, 75 is 75 mM AlCl ₃ , 100 is 100 mM AlCl ₃ , UV is 15 minutes UV, P1 is promoter 1, P2 is promoter 2, P3 is Promoter 3, P4, promoter 4, W means wild type.

The present invention relates to a new vector system using a promoter of a gene involved in resveratrol biosynthesis, and more particularly to a new vector system developed using a promoter of a stilbene synthase gene whose activity is increased by various environmental signals.

Gene expression in eukaryotic systems is regulated through complex interactions between various cis-acting and trans-acting regulatory elements. Cis-acting elements are defined as DNA sequences adjacent to a gene that directly or indirectly regulate the expression of a gene by regulating the binding of trans-acting elements called various transcription elements. Promoters are one of the most important cis-acting elements and are generally considered to be nucleotide sequence regions located upstream of the 5 terminus of the transcription initiation site of a gene. The promoter contains a binding site for RNA polymerase II and initiates transcription of the gene. The promoter also contains several well-conserved sequence elements, such as a CAAT box near base position −70 and a TATA box near base position −30 associated with the transcription initiation site (+1) (Joshi, 1987). TATA boxes are important for accurately positioning the onset of transcription. CAAT boxes are not present in all promoters, but in some promoters with GC-rich regions replaced. In addition to these base elements, the promoter also includes additional sequences that respond to environmental signals and physiological conditions such as dry stress, heat shock, cold stress, nutrient availability, light and ionic strength. In addition, some promoters include sequence elements that direct tissue-specific gene expression or cell-specific gene expression. In addition, promoters can express beneficial genes in important agricultural plants by regulating gene expression by conditions such as expression level, tissue specificity or cell-specificity, developmental stage dependence, and responsiveness to specific environmental stimuli. It is important.

On the other hand, resveratrol (resveratrol) is directly known in plants by the Pitot aleksin (phytoalexin) protective substances that inhibit the growth of invading fungi from the outside, extremely there is generated only some plants so far is a poly goneom coarse avoid conflict (Polygonum cuspidatum) , Eucalyptus, spruce, Scottish pine , grapes, peanuts and lilies. In these plants, resveratrol is synthesized from malony-CoA and p-coumaroyl-CoA by stilbene synthesis and has been reported to induce its synthesis from external environmental stresses such as fungi, ultraviolet light and ozone (Schoeppner and KindI H, 1979; Fliegmann et al., 1992; Fritzemeier et al., 1983; Vornam et al., 1988). In recent years, multifaceted studies on the molecular and genetic levels of the biosynthetic mechanism and regulation of the material have been made. As a result, genes involved in biosynthesis of this substance were isolated from pine, grape, and peanut (Sparvoli et al., 1994; Preisig-Muller et al., 1999; Schubert et al., 1997). A great deal of information has been accumulated, paving the way for the use of genes.

On the other hand, research on resveratrol in grapes has been started for a long time, and the antistress action of this compound is well known (Langcake et al., 1979). In the past, synthetic genes using antistress action and stress resistance of resveratrol have been introduced into specific crops to develop disease resistant transformant crops such as antibacterial, antibacterial, antiviral, and antiworms (Kindle et al., 1999; Hain et al. , 1998; Schroder et al., 2000). However, since the recent discovery of trans-resveratrol in wine and the fact that adequate wine intake lowers mortality from coronary heart disease, many experimental results have been published on the pharmacological effects of resveratrol. Siemann and Creasy, 1992). In particular, reports have been noted that ingestion of red wine increases blood antioxidant activity and thereby inhibits the production of oxidative LDL (Maxwell et al., 1994; Fuhrman et al., 1995). Most recently, many studies on the anti-mutation and anticancer activity of this substance have been reported (Carbo et al., 1999; Clement et al., 1998; Huang et al., 1999).

However, resveratrol, which is known to have anti-cancer and anti-oxidant activity, is a promising natural functional resource that has been identified as a promising natural functional resource, but it can still be used as a functional food material and a new drug through various studies. Very little research has been done on genes involved in resveratrol biosynthesis. In this regard, a stilbene synthase gene, which is a resveratrol biosynthetic gene, has been isolated from grapes to disclose the nucleotide sequence of the protein coding site (DE C12N 15/52 DE3873672; US C12N 15/10 US60293196; EP A01H 1/00 EP9111002). Although a basic study on the activity of the promoter has been carried out to disclose a promoter site induced by pathogens and ozone (WO A01H 5/00 WO9825257), this is still a molecule for resveratrol biosynthetic genes such as expression regulation mechanism of the gene. Biological research is very poor, and there is no research on the development of a vector system using a promoter of this gene.

The present invention has been devised in view of the above, resveratrol synthesis in which the activity is increased by a variety of environmental signals as a prerequisite for the search for the optimal environmental factors that can enhance the content of resveratrol in major grape varieties of Korea Using a cis-element in the promoter of the stilbene synthase gene, a gene involved, a new vector system was developed that can optimally express desired genes more effectively than the existing plant expression vector system.

Accordingly, an object of the present invention is to provide a new vector system comprising a promoter region of stilbene synthase gene specifically expressed by various environmental factors causing environmental stress.

The object of the present invention is to obtain four different genomic clones containing the promoter region of the stilbene synthase gene in grape grapes, and then amplify only the promoter therefrom to produce the GUS and fusion genes, using the Agrobacterium tobacco After transforming to the tobacco transformants treated with various environmental stress to examine the degree of GUS expression according to confirm the development of the vector system for transformation and environmental factor search system, and then to the promoter site for This was accomplished by investigating sites having meaningful functions as promoters through analysis of the deletion series and again confirming the potential as an environmental factor search system from the present invention.

Hereinafter, the configuration of the present invention will be described.

The present invention is to prepare a GenomeWalker library by cleaving genomic DNA isolated from the skin of giant grape grapes with restriction enzymes of EcoR V, Pvu II, Dra I, and PCR-based DNA walking to include a promoter region of the stilbene synthase gene 4 Obtaining two different genomic clones; From the amplification of only the promoter to produce a GUS and a fusion gene using it to transform Agrobacterium, and then to transform the tobacco by using a variety of stress on the tobacco transformant to analyze the degree of GUS expression ; A deletion series was prepared from the longest of the four promoters, transformed into Agrobacterium, transformed Arabidopsis with transformed Agrobacterium, and then treated with various environmental stresses to the Arabidopsis. The useful site of the promoter is identified by histological and fluorescence analysis of GUS activity and compared with the other three promoters.

Promoters provided by the present invention are induced very high in response to various environmental factors causing environmental stress, such as UV, AlCl ₃ , wound, yeast extract. In this regard, the present invention can be used to manipulate genes, in particular, to express genes at certain levels under standard conditions and to be further induced by various environmental stresses. In addition, according to the present invention, plants can be manipulated in order to mass produce biologically important substances, promote growth and development, or increase adaptability to environmental changes. For example, after placing any desired gene under the control of the promoter of the present invention in an appropriate expression cassette, the recombinant cassette can be introduced into host cells by various transformation methods well known in the art to produce environmental stress resistant plants. It is possible to mass-produce useful substances by treating various environmental stresses on the plants.

Hereinafter, the configuration of the present invention will be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

Example 1 Procurement of Promoter through Library Preparation and PCR-based DNA Walking

Isolation from the skins of grapes (Vitis vinifera LX Vitis labrusca L. cv kyoho) harvested after flowering from the grapes of Cheongan in Cheonan to clone the promoters that regulate the expression of resveratrol biosynthesis genes, ie stilbene synthase genes. One 2.5 μg genomic DNA was cut with restriction enzymes of EcoRV, PvuII, and DraI (Clontech, Universal GenomeWalker kit), and then extracted with the same amount of phenol / chloroform. The supernatant obtained by centrifugation was mixed well by adding 2 times ethanol, 1/10 volume of 3 M sodium acetate, 20 ㎍ glycogen and centrifuged at 10,000 × g for 10 minutes to collect DNA. The pellet formed was washed with 80% ethanol, dissolved in TE buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA), stored at -20 ° C, and stored in a small amount of digested DNA in the GenomeWalker Adaptor primer (Clontech. Universal GenomeWalker kit). Ligation was induced at 16 ° C. for more than 16 hours. Finally, the reaction was stopped by standing at 75 ° C. for 5 minutes and used as a substrate for PCR.

The prepared GenomeWalker library was subjected to primary PCR under the following conditions (deionized H ₂ O, 1/10 vol. 10X Tth PCR buffer, 1.1 mM magnesium acetate, 0.2 mM dNTP, 10 μM adatpor primer 1, 50X Advantage Genomic polymerase) Mix, 10 μM gene specific primer 1, each GenomeWalker library; 7 cycles: 94 ° C 30 seconds, 72 ° C 3 minutes; 32 cycles: 94 ° C 30 seconds, 67 ° C 3 minutes, 67 ° C 7 minutes). The formed PCR product was diluted to 1/10 and the primers were changed under the same conditions to carry out the secondary PCR. Each product obtained by PCR was identified on a 1% agarose gel and then subcloned using the pCR2.1-TOPO vector (Invitrogen).

As a result, four different genomic clones containing the promoter region (hereinafter, referred to as promoter 1, promoter 2, promoter 3, and promoter 4) were obtained (FIG. 1,2). The DNA sequences between the ORFs of these clones were 100% identical and the promoter regions had at least 90% similarity (FIG. 2). Analysis of the DNA nucleotide sequence of the longest promoter 1 of the 4 clones revealed a 95 bp untranslated leader sequence and the first codon (M) of the coding region. In addition, TATA box, 33 bp away from the start of transcription, GATA motif, GCC-box reacting to ethylene, CAAT, HES (heat response element, position at -317, -112, -98), WUN (wound response element) , position at -665, -583, -453, -370, -265), G box (position at -426, -294, -151, -105), GT 1 (GGTTAA, position at -691, -479, -372) and AS-1 (position at -89) were included, and cis-acting elements such as I-box, AE-box, and GAG motifs responding to light were distributed throughout the promoter. There were two repeating structures -702--532 (named X1) and -490--319 (named X2). The repeating structure was 98% identical in sequence and there was one G box in X2 and two GT 1s by 2% difference (FIG. 3).

[Example 2: Development of vector system for transformation and environmental factor search using promoter region of stilbene synthase gene]

Vector systems that regulate the transcription of useful genes in plants are usually regulated by the CaMV 35S promoter. Such a promoter is known as a strong promoter that is expressed in all tissues of the plant, but in the present invention, in order to develop a vector system capable of optimally expressing a desired gene more effectively, the promoter obtained in Example 1 is fused with a reporter gene, GUS, to model plant. Phosphorus tobacco was transformed. The transgenic tobacco was then treated with various environmental stressors to determine the degree of GUS gene expression and resveratrol synthesis.

First step. Construction of Promoter-GUS Fusion Gene of Stilbene Synthase Gene

PCR primers were newly prepared to amplify and use only promoters from genomic clones subcloned into the pCR2.1-TOPO vector (Invitrogen). The primer was prepared by adding the XbaI restriction enzyme position to the 5 'end sense and the Bam HI restriction enzyme position to the 3' end sense, and the product obtained by amplification with the primer was subcloned into the pCR2.1-TOPO vector (Invitrogen). After treatment with the restriction enzymes, agarose gels were suspended and only the fragments were subjected to elution (Viogene, DNA.RNA extraction kit) and dissolved in a small amount of TE buffer. The prepared DNA sample was treated with the same restriction enzyme and added 1 unit of T4 DNA ligase with pBI IOI.1 (CLONETECH) and the vector: insert ratio of 1: 8 to induce a reaction for 16 hours at 16 ° C and transfect E. coli . Switched.

Second step. E. coli Transformation

E. coli transformation was performed based on the method provided by the TOPO TA Cloning Kit (Invitrogen). TOPO TA cloning is characterized by the ability to clone PCR products amplified by Taq-polymerase in minutes without the addition of ligase or restriction enzyme sites. The amplified PCR product was mixed with the pCR2.1-TOPO vector and allowed to stand at room temperature for 5 minutes, and then left on ice to terminate ligation. A portion of the ligation mixture is mixed with 50 μl of competent cell (TOP 10) and left on ice for 30 minutes, followed by 250 μl of SOC liquid medium (2% tryptone, 0.5% yeast). extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl _2, 10 mM MgSO _4, 20 mM glucose) Incubated at 200 rpm for 1 hour at 37 ℃. 100 μl of the culture solution was plated with X-gal in LB solid medium containing 50 μg / ml of ampicillin and incubated at 37 ° C. for 16 hours or more, whereby the insert was transformed. White colonies were selected.

Third step. Agrobacterium Agrobacterium ) Transformation

After transforming into E. coli , the construct (construct) was confirmed, and 1 μg of DNA was isolated and Agrobacterium tumefaciens LBA 4404 was transformed by freeze-thaw method. Competent cells were placed on ice and 1 μg of the corresponding construct DNA was added. The cells were left on ice for 5 minutes, reacted with liquid nitrogen for 5 minutes, and 37 ° C. for 5 minutes. 1 ml of YMB liquid medium was mixed and shaken at 27 ° C. and 170 rpm for 2-4 hours, and then plated in MS solid medium containing kanamycin and cultured at 27 ° C. for 2 days to obtain transformed colonies. . Transformed colonies were grown in YMB liquid medium, extracted and confirmed DNA, and used for tobacco transformation.

Fourth step. Tobacco transformation

Tobacco ( N. tabacum cv Xanthi ) transformation was carried out using the leaf disc method. Tobacco seeds were germinated to take leaves about 6-7 cm in diameter and then sterilized in 0.1% bleach solution for 10 minutes and washed sequentially in sterilized distilled water for 7 minutes, three times. The tobacco leaves were cut to about 1 cm in diameter with sterilized razor blades and then soaked in Agrobacterium cells containing the construct for 10-30 minutes to induce tobacco leaf transformation. Tobacco leaves incubated in Agrobacterium cells were dried in sterile 3MM paper and 6-7 tobacco sections per plate were healed in MS-callus induction medium with leaf veins upward, followed by two days of cancer growth. plant growth chamber) was incubated for 12 hours at 27 ℃.

Kanamycin-resistant callus was divided into 100 mg / L kanamycin and 500 mg / L cefotaxime, and 0.2 mg / L α-naphthalene acetic acid (NAA) and 2 mg / L of 6-benzyl aminopurine (BA) was selected from Murashige and Skoog medium (Duchefa, MS including vitamins, 30% sucrose, 0.8% agar pH 5.6-5.8). Callus formed was as root induction medium after stem induction in a medium containing 100-200 mg / L kanamycin and 500 mg / L cefotaxime, 0.1 mg / L NAA and 1 mg / L BA (200 mg / L). L kanamycin, 0.1 mg / L NAA) was transferred. Individuals whose roots were grown were transferred to soil and grown in a plant growth chamber (27 ° C., 12hr / day), and RNA was extracted from each plant to confirm whether a desired gene was expressed.

5th step. Stress Treatment on Tobacco Transformants

In order to confirm the expression level of the reporter gene GUS in tobacco containing the promoter, a part of the leaves were taken from each tobacco, and artificial wounds and UV (304 nm, 20 min) were treated and GUS expression was confirmed. As a result, the most expressive transgenic subjects were selected, seeded, germinated in MS medium with kanamycin (200 mg / L), and seedlings were placed 15 cm from UV (304 nm). After treatment for 24 minutes, the mixture was left at room temperature for 24 hours or treated with 75 mM AlCl ₃ in 0.1% and 0.5% yeast extract solution for 24 hours.

Step 6. GUS Dyeing

 Tissues containing the promoter-GUS fusion gene of the stilbene synthase gene cultured in MS minimal media for two days were divided into two groups, and only one group was treated with UV for 20 minutes. UV treated and untreated tissues were treated in GUS solution (50 mM phosphate buffer (pH 7.4), 3 mM potassium ferricyanide, 3 mM potasium ferrocyanide) containing 1 mM X-glucuronic acid (X-gluc) for 24 hours at 37 ° C. Reacted. Dehydration was left in 50%, 70%, and 90% ethanol for 30 minutes each, and finally put in 70% ethanol to completely remove the pigment and confirmed GUS expression.

(result)

In Example 1, it was found that cis-element related to wound, pathogen, tobacco, drought, heat stress, low temperature, etc. existed in the longest promoter among four promoters of stilbene synthase gene. Therefore, in order to confirm the function of these elements and the possibility of using the new vector system as a promoter, the promoter-GUS fusion gene construct (FIG. 4) was first transformed into tobacco using Agrobacterium.

As a result, the tobacco transformants did not normally express GUS (or very poor), but it was confirmed to express GUS in response to external stress such as UV and wounds. In addition, the amount of expression showed a quantitative difference among the tobacco transformants. Therefore, the seeds with the best expression among the transformants secured were selected and the seeds were collected, seeded by seeding, and seedlings were seeded to control the expression of the promoter during seedling stages, especially ultraviolet light, AlCl ₃ , and yeast extract. The response pattern was analyzed.

The results are shown in FIG. Referring to FIG. 5, in the wild-type tobacco, the GUS gene was not expressed at all in any region. On the other hand, the transformants showed expression in the roots even when stress was not treated. However, treatment with UV, AlCl ₃ , and yeast extract induced GUS gene expression. When treated with UV, it was strongly expressed in roots and leaves, and best in 0.5% of yeast extracts. In the case of AlCl ₃ , expression was better induced at 75 mM than at 100 mM. This slightly different induction pattern suggests that different receptors or intracellular signal transduction factors recognize different stress treatments.

Grapes have been reported to induce the expression of stilbene synthase genes in leaves and fruits under stress, such as UV or fungal infections. From the present invention using a transformed tobacco, which is a model plant, it can be seen that the expression of stilbene synthase gene is induced in leaves and stems by environmental factors such as UV. These results suggest that analyzing the response of the transformant can explore various environmental factors that induce the synthesis of resveratrol. In addition, by separating the elements related to the response to the environmental stress from the promoter region of the gene and characterizing it, it shows that the expression of resveratrol synthetic gene can be significantly induced. Therefore, we tried to develop a system for identifying useful sites by performing deletion analysis of promoter sites and searching for environmental factors that induce resveratrol synthesis. As well as using the cis-element (cis-element) of the promoter to create a new vector system different from the existing CaMV-35S promoter.

[Example 3: Environment Factor Search through Deletion Analysis of the Promoter of the Invention]

First step. Creation of the deletion series

5 μg of the DNA cloned from the pCR2.1-TOPO vector (Invitrogen) with stilbene synthase gene promoter 1 was treated with Sac I and Nco I and then treated with Exo III and reacted at 37 ° C. at 30 sec intervals, followed by Cleno enzyme ( Klenow enzyme) and dNTP were added to ligation. Then, transformed into Top10 CP cells to obtain 24 constructs. Ten constructs were selected and transformed into Arabidopsis and treated with abiotic stresses and biological stresses.

Second step. Agrobacterium Transformation

Agrobacterium (Agrobacterium) of a single colony (Single colony) 3 ㎖ of _{YMB (sol A: K 2 HPO} 4 9.9 g / 100 ㎖, sol B: MgSO 4 .7H 2 O 20 g / 100 ㎖, sol C: NaCl 9.9 g / 100 ml sol D: 10 g / L mannaitol, 1 g / L yeast extract, mix 5 ml of sol A, 1 ml of sol B, 1 ml of sol C, and sol D per 1 liter). Inoculation was incubated overnight at 27 ° C. at 170 rpm. The shake culture was diluted 100-fold in 50 ml of YMB liquid medium, shake-cultured at 27 ° C. at 170 rpm for 2-3 hours, grown to OD 660 nm of about 0.5, and centrifuged at 3000 × g for 20 minutes. The cells obtained through centrifugation were washed with TE buffer, and then dissolved in a small amount of YMB liquid medium. 500 µl portions were stored at −70 ° C. and used as a competent cell.

Agrobacterium tumefaciens GA3101 was transformed by the freeze-thaw method. Competent cells were placed on ice, 1 μg of the corresponding construct DNA was added, left on ice for 5 minutes, and then reacted with liquid nitrogen for 5 minutes and 37 ° C. for 5 minutes. 1 ml of YMB liquid medium was mixed and shaken for 2-4 hours at 170 ° C. and 170 rpm, and then plated in MS solid medium to which kanamycin was added and incubated at 27 ° C. for 2 days to obtain transformed colonies. Transformed colonies were grown in YMB liquid medium and confirmed by colony PCR and transformed into Arabidopsis.

Third step. Arabidopsis Arabidopsis ) Transformation

Agarobacterium transformed with the construct was transformed into the Arabidopsis ecotype Col-0 grown at 23 ° C. for about 2 weeks using the flowdip method. Seeds were again selected from MS medium containing kanamycin and used for Gus assay.

Fourth step. Various stress treatments

The leaves of Arabidopsis (Col-0) grown for 4 weeks at 23 ° C were exposed to UV for 15 minutes, soaked in DW for 24 hours, and soaked in 0.1%, 0.5% yeast extract solution and 75 mM, 100 mM AlCl ₃ for 24 hours. Used for GUS analysis and fluorescence assay.

5th step. Histochemical and Fluorescence Analysis of GUS Activity

Histochemical analysis showed that 1 mM X-glucuronic acid was divided into UV-treated, yeast extract-treated, AlCl _3- treated and untreated tissues from young plants of Arabidopsis with stsy promoter-GUS grown in kanamycin-added MS medium for 7 days. The reaction was performed at 37 ° C. for 16 hours in a GUS solution containing (X-gluc) (50 mM phosphate buffer (pH 7.4), 3 mM potassium ferricyanide, 3 mM potasium ferrocyanide). Dehydration was left in 50%, 70%, and 90% ethanol for 30 minutes each, and finally put in 70% ethanol to completely remove the pigment and confirmed GUS expression. In addition, after growing Arabidopsis for 4 weeks, the leaves were collected and treated with UV and AlCl ₃ , yeast extract, MeJA, SA, BA and histochemical analysis was performed in the same manner.

Fluorometric assay immersed stressed Arabidopsis leaf in 150 μl of extraction buffer, crushed with a homogenizer, run in a centrifuge at 4 ° C for 10 minutes, and then centrifuged again. It was. Protein was quantified by the Bradford assay (Bio-Rad kit), and then mixed with 2 μg of protein and 100 mM X-gluc and reacted at 37 ° C. for 30 minutes, followed by a fluorometer (Bio-Rad). Gus activity was measured by.

(result)

Histochemical GUS Activity Assay

In Example 1, four different stilbene synthase gene expression regulation promoters, that is, 944bp, 732bp, 643bp, 413bp promoters were obtained. In the longest of these promoters 1, we used ExoIII to randomly delete 5 'to determine which region of the promoter responds to a particular stress, as well as the function of each putative cis-acting element, -801, -722, -601, -513, -406, -308, -258, -200, -138 and -75 promoters were obtained (FIG. 6). This was transformed into Arabidopsis by fusion with GUS, and the To plants transformed with the respective promoters were treated with UV 15 minutes, 0.1% and 0.5% yeast extract, 75 mM and 100 mM AlCl ₃ , and then subjected to histochemical GUS analysis.

The results are shown in FIG. Referring to Figure 7, it can be seen that the expression of promoter 1 is induced by all the above treatment even in Arabidopsis. When stress was not treated (control, a in FIG. 7), the expression at the root was hardly seen at the -406 promoter, 0.1%, 0.5 compared to the case of treatment with 75 mM, 100 mM AlCl ₃ (e, f in FIG. 7). When treated with% yeast extract (c, d in Fig. 7) the -513 ~ -308 promoter was a strong expression of both GUS in the roots and leaves. In addition, it was also observed that GUS was expressed strongly in the UV 15-minute treatment (FIG. 7B). Therefore, in the promoter 1 of the stilbene synthase gene, the -406 ~ -308 region is the portion that reacts to AlCl ₃ , the -308 ~ -258 region is the portion that is involved in the UV reaction, and the -308 ~ -200 region is the yeast extract. It was supposed to be a reaction area. However, it was difficult to identify the amount of induction by the intensity of the color.

Fluorescence GUS Activity Assay

Fluorescence GUS analysis showed that promoter 1 was induced in 15 minutes of UV, yeast extract, and AlCl ₃ treatment. Especially, -801 promoter was induced 5 times by UV and 3 times by 0.5% yeast extract. . However, AlCl ₃ treatment induced less promoter 1 than control. In the case of the -722 promoter, it was induced 15 times by UV and 8 times by yeast extract. As the promoter was deleted from -801 to -601, the stress-induced amount also tended to decrease, but AlCl ₃ was found to express greater than that of complete promoter 1 (-801) at the -513 and -406 promoters. Could.

The above results can be thought of as being related to the repeat structure in the promoter. In the absence of X1 when treating yeast extract (compared to -801 and -513 promoter), GUS activity decreased by 4 times, and 2 times without X2. Decreased. UV treatment, however, was about two times less in the absence of X1 and six times less in the absence of X2. Therefore, in the treatment of UV and yeast extract, the repeat structure was thought to play a role in regulating the amount of stilbene synthase gene promoter expression. However, in the case of treatment with AlCl ₃ , the repeat structure (X1) is considered to play a role as a suppressor (FIG. 8).

Comparison of promoter 1 and promoters 2, 3, 4

Comparing the three promoters obtained by PCR based DNA walking in addition to promoter 1, promoters 1 and 2 have similar nucleotide sequences of 99%, and when the transcription start site is -1, promoter 1 is -322 and promoter 2 is -323. , Promoter 3 has -363, promoter 4 has a common sequence of 4 promoters from -331 region. Promoter 1 has a repeating structure of X1, X2, and promoter 2 has X2, but not in promoters 3 and 4. And comparing the results obtained from the deletion series experiment of promoter 1 with the nucleotide sequences of promoters 2, 3, and 4, promoters -407 ~ -309 regions, promoters -421 ~ -349 regions, promoters 4 -338 ~ -317 region of the promoter 1 of the -309 ~ -201 region, the promoter of the promoter 2 3 same as the part that responds to the AlCl ₃ -349 ~ -222 region, -317 ~ -220 region of the promoter 4 The region and nucleotide sequence related to this yeast extract of promoter 1 are identical.

The stilbene synthase gene promoters 1, 2, 3, and 4 were fused with GUS to transform Arabidopsis and subjected to stress, followed by histochemical GUS analysis and fluorescence GUS analysis. The results are shown in FIG. In FIG. 9, promoter 1 and promoter 3 showed similar GUS activity. Promoter 3 is difficult to analyze in the results of the deletion series experiment of Promoter 1 because the 5 'base sequence is different from Promoter 1, but it suggests the possibility of using Promoter 3 in an induction vector system. Promoter 3 may be analyzed by gel mobility shift assay by comparison with transcription factor or cis-element.

Taken together, the deletion series of Promoter 1 found that the regions reacting differently to UV, yeast extract, and AlCl ₃ were different. In addition, UV or yeast extract was better than AlCl ₃ as an effective inducer in the normal promoter (-801). However, if AlCl _{3 was} also cut from -801 to -514 and from the -513 promoter, it could be seen that a lot of induction. Therefore, it is possible to construct a recombinant promoter by selecting the portion of which expression is best induced by each external environmental stress, and to construct a system that induces strong expression by inducing the expression of the recombinant promoter only when necessary effectively.

As is apparent from the above Examples and Experimental Examples, the vector system of the present invention using the promoter of stilbene synthase gene whose activity is increased by various environmental signals is more optimal than the conventional plant expression vector system. It provides an excellent effect that can be expressed, and suggests a method for mass production of resveratrol having anticancer and antimicrobial activity by enabling the search of an optimal environmental factor that highly enhances the resveratrol content in major grape varieties in Korea. In addition, it is a very useful invention because it can be used to produce an environmental stress-resistant transgenic plant.

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Claims (7)

An expression vector system specifically expressed in external environmental stress, comprising a promoter of a stilbene synthase gene derived from giant grapes having the nucleotide sequence of SEQ ID NO: 1. The expression vector system of claim 1, wherein the external environmental stress comprises any one or more of UV, yeast extract, and AlCl ₃ . An expression vector system specifically expressed in external environmental stress, comprising a promoter of a stilbene synthase gene derived from giant grapes represented by a nucleotide sequence having at least 90% similarity with the sequence of SEQ ID NO: 1. Expression vector system specifically expressed by UV and yeast extract, characterized in that it comprises a recombinant promoter is deleted from the base position of -801 to -723 in the base sequence of SEQ ID NO: 1. Expression vector system specifically expressed by AlCl ₃ characterized in that it comprises a recombinant promoter deleted from the base position of -801 to -514 in the base sequence of SEQ ID NO. An expression vector system comprising a recombinant promoter of a stilbene synthase gene comprising a nucleotide sequence at a position of -308 to -258, which is a UV-responsive site, in the nucleotide sequence of SEQ ID NO: 1. An expression vector system comprising a recombinant promoter of a stilbene synthase gene comprising a nucleotide sequence at a position of -308 to -200, which is a site that reacts to a yeast extract in the nucleotide sequence of SEQ ID NO: 1.

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