KR100452431B1 - Expression cassette and plasmid for strong constitutive gene expression and the use thereof - Google Patents

Abstract

The present invention provides an isolated nucleic acid construct comprising a potent structural promoter of a plant capable of binding to a heterologous gene encoding a desired polypeptide in a transgenic plant tissue. Such constructs, when used as constructs for heterologous coding sequences in chimeric genes, allow high expression of desirable polypeptides in plant root tissue and plant leaf tissue. A vector incorporating any chimeric gene into the construct can be introduced into plant tissue, thereby intentionally modifying the plant to produce foreign components in the plant.

Description

Expression cassette and plasmid for strong constitutive gene expression and the use}

The present invention relates to nucleic acid constructs comprising specific promoters of structural non-tissues for the high expression of desirable polypeptides in plant cells and their use, in particular that can be structurally expressed at high levels in transgenic plant root tissues and leaf tissues. It relates to a promoter with a gene. Gene expression by this construct is much more potent than the 35S promoter. The present invention is preferably an example of the use of the Arabidopsis TPS3 promoter to structurally regulate gene expression to produce converted plants.

In breeding, conventional breeding has a problem in that the genotype to be introduced is limited to relative breeding, and it takes a long time to obtain the intended hybrid. However, with recent developments in life sciences such as genetic engineering, preferred genes can be introduced directly into plants, and such systems are expected to overcome the problems of conventional breeding. In addition, the use of plant genetic engineering is very beneficial for producing plants with improved properties. In order to improve resistance to disease, insects and environmental stress in plants or to enhance the nutritional properties of transgenic plants, the preferred gene must be bound to a strong structural promoter that regulates gene expression and the gene is under the control of the promoter. Allow expression. In addition, the economic production of biologically active polypeptides is important in the manufacture of pharmaceutical and nutritional products and other specific chemicals in the biological cultivation of plants. Recombinant DNA techniques using recombinant plant cells as expression hosts generated from recombinant nucleic acid constructs comprising strong structural promoters are particularly useful as a means for producing large amounts of polypeptides.

Several promoters have been identified that are used to express heterologous genes in plants. The most widely used promoter is the 35S promoter of cauliflower mosaic virus (CaMV35S promoter). The 35S promoter is a strong structural promoter that causes gene expression in all tissues of transgenic plants. Other structural promoters identified include promoters derived from the mannopine synthetic gene of agrobacterium tumefaciens T-DNA and nopaline. In particular, in order to produce recombinant polypeptides for commercial use, the expression of the desired polypeptide in the host cell must be high. Therefore, there is a need for expression control signals that enable high levels of strong structural expression of desirable polypeptides in plant tissues that maintain expression properties. The use of such structural promoters enables the genetic engineering of commercially important crops. The present invention has completed this.

The complete sequence of cDNA of the Arabidopsis TPS3 gene has been reported to consist of 2,583 bases, and their amino acid sequences comprising 861 residues are also deduced (see Genebank AC004473). However, with respect to the promoter site that regulates the transcription of the TPS3 gene, reports and / or inventions have identified the Arabidopsis TPS3 promoter that maintains expression characteristics in the root and leaf tissues of the transgenic plant and causes very strong and structural gene expression of the desired gene. Has not been to date.

Therefore, according to the present invention, preferred foreign genes and terminators can be linked to the isolated Arabidopsis TPS3 promoter site and introduced into the plant, so that the preferred gene can be expressed in the transgenic plant tissue. Therefore, the promoter region can express a desired gene for biological or abiotic stress-related transcription and plant improvement and can also be used to produce biologically active ingredients in plant tissue.

1 is a schematic of plasmid (a) pLES99010 comprising a 1.0 kb Arabidopsis TPS3 promoter (SEQ ID NO: 1) for E. coli modifications (a) pLES99010 and plasmid (b) pLES99011 comprising a 2.0 kb Arabidopsis TPS3 promoter (SEQ ID NO: 2) Explanation is shown.

FIG. 2 shows a schematic illustration of plasmids (a) pLES99014 comprising a 1.0 kb Arabidopsis TPS3 promoter (SEQ ID NO: 1) for plant modification and (b) pLES99015 containing a 2.0 kb Arabidopsis TPS3 promoter (SEQ ID NO: 2). It is shown.

Figure 3 shows expression of GUS reporter gene activity by the Arabidopsis TPS3 promoter or CaMV 35S promoter. Developing seedlings grown for 5, 10 and 21 days in vertically positioned media were assayed in X-Gluc solution for 1 to 3 hours.

Figure 4 shows the quantitative activity of the GUS reporter gene by the Arabidopsis TPS3 promoter or CaMV 35S promoter. Developing seedlings grown for 5, 7, 10 and 14 days in vertically positioned media were assayed in 4-MUG solution.

The present invention provides an isolated nucleic acid construct comprising a plant promoter capable of binding to a heterologous gene for expression at the highest levels of the plant's root and leaf tissues. The plant promoter is an arabidopsis gene encoding trehalose-6-phosphate synthase that is involved in trehalose metabolism and contains a stronger structural promoter than the CaMV 35S promoter. Also provided by the present invention is an expression vector comprising an arabidopsis TPS3 promoter capable of binding to a heterologous gene encoding a preferred polypeptide.

The expression vector may further comprise other components, such as a selection marker. The construct may also include the origin of a replication sequence that functions in a plant or other host cell. As a preferred structure of the invention, the plasmid pLES 99010 was deposited in the Korean Collection for Type Cultures (KCTC) located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea (305-333) under the Budapest Treaty. Deposited 28 days. Plasmid pLES 99011 was deposited with the Korean Type Culture Collection (KCTC) under accession number KCTC 0812BP under the Budapest Treaty. Plasmid pLES 99014 was deposited with the Korean Type Culture Collection (KCTC) under accession number KCTC 0813BP under the Budapest Treaty. Plasmid pLES 99015 was deposited with the Korean Type Culture Collection (KCTC) under accession number KCTC 0814BP under the Budapest Treaty.

The present invention also provides a plant cell comprising an expression cassette comprising the Arabidopsis TPS3 promoter capable of binding to a heterologous gene. The expression cassette may be integrated into the genome of the host cell or may be present independently in a replicating plasmid. The expression cassette can be used to structurally regulate gene expression, which is capable of genetic manipulation of plants and which is desirable for plants of commercial importance. Preferred plant cells are dicot and monocot species.

The present invention has conducted extensive research to find a promoter capable of functioning in plant tissues, and completed the present invention in which the desired gene can be expressed very strongly structurally.

The present invention provides expression cassettes and vectors useful for structurally expressing high levels of desirable polypeptides in plants. The promoters and vectors of the present invention are particularly suitable for protein expression in plant hosts, including soybeans and rice, for pathogen resistance, environmental stress tolerance, or plant biofarming to obtain high levels of recombinant protein expression. The promoter provides higher levels of expression than that provided by the CaMV 35S promoter.

General experimental procedures required in this application are described in Sambrook et al., Molecular cloning; A Laboratory Manual (2 ^nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein are used to perform and test the present invention, more preferred methods and materials are described.

The term “nucleic acid”, unless otherwise limited, refers to deoxyribonucleotides or ribonucleotides in single- or double-helix form and includes known analogs of the original nucleotides that bind the nucleic acid in a manner similar to naturally occurring nucleotides. . Unless specifically stated, certain nucleic acid sequences include their complementary sequences.

The term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (such as a promoter, signal sequence or transcription element binding site) and a second nucleic acid sequence, wherein the expression control sequence corresponds to the second nucleic acid sequence. Affects transcription and / or translation of the nucleic acid.

The term "recombinant" as used in connection with a cell refers to the cell replicating the heterologous nucleic acid or expressing a peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can also express genes found in the cell's original form, but modified genes can be reintroduced into cells by artificial methods.

As used herein, a "heterologous gene" is a gene derived from a foreign species or from the same species and modified from its original form. Thus, heterologous genes that can bind to a promoter are from a different source from the form from which the promoter is derived, and a promoter modified from the original form if it is from the same source. For example, the trohalose-6-phosphate synthase gene promoter can bind to a structural gene encoding a polypeptide different from the original trehalose-6-phosphate synthase. Modifications of heterologous sequences can occur, for example, by treating DNA with restriction enzymes that produce DNA fragments that may be bindable to a promoter. Site-directed mutagenesis is also useful for modifying heterologous sequences.

Here, the promoter means a promoter capable of controlling the expression of the protein in plant cells when the gene of the desired protein is fused downstream of the promoter. In addition, the promoter of the present invention can be further modified by binding to other transcription-translational active sequences.

An "expression cassette" is a generated or synthetic nucleic acid construct having nucleic acid elements that can affect the expression of a structural gene in a host suitable for such sequence. The expression cassette optionally comprises at least promoters a transcription termination signal. In general, expression cassettes include a nucleic acid to be transcribed and a promoter (eg, the Arabidopsis TPS3 promoter). Further factors affecting expression can also be used as described herein. For example, the expression cassette may also include a nucleotide sequence that encodes a signal sequence that directs the secretion of the expressed protein from the host cell. To select the modified cells that make up the construct, a selection marker gene can be conveniently included in the expression cassette. The person skilled in the art can see that this vector component can be modified without a substantial impact on its function.

The present invention includes expression of genes in nucleic acid constructs and modified plant cells. Molecular cloning techniques for this purpose are known in the art. Various cloning and in vitro propagation methods suitable for recombinant nucleic acid constructs such as expression vectors are well known to those skilled in the art. Examples of techniques and guidance known to people with skills through many cloning practices are based on Jose M. Martinez-Zapater and Julio Salinas, Arabidopsis Protocol, Human Press.

A first aspect of the invention relates to a nucleic acid construct comprising the nucleotide sequence of a promoter (SEQ ID NO: 1) from the Arabidopsis trehalose-6-phosphate synthase (AtTPS3) gene. Identification of promoters with desirable properties has been accomplished in several ways. For example, genomic or cDNA libraries can be prepared from materials of known TPS3 genes such as Arabidopsis, Myrothamnus flabellifolius, and yeast. Libraries from certain species are screened with oligonucleotide probes designed to contain sequences complementary to coding regions of other known genes. Alternatively, oligonucleotide probes can be designed based on amino acid sequence information obtained from purified TPS3 protein. Oligonucleotide probes based on amino acid sequences can be modified or biased for selective codons of material plants. Oligonucleotide primers can be designed for use in reverse transcription-polymerase chain reaction (RT-PCR) to amplify PCR fragments for cloning and sequencing. The sequence of the PCR fragments can be compared with known coding regions of the desired genes to identify corresponding clones. For example, cDNA of pathogen response genes is obtained by RT-PCR using total RNA isolated using pathogen infected plant tissue and gene specific primers.

The cDNA is then used as a bait to isolate promoter sites using genome restriction libraries made from plant tissues in a continuous PCR reaction with primers located inside. PCR amplification products are cloned and sequenced. The promoter region may be original, homologous to the host or foreign, or heterologous to the host that was intended to be used. The term "foreign" means that the transcription start region is not always found in the plant host into which it is introduced. Arabidopsis trehalose-6-phosphate synthase 1 gene (Blazquez et al., 1998. The Plant Journal 13 (5): 685-689) or Saccharomyces cerevisiae trehalose-6-phosphate synthase 2 gene (De Virgiolio et al., 1993 Eur. J. Biochem. 212: 315-323) is a specific example of a gene from which the promoter used in the present invention can be obtained. The construct may comprise a base sequence derived from SEQ ID NO: 1 having a deleted, inserted or substituted base so long as the resulting sequence effectively maintains expression control activity with the promoter. In addition, such sequences with deleted, inserted or substituted bases essentially have the same function as the base sequence as long as they effectively maintain the promoter activity.

The nucleotide sequence of the most preferred promoter is shown in SEQ ID NO: 1. As shown, the promoter is inserted at the XbaI site of the pLES99014 expression vector. The TATA box sequence of the promoter is at nucleotides 918-923. The SmaI site is located just 3 'of the pLES99014 sequence of the XbaI site to facilitate insertion of the gene for expression downstream of the promoter.

The nucleotide sequence of a preferred promoter fragment can be synthesized by well known techniques as described in the technical literature (Carruthers et al., 1982. Cold Spring Harbor Symp. Quant. Biol. 47: 411-418). Complementary strands can be synthesized and the strands annealed together under appropriate conditions to obtain double stranded DNA fragments.

A second aspect of the invention relates to a vector that binds to an expression cassette used as an expression cassette or expression vector. Plasmids comprising the promoter of the invention are assembled to have restriction enzyme sites for deleting or inserting the desired gene downstream of the promoter of the invention. Preferred genes may be heterologous to the promoter. In addition, plasmids are prepared by binding the desired structural genes to these promoters for the expression of the desired proteins in plant roots and leaf tissues, and also automatically include chimeric genes that are cloned, inherited and the like. The promoters of the present invention can express desired proteins in plant roots and leaf tissues, and the GUS reporter genes of the present invention are desirable for resistance to biological / abiotic stresses or to enhance the production of biologically active ingredients in transgenic plants. Can be deleted and replaced by genes.

The nucleotide sequence of interest can be inserted downstream from the promoter under the promoter control of the invention. The nucleotide sequence of interest allows altering the production of endogenous products or functionally encoding novel gene products to modify phenotypic properties. The nucleotide sequence may have a sequence that is complementary to any open reading frame or genomic sequence that encodes an enzyme, and the genomic sequence may be any other sequence where the open reading frame or complementary sequence inhibits transcription, messenger RNA processing. The nucleotide sequence of interest can be a synthesis of the original origin or a combination thereof. Depending on the nature of the nucleotide sequence of interest, it would be desirable to synthesize a sequence with plant selective codons.

The vectors also include a selection marker gene for selecting host cells with the desired construct. Such genes encode proteins necessary for the survival or growth of modified host cells grown in selective culture medium. Host cells that are not modified with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that are resistant to antibiotics or other toxins such as kanamycin and hygromycin. Selection marker systems are well known to those skilled in the art and are described in Sambrook et al. The preparation of the preferred vectors comprising one or more of the above mentioned components uses standard methods according to those described in the references mentioned above.

The vector of the invention can be used in many plant host cells. Examples of plants include beans, tomatoes, potatoes, tobacco, rice, corn, wheat, barley, strawberries and rapeseed. These examples are illustrative rather than limiting. Depending on the host cell used, modifications can be made using standard methods suitable for such cells.

The promoters of the invention are used to express any desired protein in plant host cells at very high levels. The protein may be homologous to the plant host cell or is preferably heterologous to the host cell. For example, the promoter can be used to express mammalian, fungal and plant proteins at very high levels. Typical proteins that can be expressed using such promoters include trehalose-6-phosphate synthase, ABA-responsive element binding factor (ABF), pathogen-responsive gene (R gene), carbohydrate metabolizing enzyme, carotene synthetase, flavonoid synthase , Growth hormones, insulin, interleukin, colony stimulating factor and the like. The enzymes mentioned above are not exclusive but are examples used to obtain transcription of any nucleic acid expression capable of binding to the promoter of the invention. In host plant cells into which the promoter has been introduced, plant roots and leaf tissues are preferred in that they significantly increase the expression of foreign genes. Therefore, transduction of an expression vector comprising said promoter into plant cells with reproducibility may engage in grains such as genie rice with commercially and agriculturally desirable properties of biological / abiotic resistance and / or high plant productivity. Provide an effective method for the technician.

The constructs of the present invention provide a transgenic plant that is structurally resistant to infection and stress or that enhances the production of the active ingredient. In particular, plant cells modified with the constructs of the present invention can be regenerated by conventional plant tissue culture techniques (Jose M. Martinez-Zapater and Julio Salinas, Arabidopsis protocol , Human press, 1998). Expression of the DNA sequence of interest is important because it is expressed structurally, particularly at stress sites such as root and leaf tissue. The construct provides control of expression of the endogenous product as well as the production of the exogenous product in the plant. The DNA construct is introduced into a plant cell host for binding to and transcription of the genome. In this method, moderately high expression of RNA and polypeptides can be achieved over many years. Confirmation of very strong and structural expression of a preferred gene can be obtained in modified plants or plant tissues with the invention.

Trehalose-6-phosphate synthetase 3 (TPS3) was synthesized from glucose-6-phosphate and uridine-diphosphoglucose of α-D-glucopyranosyl α-D-glucopyranoside (non-reducing disaccharide prehalose). Promote synthesis The lack of reducing ends interferes with heat, pH and Maillard's reactions (reactions between carbohydrates and amino acids that discolor during potato processing). In addition, trehalose forms a glassy structure after dehydration and has a strong stability effect in biological structure. Because of this property, there is an increasing interest in trehalose as an additive to means of treating environmental stress resistance in crops and to stabilizers and to cosmetics and pharmaceuticals for preserving dry or frozen food.

The complete cDNA sequence of the TPS1 gene derived from Arabidopsis has been reported (Blazquez et al., The Plant Journal (1998) 13: 685-689). However, for promoter sites that function to regulate the transcription of the TPS3 gene, there have been no reports of promoter gene isolation and their use. With this knowledge, the present invention can be used to express foreign genes in agriculturally important plants. The constructs of the present invention are activated and remain active within 15 minutes after expression. Therefore, the rapid gene expression induction and intensity provided by the constructs of the present invention cause the effect of mechanisms that cause or greatly increase plant resistance responses to pathogens and defend against abiotic stress (eg, environmental stress response enzymes). It is particularly advantageous, as it is desirable to increase or to produce biologically active ingredients (eg, enzymes that produce biologically active substances). In this method, the DNA sequence of interest is structurally expressed in transgenic plant roots and leaf tissues, and the specificity can avoid the possibility of having a deleterious effect on the plant in which the structural expression of such sequence occurs.

Preferred effects on plant resistance can be achieved, for example, by expressing resistance genes to fungi or nematodes. Examples of genes that can be expressed for enhanced disease resistance under the controlled inhibition of the constructs of the invention are described in Hammond-Kosack, KEand Jones, JGD ("Plant disease resistance genes", Annu. Rev. Plant Physiol. (1997) 48: 575-607).

In addition, the ability to obtain high levels of transgene expression is of considerable importance in "molecular farming," where plants produce industrial or pharmaceutical polypeptides and other biopolymers that are foreign to the plant. Used. The promoters of the present invention are also expected to benefit from use in well known genetic constructs for developing rapid and cellular level screens that are sensitive to environmental and agricultural monitoring.

With attempts to isolate the promoter region of the TPS gene that is actively expressed in plant roots and leaf tissues, it can be used to improve plant disease resistance, to improve environmental stress tolerance or to produce biologically active ingredients in plant root and leaf tissues. The present inventors studied to achieve this problem, and as a result, the present invention was completed.

The following examples illustrate the preparation of expression cassettes, in particular DNA sequences comprising regulatory sequences of potent and structural gene expression in plant root and leaf tissues than the CaMV35S promoter. Introduction of the sequence into the plasmid is also described in that modification of the plant cell is possible. In addition, the regeneration of transgenic plants and the functional studies of expression cassettes in transgenic plants are shown. Those skilled in the art will recognize that substitutions and changes are possible in the components, conditions and procedures presented herein without departing from the scope and intent of the protocol.

(Example)

When explaining the embodiments of the present invention in detail as follows. However, the present invention is not limited to the embodiment.

Example 1

Promoter Separation

1) Isolation of Total RNA from Arabidopsis thaliana

10 g of leaves of Arabidopsis thaliana , a month old, are cooled with liquid nitrogen and powdered using a grinder. The powder was transferred to a cold Corex tube to which 10 ml of cold ice extraction buffer (pH 7.0) containing 20 mM Tris, 0.5 mM EDTA, 0.1 M LiCl and 1% SDS was added and heated at 65 ° C. for 10 minutes. The suspension was centrifuged at 15,000 rpm for 20 minutes at 20 ° C. The upper part was collected. Total RNA was precipitated by addition of 0.5 volume of phenol and chloroform followed by treatment with 2M LiCl. After dissolving with water, total RNA was precipitated by adding 2 volumes of ethanol at -20 ° C and centrifuging at 42,000 ° C for 10 minutes. All glassware and solutions were previously treated with 0.1% diethylpyrocarbonate and sterilized.

2) Isolation of mRNA from Arabidopsis thaliana

Poly-A ⁺ RNA was isolated and purified with Oligotex ^™ mRNA midi kit (QIAGEN, Germany) according to the manufacturer's instructions. After total RNA was heated at 65 ° C. for 5 minutes, total RNA was loaded onto an Oligotex ^™ column equilibrated with 2 × binding buffer. Oligotex ^™ columns were washed twice with wash buffer and treated with 20 μl of elution buffer. The obtained Poly-A ⁺ RNA was precipitated by centrifugation at 12,000 rpm and 4 ° C for 10 minutes by adding 2.5 volumes of ethanol at -20 ° C. Precipitated Poly-A ⁺ RNA was washed with 70% ethanol and dissolved in 20 μl of TE buffer containing 10 mM Tris-Cl, 1 mM EDTA at pH 8.0. The concentration of Poly-A ⁺ RNA was determined spectroscopically at 260 nm. All procedures were performed wearing surgical rubber gloves to minimize nuclease contamination in mRNA preparation.

3) Synthesis and Cloning of cDNA from Arabidopsis thaliana

CDNA libraries from Arabidopsis thaliana were prepared with the Uni-ZAP ^™ cDNA Library Kit (Stratagene, USA) according to the manufacturer's manual. First-strand cDNA was synthesized from the reaction mixture of 5 μg poly-A ⁺ RNA, oligo (dT) _12-18 , murine reverse transcriptase, dNTP, BSA and DTT. To synthesize double-stranded cDNA, the first-strand synthesized blunted the cDNA ends at 16 ° C. for 3 hours, including E. coli RNase H, E. coli DNA polymerase I and dNTP, and 10 minutes Treated with dNTP mixture with Pfu DNA polymerase at 65 ° C.

4) Binding to Uni-ZAP ^TM ZAP Vectors of cDNA

The ends of the synthesized cDNAs are coupled with EcoRI adapters to insert cDNAs into the vector. The synthesized cDNA was reacted overnight at 12 ° C. by adding EcoRI adapter, ATP and T4 DNA ligase, and further reacted at 37 ° C. for 30 minutes by adding T4 DNA kinase and ATP for binding of EcoRI adapter. cDNA was purified by Sephacryl S-500 spin column and its signal was confirmed by 1.0% agarose gel electrophoresis. The 1kb of cDNA portion was used for further experiments. Insertion of cDNA was obtained by reacting a mixture containing 200 ng of cDNA, 1 μg of vector DNA (Stratagene), and T4 DNA ligase at 4 ° C. for 48 hours.

5) Preparation and Amplification of cDNA Library

Uni-ZAP ^™ vector DNA was packaged using Gigapack II packaging extracts containing phage ghost (Stratagene). The packaging extract was reacted by adding the recombinant vector at 22 ° C. for 2 hours. The reaction solution was adjusted to a final volume of 500 μl with buffer supplemented with 10 μl chloroform and stored at 4 ° C. until ready or used. Total plaque-forming units (pfu) of the recombinant cDNA library were obtained from 10 ⁻² to 10 ⁻⁶ fold-diluted solutions. 200 μl of E. coli strain XL1-Blue MRF ′ was incubated with cDNA library for 15 minutes at 37 ° C. and applied at 10 ⁶ pfu on a 150 mm diameter plate. Plates supplemented with 5 ml of SM buffer were incubated at 4 ° C. overnight after reacting the plates at 37 ° C. for 12 hours. Supernatants were obtained from the reacted SM buffer by centrifugation at 12,000 rpm for 10 minutes and stored in 100 μl chloroform at −4 ° C. Plaque-forming units were calculated as described above.

6) Polymerase Chain Reaction for Promoter Cloning

Large amounts of total phagemid DNA production were also made by PCR amplification to isolate partial cDNA fragments for further library screening. Phage stocks containing cDNA libraries were transferred from E. coli XL1-B MRF ′ cells to XL1B SOLR cells via ExAssist helpers in the form of plasmids. Using this phagemid prep as a template, 5 μl of 10 × Taq buffer, ₂ μl of 25 mM MgCl _2, 2 μl of 10 pmol each primer, 4 μl of 2 mM dNTPs, cDNA using a combination of vector primer and two degenerate primers PCR was performed at 95 ° C./30 sec, 50 ° C./30 sec, 72 ° C./30 sec 30 cycles with a cocktail mixture comprising 0.25 μl pool and 33.75 μl water.

7) Selection of cDNA Library

As host cells for phage attachment, E. coli XL1-Blue MRF ′ was incubated in 50 ml of LB medium with 20% maltose, 0.5 ml of 1 M MgSO ₄ and 50 μg / ml tetracycline. The cell culture was suspended with ₁₀ mM MgSO ₄ to make OD 0.5 at A ₆₀₀ . After incubating E. coli XL1-Blue MRF 'containing the cDNA library for 37 ° C. for 30 minutes, the culture solution containing 0.7% LB agar was further cultured on 1.5% LB agar plate for 37 ° C. for 12 hours. 5 x 10 ⁴ cells were used in the first selection and 1 x 10 ³ cells were used in the second selection. Phage plaque formed plates were maintained at 4 ° C. for 1 hour and replicated with Hybond-C membrane (Amersham, USA).

8) PCR Cloning of 1.0Kb TPS3 Gene Promoter

To clone the TPS3 promoter, a set of gene specific primers was designed based on the Arabidopsis thaliana BAC T13D8 genomic sequence, including the trehalose-6-phosphate synthetic parent animal, T13D8.4. Approximately 1.0 Kb of the TPS3 promoter was derived from genomic DNA using T13P3 (5'-TCCAAATGATTTTGACCCCAT-3 ') [SEQ ID NO: 3] and T13P5-2 (5'-GTGTTCATTTGATAGA GTCTA-3') [SEQ ID NO: 4] primers. Amplified by polymerase chain reaction (PCR). The PCR reaction mixture contained 500 ng of genomic DNA, 5 μl of 10 × Taq polymerase buffer (pH 8.0), 1 mM MgCl ₂ , 200 mM dNTPs, 20 pmole of each primer and 5U Taq polymerase. The reaction was started without the enzyme and amplified according to the following conditions: 1 cycle of 95 ° C. for 5 minutes with post-denaturation; 3 cycles of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 1 minute; 1 cycle of 72 ° C. for 10 minutes with extension in the Perkin-Elmer 9800 amplifier. The main product amplified from the PCR reaction was gel-purified using the QIAquick gel extraction kit (Qiagen, USA) and subsequently cloned into a pGEM-T easy vector (Promega, USA) and plasmid according to the manufacturer's instructions Construct pLES99010 (FIG. 1A) was generated and sequenced. Clones comprising the 1 kbp upstream sequence (SEQ ID NO: 1) were designated as the 1 kb TPS3 promoter.

9) PCR Cloning of 2.0Kb Gene Promoter

Approximately 2.0 Kb of the TPS3 promoter was polymerized from genomic DNA using T13P3 (5'-TCCAAATGATTTTGACCCCAT-3 ') [SEQ ID NO: 3] and T13P5-1 (5'-CGACGGCATTAACATAAACC-3') [SEQ ID NO: 5] primers. Amplified by enzyme chain reaction (PCR). PCR reactions were carried out using such buffers and amplification conditions. The main product amplified from the PCR reaction was cloned into pGEM-T easy vector (Promega, USA) and sequenced by generating plasmid construct pLES99011 (FIG. 1B) according to the manufacturer's instructions. Clones comprising the 2 kbp upstream sequence (SEQ ID NO: 2) were designated as the 2 kb TPS3 promoter.

Example 2

Preparation of Expression Vectors

A) Preparation of TPS3 Promoter-GUS Reporter Gene Construct and Expression Vector

The DNA fusion construct of a promoter with a reporter gene encoding β-glucuronidase (GUS) was constructed as follows. For convenient cloning into the binary vector pBI101, plasmids pLES99010 and pLES99011 were digested with EcoRI and subcloned into the EcoRI site of pBluescript KS. These clones were named pLES99012 and pLES99013, respectively. pLES99012 was digested with XbaI and EcoRV to form a 1.0Kb TPS3 promoter, subsequently subcloned into the XbaI / SmaI site of pBI1101.2, resulting in a pLES99014 expression vector (FIG. 2A). For the pLES99015 expression vector construct (FIG. 2B), pLES99013 was digested with HindIII and SmaI and subcloned into the HindIII / SmaI site of pBI101.2. The accuracy of all constructs was verified by sequencing. Vectors pLES99014 and pLES99015 containing the TPS3 promoter / GUS gene fusion are Agrobacterium tumefaciens bacteria GV3101, respectively, Jose M. Martinez-Zapater and Julio Salinas, Aribidopsis protocol (Humana press, 1998) Was introduced by.

Example 3

Generation of Transgenic Plants and Evaluation of TPS3 Promoter Function in Transgenic Plants

A) Transformation of Arabidopsis plants

The vectors pLES99014 and pLES99015 were selected from the group of Arabidopsis thaliana (L.) Heynh., Eco Col. according to Jose M. Martinez-Zapater and Julio Salinas, Agrobacterium-mediated vacuum infiltration according to the Aribidopsis protocol (Humana press, 1998). Is introduced as -0. To select kanamycin resistance-positive genetic variants, T ₁ seeds were sterilized with 70% ethanol and 50% household bleach containing Tween 20 and then washed four times with sterile distilled water. Seedlings are grown in a vertical or horizontal direction on Murashige and Skoog (MS) medium agar plates containing 1% sucrose and 50 mg / ml kanamycin. The plants were grown at 22-24 ° C. for 16 hours of light cycle. Transformants with a single gene inserted were demonstrated by RT-PCR method using T ₂ seedlings, and their homozygotes were assayed for GUS activity.

B) GUS Analysis

Positive transformant seedlings grown on flat plates to measure TPS3 function were stained with 5-bromo-4-chloro-3-indole-β-D-glucuronide (X-Gluc) 1 An hour or 16 hour essay. Transformants seedlings or seedlings were GUS reaction buffer (2 mM 5-bromo-4-chloro-3-indole-β-D glucuronide (X-Gluc), 1% dimethylformamide, 0.1 mM potassium ferricyanide , 0.1 mM Potassium ferrocyanide, 1 mM EDTA, 50 mM sodium phosphate buffer, pH 7.0) and soaked by simple vacuum. Tissues were assayed at 37 ° C. for 1-3 hours. After the assay, seedlings or seedlings were washed for 1 hour with 70% ethanol and washed for at least 48 hours with 100% ethanol to make it more visible (Figure 3).

Five kanamycin resistant Arabidopsis lines were analyzed for induction of GUS activity. Three lines showed strong induction of GUS expression in culture after 15 minutes and two lines showed mild induction. The intensity of induction was determined by visual observation of the relative degree of color after GUS staining.

references

A Laboratory Manual (2nd Ed.), Edited by J. Sambrook and D.W. Russel, Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;

Arabidopsis Protocols, edited by Jose M. Martinez-Zapater and Julio Salinas, Humana press, 1998;

Blazquez et al., 1998. The Plant Journal 13 (5): 685-689;

De Virgilio et al., 1993. Eur. J. Biochem. 21: 315-323;

Carruthers et al., 1982. Cold spring Harbor Symp. Quant. Biol. 47: 411-418;

Hammond-Kosack, K.E. and Jones, J.G.D. Annu. Rev. Plant Physiol. 1997, 48: 575-607;

The effect of the present invention is to provide an isolated nucleic acid construct comprising a potent structural promoter of a plant capable of binding to a heterologous gene encoding a desired polypeptide in a transgenic plant tissue, wherein the construct is a heterologous coding sequence in a chimeric gene. When used as a construct for, high expression of the desired polypeptides in plant root tissue and plant leaf tissue can be achieved. After any chimeric gene can bind to the construct, the resulting vector can be introduced into plant tissue, thereby intentionally transforming the plant and causing the resulting plant to produce foreign components therein.

<110> Korea Kumho Petrochemical Co., Ltd. <120> Expression cassette and plasmid for strong constitutive gene expression and the use <130> KP-556 <160> 5 <170> Kopatent In 1.71 <210> 1 <211> 1013 <212> DNA <213> Arabidopsis thaliana <400 > 1 gtgttcattt gatagagtct aaaatcacca cttacattga ctaaccccaa aaatttgtcc 60 taaacactaa tcaatccgaa gattataaaa tgtcgacaaa gaaaataaaa taaaatagga 120 gtctatgagt cgatggaact tatccaacta aactttaata agaatgaacc aaactcaatt 180 ttgtaataat atttaaaata cgtttttact ctctcccaga atatcccatc tctctgattc 240 caattcctcc tctctctctt tcgtctctct acttcgaatt gattgtttta accgccggtg 300 atcggagcaa actctctgac ggtgaccgcc ggtgatcatt ttttgtgaac ttcacttttg 360 gtaagcagct ttgacttttg ttcataatcc ttcacttttc cttttcaatt ttgctttttc 420 ctctgatctg atcttagtta ttaagctgtt gaatcgcatg atcctttctt ctctgattga 480 ttatttctaa tccttcatgg gttttatgac gaatcgtgtc ttagggattg gttttttctg 540 ggttgggttt ctaaatttgt tggtctaaga acagtgaaat tgttgtttca 600 attctacact tctagtgatt gaactgttat ggaaaattac aaaatatgtg gctctgcttt 660 catttaaatt caatcatcat catctctgct acttgatgat caggtcacct tctctgtgtt 720 ttgtaatctc cataacgtaa gagtttgttt gatattgcaa ttttgaataa gatgattagc 780 atccaatcca atagctgatg ttgaaaccta actctgtaaa gactttaaat ttgttttctc 840 tggctttgtg ctcgttgaag ggtaacctaa agatttgacc tttttgatcc aacacctctc 900 tttttgtact gtaggtgtat aaagcatagt tcgttgggaa ctttttttta cagctttgga 960 gtaactacct ttcatggttt ttggttccaa caatggggtc aaaatcattt gga 1013 <210> 2 <211> 1943 <212> DNA <213> Arabidopsis thaliana <400> 2 cgacggcatt aacataaacc ataacgctag aaatcttaaa gtattctaat agcctaattt 60 ggtttttatt agaattaaaa ccctacataa tcctggatgt gcgtaacgta gtagttgagc 120 tctattctta tacaccagta aagtaaataa ataaaaagaa cagaaaattt catgagcatg 180 cagtagaaaa atcagtcgaa tgttataaaa atagggttac agctaagttc gacgaggtgc 240 tatttcattg gatatcacga aacttgatcg tcaatatgtt ttctttttgt cttttttg tt 300 tttgggtcaa cagatgtata aatgtatttg tatatagctt acatgaagaa cacttggtta 360 gctaaaaaga aaagctaaaa gaaacaaaag aggaggcagc taaaaaacga aaaaagagcc 420 agcattatag atgagcctct tcgtgagcaa gtgagtcgtt tattgtaaga tttgcctctt 480 ctaatttttt tctttaaagc cttataattg tatttaaaca aatatcttca taagaaaaaa 540 tatcaaatag atatcaatgt acttttcttt gaaataggat ttcgctatta atccttattt 600 taacattcat ataactgacc aattatattg gtatacagaa aaaaagaatt gtatttgttt 660 ttttcacatt ttagtaaatc tcattttttt ctttctaaat tatgattaat atgaaaatta 720 aaatagaaag tgacatgtcc ttattttaac tccttttatt tcattcattg gtttaatgtt 780 atttacctaa ggatactaat cattggtttt tgaaagataa caataaaaat tcattccaaa 840 catgtggtgc aattatatat aattacaaag tggatagagt atagaactac aaaatggata 900 gagtttagtt ataacaacta atttaaaaaa gtgttcattt gatagagtct aaaatcacca 960 cttacattga ctaaccccaa aaatttgtcc taaacactaa tcaatccgaa gattataaaa 1020 tgtcgacaaa gaaaataaaa taaaatagga gtctatgagt cgatggaact tatccaa cta 1080 aactttaata agaatgaacc aaactcaatt ttgtaataat atttaaaata cgtttttact 1140 ctctcccaga atatcccatc tctctgattc caattcctcc tctctctctt tcgtctctct 1200 acttcgaatt gattgtttta accgccggtg atcggagcaa actctctgac ggtgaccgcc 1260 ggtgatcatt ttttgtgaac ttcacttttg gtaagcagct ttgacttttg ttcataatcc 1320 ttcacttttc cttttcaatt ttgctttttc ctctgatctg atcttagtta ttaagctgtt 1380 gaatcgcatg atcctttctt ctctgattga ttatttctaa tccttcatgg gttttatgac 1440 gaatcgtgtc ttagggattg gttttttctg ggttgggttt ctaaatttgt tggtctaaga 1500 acagtgaaat tgttgtttca gtgatctcag attctacact tctagtgatt gaactgttat 1560 ggaaaattac aaaatatgtg gctctgcttt catttaaatt caatcatcat catctctgct 1620 acttgatgat caggtcacct tctctgtgtt ttgtaatctc cataacgtaa gagtttgttt 1680 gatattgcaa ttttgaataa gatgattagc atccaatcca atagctgatg ttgaaaccta 1740 actctgtaaa gactttaaat ttgttttctc tggctttgtg ctcgttgaag ggtaacctaa 1800 agatttgacc tttttgatcc aacacctctc tttttgtact gtaggtgtat aaagca tagt 1860 tcgttgggaa ctttttttta cagctttgga gtaactacct ttcatggttt ttggttccaa 1920 caatggggtc aaaatcattt gga 1943 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 3 tccaaatgat tttgacccca t 21 <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 gtgttcattt gatagagtct a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> Artificial sequence <400> 5 cgacggcatt aacataaacc 20

Claims (21)

An isolated promoter that functions in a plant with the nucleotide sequence represented by SEQ ID NO: 1 (about 1 kbp) In an isolated promoter functioning in a plant having the nucleotide sequence represented by SEQ ID NO: 2 (about 2 kbp), i) isolated from Aribidopsis thaliana, ii) SacI (0.12 kb), BspHI (0.17 kb), SauI ( 0.78 kb) and a restriction enzyme site for SalI (1.0 kb), i) an isolated promoter characterized by comprising the nucleotide sequence of SEQ ID NO: 1 delete delete The chimeric gene comprising the plant promoter of claim 1 and a heterologous nucleic acid encoding a preferred polypeptide, wherein the promoter is a 1 kbit Arabidopsis trehalose-6-phosphate synthase 3 promoter. The chimeric gene comprising the plant promoter of claim 2 and a heterologous nucleic acid encoding a preferred polypeptide, wherein the promoter is a 2kb long Arabidopsis trehalose-6-phosphate synthase 3 promoter. delete delete delete delete delete delete delete delete delete delete In a microorganism comprising a plasmid, the microorganism is E. coli DH5 @ / pLES99010 comprising the promoter of claim 1 (deposited as KCTC 0811BP) or E.coli DH5 @ / pLES99011 comprising the promoter of claim 2 (KCTC 0812BP Deposited microorganisms characterized by fungi In a microorganism comprising a construct, the microorganism is Agrobacterium tumefaciens GV3101 / pLES99014 (deposited as KCTC 0813BP) comprising the promoter of claim 1 or Agrobacterium comprising the promoter of claim 2 Microorganisms characterized by tumefaciens GV3101 / pLES99015 (deposited as KCTC 0814BP) delete delete delete