KR101054891B1 - WMDE1 promoter expressed in meristem of rice leaves and roots, expression vector containing this promoter, transformant transformed with this expression vector, and method for producing the transformant - Google Patents

Abstract

The present invention is a rice ( Oryza The present invention relates to a pDME1 promoter gene expressed in meristems of leaves and roots isolated from sativa ), an expression vector comprising the gene, a transformant transformed with the expression vector, and a method for producing the transformant.

By using the promoter according to the present invention, it is possible to obtain a vector and a transformant capable of expressing useful cleavage genes for the development of excellent growth varieties in the meristem of leaves and roots.

Meristem, tissue specific expression, promoter

Description

{Recombinant expression vector comprising pDME1 promoter and transformant transformed ｐDME1 promoter expressed in meristem of rice leaves and roots, expression vector containing the promoter, transformant transformed with the expression vector and preparation method of the transformant therewith}

The present invention is a rice ( Oryza The pDME1 promoter gene expressed in the meristem of leaves and roots isolated from the sativa ), the expression vector containing this gene, the transformant transformed with this expression vector, and the manufacturing method of this transformant are provided.

 Rice is one of the world's most important food crops, and has been steadily increasing its yield over the past two decades, but the current population growth trend means that by 2020, more than 70% of the current crop will be produced. However, farmland where rice is cultivated is gradually decreasing due to industrialization of countries around the world, and new genetic resources, which are used for breeding, are depleted, and foreign useful genes are required to be introduced. Fortunately, the development of molecular biology makes it possible to isolate and manipulate foreign useful genes, transforming these useful genes into many plant cells, including rice, to obtain transformants incorporating new genes. Expression studies, of course, have become a good material for breeding. The production of new varieties using these transformations will not only emerge as a high value-added industry in the coming 21st century, but also solve the food problem, which is one of the biggest problems of mankind.

The rice transformation method known to date has obtained a large number of transformants in the 80's with polyethylene glycol (PEG) and electroporation in the protoplasts, and in the late 90's, the use of a gene gun has become popular. ssangjayeop Agrobacterium (Agrobacterium), which has been widely used in plant How to use has also been widely used. In addition, there are pollen method and microinjection method.

Promoters affecting the rice transformation efficiency was cloned from CaMV (Cauliflower mosaic virus), rice, corn, soybeans and potatoes, and contributed to the transformation efficiency.

  The level of marker gene expression has a very important effect on transfection efficiency. Several factors include efficient vector, suitable promoter, location in plant genome, number of copies, 3 'noncoding sequence, and codon frequency. . Promoters determine the pattern of transgenes and two types of promoters have been used, namely, constant and tissue specific. This constitutive promoter independently expresses genes in all tissues. The CaMV35S promoter is a potent and always-on, commonly used in transformation studies and widely used in rice, wheat and corn.

Gene expression in target tissues suggests the potential for crop improvement. Rice is currently used as a model for gene expression and promoter research in monocotyledonous plants, and it is very helpful in understanding the functional interactions between the already identified elements and DNA binding factors. Will be.

Studies on the transformation of rice using the gene in the starting point that the obtained transformants in japonica by using the plasma in the late 1980s using a polyethylene glycol method and electric shock method indica gun, Agrobacterium (Agrobacterium) It is reported that a transformant can be obtained. In particular, herbicide-tolerant ( bar ) genes, also known as starting genes, have been used most often for transformational research in the 1990s, after which new agricultural genes have been developed and manufactured. We use for change over.

For such tissue-specific promoters, US Pat. No. 5,808,034 (Bridges et al., Registered on Sep. 15, 1998) uses genes in the male organs of plants using expression repressors that are specifically promoted by external chemicals. Techniques for regulating fertility have been disclosed by allowing cascades of the cells to occur. In addition, US Pat. No. 6,784,289 (Ouellet et al., Aug. 31, 2004) discloses a technique for controlling expression of a translational regulator that can increase or decrease the expression of a protein involved in a host organism. In addition, promoters specifically expressed in fruit epidermal tissue (KR 813,118) and promoters specifically expressed in plant root tissues (KR 803,390) have been disclosed. In the case of rice, a technique related to a tissue-specific promoter is disclosed in Korean Patent Publication No. 2003-0081377, KR 2001-0085990 and the like.

DME gene is known as a gene showing a tissue-specific expression pattern in meristem of plants. Currently, the function of this gene is not clear, but the function of Arabidopsis DME gene is a gene that is expressed specifically in pistil before modification, especially when it is modified for seed generation. have.

Currently, there is no disclosure about a system for expressing useful foreign genes by using a promoter specifically expressed in the meristem of rice leaves and roots. These tissues are major tissues that play an important role in rice growth, and expression or regulation of useful genes in these tissues can also be directly linked to rice growth and harvest. Therefore, there is an urgent need for a technique for regulating gene expression specific to these tissues as a direct and suitable technique that can regulate physiological phenomena involved in growth through regulation of useful gene activity.

An object of the present invention is to develop a promoter specifically expressed in the meristem of the leaves and roots of rice for the development of excellent varieties.

According to one aspect of the present invention, it is possible to provide a promoter of a characteristic specifically expressed in the cleavage of the leaf tip and root of the rice comprising the nucleotide sequence represented by SEQ ID NO: 1.

According to one embodiment, a vector comprising the promoter and an operatively linked foreign gene may be provided.

According to one embodiment, an Agrobacterium transformant transformed by the vector may be provided.

According to one embodiment, a transformed plant transformed by the vector or the Agrobacterium transformant may be provided.

According to one embodiment, the transformant may be rice (Oryza sativa).

According to another aspect of the present invention, there can be provided a plant transformation method comprising the step of transforming a plant to express the foreign gene in the plant.

According to another aspect of the invention, preparing a vector comprising a promoter comprising a nucleotide sequence represented by SEQ ID NO: 1 and a foreign gene operatively linked to (operatively linked), introducing the expression vector into rice And a step of introducing the expression vector to select a rice transformant expressing a foreign gene specifically at the foliar tissue of the leaf tip and root of rice, specifically expressing the foliar tissue of the rice leaf and root A method for producing a rice transformant can be provided.

According to one embodiment, the step of introducing the expression vector to the rice is characterized in that it comprises the step of transforming Agrobacterium with the expression vector and infecting the transformed Agrobacterium rice; A method for producing a converter can be provided.

By using the promoter according to the present invention, it is possible to obtain a vector and a transformant capable of expressing useful cleavage genes for the development of excellent growth varieties in the meristem of leaves and roots.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples.

Example  1: genome DNA of PCR On by pDME1 In the promoter area Cloning  And sequencing

1) Gene expression regulatory motif search and PCR amplification of promoter region

TATA box, CAAT box, GATA box, AMYBOX2 CBFHV, POLLEN1, etc., by searching for motifs in the upstream promoter region of the OsDME gene using the PLACE (a database of Plant Cis-acting Regulatory DNA Elements) program. , DS, A computer program that scans DNA sequences for eukaryotic transcriptional elements (1999), and primers were designed to be amplified, and then PCR region of the promoter region (pDME1) was isolated.

Genomic DNA was extracted from the leaves of rice (Qiagen, DNeasy Plant Mini kit) to design a primer for amplifying the fragment of 628bp expected to be the promoter region of the OsDME gene to design a forward primer (sense primer) pDME-S: 5- ACTGGTCCTCGTTGGAAAGGCAGTA-3 '(SEQ ID NO: 2) and antisense primer pDME-AS: 5-TTCTGTAGAGGTGCAAGTCTAGATG-3' (SEQ ID NO: 3) was synthesized in a PCR reaction solution with Takara's r Taq polymerase. Gene was amplified using Applied Biosystem 9700. The PCR reaction conditions used were denatured at 95 ° C. for 10 minutes, and then repeated 25 times at 94 ° C. for 1 minute, 56 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by further treatment at 72 ° C. for 10 minutes for expansion reaction. After the PCR reaction, the amplified gene was electrophoresed in 1% agarose, and the band estimated to be the pDME1 promoter was cut and purified using a QIAquick gel extraction kit, and then used for cloning.

2) Cloning to Topo 2.1 vector (Invitrogen, TOPO TA Cloning kit)

Amplified genes were transformed, and white colony obtained by applying to X-gal-added LB medium was selected and subjected to colony PCR. As a result, the plasmid of the colonies where the band was confirmed was extracted and used for sequencing.

3) sequencing

Sequence analysis using ABI 3700 sequencer, followed by BLAST search of NCBI It was confirmed that it matches the pDME1 gene. (SEQ ID NO: 1)

Example  2: rice transformation vector ( vector Production

1) pDME1 gene carrier construction

Gateway cloning kit (Gateway® cloning, purchased from invitrogen) was used to construct the pDME1 gene carrier. For cloning, forward primers pDME1-S: 5-AAAAAGCAGGCTCCGAAATGTCTACACACT-3 '(SEQ ID NO: 4) and reverse primers pDME1-AS: 5-AGAAAGCTGGGTATTCTGTAGAGGTGCAAG-3' (SEQ ID NO: 5) were synthesized. These primers were put into a PCR reaction and denatured at 95 ° C. for 10 minutes, then repeated 30 times at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 68 ° C. for 2 minutes, and then expanded to 72 ° C. for 10 minutes to amplify the pDME1 gene. Denature the primers attB1: 5-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '(SEQ ID NO: 6) and attB2: GGGGACCACTTTGT ACAAGAAAGCTGGGT-3' (SEQ ID NO: 7) to reattach the adapter primers to the amplified genes. After repeating twice at 94 ℃ 30 seconds, 45 ℃ 3 seconds, 68 ℃ 2 minutes conditions, repeated 19 times at 94 ℃ 3 seconds, 55 ℃ 3 seconds, 68 ℃ 2 minutes conditions expansion reaction at 68 ℃ 10 minutes By attaching adapter primers to both ends of the pDME1 gene, amplification was completed.

2) Construction of pBGWFS7 vector for promoter expression

Attach attB-adapter primer (Gateway® cloning kit, Invitrogen) to pDME1 gene and mix with 2µl of BP Clonase II enzyme mix for more than 1 hour at 25 ℃ (http://www.invitrogen.com/site/us /en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/GatewayC-Misc/Protocols.html#bp) and introduce it into the pDONR221 vector (Invitrogen) and again with 2 μl of LR Clonase II enzyme mix. Mix and react at 25 ° C for at least 1 hour (http://www.invitrogen.com/site/us/en/home/Products-and- Services / Applications / Cloning / Gateway-Cloning / GatewayC-Misc / Protocols.html # bp) was introduced into the target vector pBGWFS7 vector (PSB, Plant System Biology). After confirming the gene introduced by colony PCR and sequencing, it was transformed by Agrobacterium infection to transform the rice.

Figure 1 shows a schematic diagram of the vector for transforming rice produced through the gateway system.

To transform the finished vector into Agrobacterium (LBA4404), freeze and thaw were repeated 2-3 times, and then transformed by heat shock at 37 ° C., followed by YEP medium ( Yeast 10g, NaCl 5g, peptone 10g, Agar 15g / 1L) overnight culture (overnight) was confirmed colonies.

Colonies confirmed to be transformed by colony PCR were prepared using AB medium (AB buffer (K ₂ HPO ₄ 60 g, NaH ₂ PO ₄ 20 g / 1 L), AB Salts (NH ₄ Cl 60 g, MgSO ₄ 7H _2). O 6g, were cultured in _{KCl 3g, CaCl 2 2H 2 O} 0.265g, FeSO 4 .7H 2 O 50mg / 1L), Glucose 5g / 1L).

Example  3: Rice Transformation with Agrobacterium

1) Induction of rice callus

Agrobacterium-induced rice transformation was performed by washing seeds in lacs to induce callus of rice in MS medium (Duchefa Murashinge and Skoog vitamin containing medium), and then drying it appropriately to add 2,4-D hormone to MS medium. In the cultured medium. After induction of callus by cancer culture for 3-4 weeks at 27 ° C., embryogenic callus was sub-cultured in fresh medium to which 2,4-D hormone was added to MS.

2) Agrobacterium Infection

Embryonic callus and Agrobacterium transformed genes were incubated in AB liquid medium for 20 min disinfection and then cultured for 3 days. Cefotaxime was added to sterile water, washed until complete removal of Agrobacterium, and then cultured for 3 weeks in a medium containing cefotaxime and ppt (phosphitothricin, phosphinothricin) in MS medium. Callus which survived browning in the MS medium containing cefotaxime and ppt was transferred to MS medium containing cefotaxime and ppt and cultured for 2 weeks. To induce shooting in callus, the cells were transferred to medium containing MSR-cefotaxime and ppt, and cultured for 4 weeks.

3) seed harvesting and posterior fixation

After about 5-7 days, the obtained transformant (T ₀ ) was grown in a greenhouse, harvested seeds, and selected for resistance to Basta (= ammonium glufosinate) for later fixation. Transformants for pDME1 promoter analysis were expressed by GUS staining in T ₂ generation seeds.

Figure 2 shows a plant obtained through Agrobacterium infection as described above.

Example  4: pDME1  Of promoter GUS  Expression analysis by staining

Through histochemistry assay of the pDME1 promoter region by GUS staining, the GUS reporter gene was expressed in leaf meristem from 3 days after germination and strongly in meristem of root from 6 days before and after. It was confirmed to be expressed.

Figure 3 is a plant showing a color reaction due to the expression of the GUS gene before and after 3 to 6 days after germination.

Example  5: pDME1  Root Entourage Expression Analysis of Promoters

As a result of observing the expression of the pDME1 promoter region transformed in rice, it was confirmed that the pDME1 promoter was strongly expressed in the root at the time of entourage from 6 days after germination.

Figure 4 shows that GUS is strongly expressed in the root aides on the 6th to 7th day of germination.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

FIG. 1 shows a schematic diagram of a vector for transforming rice prepared using a gateway system.

2 shows transgenic plants obtained by Agrobacterium infection transformation.

Figure 3 shows the expression pattern before and after a few minutes by GUS staining of the pDME1 promoter.

4 shows the expression pattern at the root of the pDME1 promoter.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Recombinant expression vector comprising pDME1 promoter and          transformant transformed therewith <130> NPF-15281 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 628 <212> DNA <213> Oryza sativa <400> 1 gaaatgtcta cacactttta aactagtgtt gtggtttgca agggatattc cttaaattat 60 ggaatttatt attgtggttt gcaagggaat ctaatttaaa ctaaactcca ctttacactt 120 taaagtctac aatctacaca cttaaagtag atattcctta aaactaggga atctattgtt 180 gtggtttgca agggaattta gtttaaagtg tcaagtgcac tttttattgt gagggaatct 240 acatttcctt ttaagtgttg tggttcattt gttgcttcct tagaatcctc acttagagag 300 aagattgaac tatataagtt gaaacaaatt tcgctcagac tggggtttat ttgtagaggt 360 gggattttgt tggagggatt ggggtttagt tctagagtag ttgcttgatt ataccctatt 420 tttcttgctt taatattttg agaaacacaa tcagaagtgt agttgcatgc aaccaattta 480 ctatgccatt ttcattatat tgtgattagt attgatcttt tgaatagtat atgaccatct 540 aaattgcgtc cttatgatat tgtcataggt gaaaacgacg ggaagctaac tagaaccgtg 600 cttcatctag acttgcacct ctacagaa 628 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pDME-S <400> 2 actggtcctc gttggaaagg cagta 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pDME-AS <400> 3 ttctgtagag gtgcaagtct agatg 25 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pDME1-S <400> 4 aaaaagcagg ctccgaaatg tctacacact 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pDME1-AS <400> 5 agaaagctgg gtattctgta gaggtgcaag 30 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB1 <400> 6 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB2 <400> 7 ggggaccact ttgtacaaga aagctgggt 29  

Claims (8)

Promoter of the characteristic is expressed specifically in the meristem of the leaf tip and root of rice comprising the nucleotide sequence shown in SEQ ID NO: 1. A vector comprising the promoter of claim 1 and a foreign gene operatively linked thereto. Agrobacterium transformants transformed with the vector of claim 2. A transformed plant transformed with the vector according to claim 2 or the Agrobacterium transformant according to claim 3. The transformed plant according to claim 4, wherein the transformed plant is an Oryza sativa transformant. A method of transforming a plant, comprising the step of transforming the plant with the vector of claim 2 to express the foreign gene in the plant. Method of producing a rice transformant specifically expressed in the meristem of the leaf tip and root of rice comprising the following steps: Preparing a vector comprising a promoter comprising a nucleotide sequence represented by SEQ ID NO: 1 and a foreign gene operatively linked thereto; Introducing the expression vector into rice; And Selecting a rice transformant is introduced to the expression vector is specifically expressed in the meristem of the leaf tip and root of the rice. The method of claim 7, wherein the introducing of the expression vector into the rice comprises: transforming Agrobacterium with the expression vector; And infecting the transformed Agrobacterium with rice.

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