KR101185845B1 - Recombinant expression vector comprising pLim3 promoter and transformant transformed therewith - Google Patents

Abstract

The present invention is a rice ( Oryza The pLim3 promoter gene expressed in pollen of sativa ), the expression vector containing this gene, the transformant transformed with this expression vector, and the manufacturing method of this transformant are related.

By inducing pollen specific expression using the promoter according to the present invention, it is possible to reduce the time and labor required to induce gene male infertility and to produce superior seed, which is a large hybrid.

pLim3, pollen, rice, transformant

Description

Recombinant expression vector comprising pLim3 promoter and transformant transformed therewith, an expression vector comprising the promoter, an expression vector comprising the promoter, a transformant transformed with the expression vector, and a method for producing the transformant

The present invention is a rice ( Oryza sativa ) relates to a pLim3 promoter gene expressed in pollen, an expression vector comprising the gene, a transformant transformed with the expression vector, and a method for producing the transformant.

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, the agricultural land where rice is cultivated is getting smaller and smaller as the industrialization phenomenon of each country in the world, and the new genetic resource which is the subject of breeding is depleted and the introduction of the foreign useful gene is required. 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 this transformation will not only lead to a high value-added industry in the coming 21st century, but will also solve some of the food problems that are the biggest problem of humanity.

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. 7,365,185 (Boukharov et al., April 29, 2008) discloses a technique for regulating 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.

Lim gene is known as a gene showing a tissue-specific expression pattern in plants. The OsLim gene in rice is not yet known to function correctly in rice, but has been shown to function in the LIM domain in Arabidopsis, cotton and sunflower. The LIM domain contains zinc finger motifs, which are commonly included in transcriptional regulator genes, which are involved in the growth of organisms. In particular, it is known as a gene expressed in cytoskeleton tissue such as actin during germination of pollen.

A promoter specifically expressed in pollen of rice and a system for expressing useful genes using the same have not been disclosed yet. These tissues are major tissues that play an important role in rice reproduction, and expression or regulation of useful genes in these tissues can be directly linked to the production of superior new varieties of rice. Therefore, there is an urgent need for a technique for controlling gene expression specific to pollen as a direct and suitable technique that can regulate physiological phenomena involved in growth through regulation of activity of useful genes.

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

According to one aspect of the present invention, it is possible to provide a promoter specifically expressed in a pollen of rice consisting of the nucleotide sequence represented by SEQ ID NO: 1.

According to an aspect of the present invention, it is possible to provide a vector comprising the promoter and a foreign gene operatively linked thereto.

According to one aspect of the invention, it is possible to provide an Agrobacterium transformant transformed by the vector.

According to an aspect of the present invention, it is possible to provide a transformed plant transformed with the vector or the Agrobacterium transformant.

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

According to another aspect of the present invention, there may be provided a method for transforming a plant comprising the step of transforming the 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 A method for producing a rice transformant specifically expressed in a pollen of rice, comprising the step of introducing the expression vector and the step of introducing a rice transformant is specifically expressed in a pollen to introduce the expression vector Can be.

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 inducing pollen specific expression using the promoter according to the present invention, it is possible to reduce the time and labor required to induce gene male infertility and to produce superior seed, which is a large hybrid.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1: genome DNA of PCR On by pLim3 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 Athb-1, POLLEN1, etc. by searching for the motif of the upstream promoter region of the Lim gene using PLACE (a database of Plant Cis-acting Regulatory DNA Elements) program After designing primers to amplify the region containing the motif of (Prestridge, DS, A computer program that scans DNA sequences for eukaryotic transcriptional elements, 1999), the promoter region (pLim3) of the OsLIM gene was isolated by PCR.

Genomic DNA was extracted from the leaves of rice (Qiagen, DNeasy Plant Mini kit) to design a primer for amplifying a fragment of 575bp expected to be the promoter region of the OsLIM gene. CATAGCAATAAAGGTTGCTCTAAACT-3 '(SEQ ID NO: 2) and antisense primer pLim-AS: 5-TATGTCAATTTGTTGCTAACAAGAA-3' (SEQ ID NO: 3) was synthesized and added to the 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 pLim3 promoter was cut out 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 pLim3 gene. (SEQ ID NO: 1)

Example  2: rice transformation vector ( vector Production

1) pLim3 Gene Carrier Construction

Gateway cloning kit (Gateway cloning, purchased from invitrogen) was used to prepare the pLim3 gene carrier. For cloning, forward primers pLim3-S: 5-AAAAAGCAGGCTTGAGGTCACATTTCTCT-3 '(SEQ ID NO: 4) and reverse primers pLim3-AS: 5-AGAAAGCTGGGTGTATGTCAATTTGTT-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 under 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 68 ° C. for 2 minutes. 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 pLim3 gene, amplification was completed.

2) Construction of pBGWFS7 vector for promoter expression

After attaching attB-adapter primer (Gateway cloning kit, Invitrogen) to pLim3 gene, it is mixed with BP Clonase II enzyme mix 2 and reacted for more than 25 to 1 hour (http://www.invitrogen.com/site/us/en/ home / Products-and-Services / Applications / Cloning / Gateway-Cloning / GatewayC-Misc / Protocols.html # bp) and introduced into the pDONR221 vector (Invitrogen), then mixed with LR Clonase II enzyme mix 2 at 25 Over 1 hour response (see http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/GatewayC-Misc/Protocols.html#lr) It 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 pLim3 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: pLim3  Of promoter GUS  Expression analysis by staining

Through histochemistry assay of the pLim3 promoter region by GUS staining, it was confirmed by this promoter that the GUS reporter gene was expressed in post- and post-flowering operations and pollen.

Figure 3 shows the expression pattern of the GUS gene expressed by the pLim3 promoter.

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 is a micrograph showing the expression pattern before and after a few minutes by the pLim3 promoter using the GUS reporter gene.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Recombinant expression vector comprising pLim3 promoter and          transformant transformed therewith <130> NPF-15282 <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 575 <212> DNA <213> Oryza sativa <400> 1 aggtcacatt tctctaaaag gccaatgaca tgcatgctgt ttaattttat catgtgtgca 60 aatggttata catttgtttg tttgtgttat tctcggtgca ctaagaatag cttggtcaac 120 ttcttcctca aaagagaaaa atgaccggct gataacaatg gcatttaaat aactcttctg 180 taaatgtctt gggctattgt tagtcactaa ctattattgc accctaaata tagtcaaaca 240 ctttctgtag gagaataacc acaaattata accatgtgag gagtctccaa agttagcatt 300 gtgatgtggc acatctaatc tcatctctct gaccatttcc tctggcttag aggtggagtt 360 agacccaagc ctcttatatt tcttccctag cttcagcctc tcccaaatca ggtttccaga 420 attgcatatt aaagcctccc atcaacttct tgcacacacc ctccctttgg ctttcatctg 480 catctattca gttgcagatt gagagccaaa gacattatct tgattctatc aggagggttg 540 tgctagcgaa ttcttgttag caacaaattg acata 575 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pLim-S <400> 2 catagcaata aaggttgctc taaact 26 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pLim-AS <400> 3 tatgtcaatt tgttgctaac aagaa 25 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pLim3-S <400> 4 aaaaagcagg cttgaggtca catttctct 29 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pLim3-AS <400> 5 agaaagctgg gtgtatgtca atttgtt 27 <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)

A promoter of pollen specific expression characteristics comprising the nucleotide sequence represented by 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. delete The transformant of claim 3, wherein the transformant is a rice transformant. delete Method for producing a rice transformant specifically expressed in pollen 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 having the foreign gene specifically expressed in a pollen of the rice by introducing the expression vector. 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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