KR20220072074A - OsRP3 promoter expressed in root vascular cylinder and uses thereof - Google Patents

Abstract

The present invention relates to a promoter for specifically inducing expression in the root of a plant and a use thereof, and more particularly, the OsRP3 promoter for inducing the expression of a target gene specifically in the root of a plant, and a recombinant expression vector comprising the promoter , It relates to Agrobacterium transformed with the recombinant expression vector, a transgenic plant, and a method for producing a transgenic plant.
The OsRP3 promoter specifically expressed in the root tissue of the plant of the present invention can be used, and the target gene can be specifically expressed in the root tissue, and by using this, the transport of nutrients, ions and water, and root morphology control are improved. It can be used to improve the agricultural characteristics of crops.

Description

OsRP3 promoter expressed in root vascular cylinder and uses thereof}

The present invention relates to the OsRP3 promoter for inducing specific expression in the centromere of rice roots, and to a use thereof.

Due to climatic abnormalities and environmental pollution, exposure of plants to abiotic stresses such as drought, high salt, heavy metals, cold damage, thermal shock and ozone is increasing. Such abiotic stress acts as a factor limiting the growth and development of crops. A major abiotic stress is known to be the result of water scarcity. In order for plants to grow, nutrients and water absorption are the most important, and the roots are playing that role. In addition, roots play an important role in plant growth by immobilizing plants, absorbing inorganic salts, secreting hormones or synthesizing their secondary metabolites. Therefore, it is possible to enhance the abiotic stress tolerance of crops and increase productivity by controlling the root nutrient, ion and water transport, root shape, hormone secretion, etc.

Rice is one of the most important food crops in the world, and has been steadily increasing its yield for the past 20 years, but is now in a situation where it has to produce an excess amount due to population growth. However, the farmland where rice is grown is gradually decreasing due to the industrialization phenomenon of countries around the world, and the new genetic resources used for breeding are depleted, so the introduction of useful foreign genes is required. Fortunately, with the development of genetic engineering technology, many studies are underway to improve the traits of plants. In particular, there is a lot of interest in technologies for obtaining a useful gene for a purpose from a transgenic plant. When expressing this useful gene for a purpose in a transgenic plant, a promoter involved in gene expression is required.

In general, a promoter is a type of regulatory region having a function of regulating the expression of a gene, that is, a nucleotide sequence that determines when, where, and to what extent a gene is expressed. In the meantime, numerous types of promoters have been revealed through constant research. Previously reported promoters are classified according to their characteristics and functions, such as a promoter for constitutively inducing expression and a promoter for inducing time- or site-specific expression.

Arabidopsis peroxidase (prxEa) was isolated as a root-specific expression promoter, and root-specific expression was confirmed. ) gene is isolated, and it has been reported that the promoter specifically induces the expression in the root, and induces the root-specific transient expression in carrots and radishes.

However, conventional promoters have the advantage of tissue-specific expression of a target gene, but have a disadvantage in that the amount of expression is insignificant. Therefore, there is a need to discover useful new promoters capable of tissue-specific and large-scale expression of target genes in plants.

Accordingly, the present inventors have made efforts to develop a new promoter capable of tissue-specific expression of a target gene in a plant, specifically, a useful promoter capable of specifically expressing a target gene in the root tissue of a plant. By isolating the OsRP3 promoter capable of expressing the gene specifically in the root tissue, the present invention was completed.

Republic of Korea Patent No. 10-2065491

In order to achieve the above object, an object of the present invention is to provide a promoter comprising the nucleotide sequence of SEQ ID NO: 1 and specifically inducing expression in the root of a plant.

Another object of the present invention is to provide a recombinant expression vector comprising a foreign gene encoding a target protein operably linked to a promoter.

In addition, the present invention aims to provide a transformed Agrobacterium ( Agrobacterium ) using the vector.

Another object of the present invention is to provide a transgenic plant transformed with the vector or the Agrobacterium.

Another object of the present invention is to provide a method for producing a transgenic plant comprising the step of transforming a plant with the vector and expressing a target protein in the plant.

In order to achieve the above object, the present invention can provide a promoter comprising the nucleotide sequence of SEQ ID NO: 1 and specifically inducing expression in the root of a plant.

The root may be a vascular cylinder.

The vascular bundle centroid may be a xylem tissue.

The promoter may be one that induces expression in the lateral root primordia of the root by Abscisic acid (ABA) or auxin.

In addition, the present invention may provide a recombinant expression vector comprising a foreign gene encoding a target protein operably linked to the promoter.

In addition, the present invention can provide a transformed Agrobacterium ( Agrobacterium ) using the vector.

In addition, the present invention may provide a transgenic plant transformed with the vector or the Agrobacterium.

In the present invention, the plant includes rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat or sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion or carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots or bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies or tulips; And it may be at least one selected from the group consisting of feed crops including ryegrass, red clover, orchard grass, alpha alpha, tall fescue or perennial ryegrass.

In addition, the present invention may provide a method for producing a transgenic plant comprising the step of transforming a plant with the vector to express a target protein in the plant.

Also, in the present invention, the target protein may be specifically expressed in the root of a plant.

In the present invention, the root may be a central vascular bundle.

In the present invention, the vascular bundle center may be a xylem tissue.

In the present invention, the target protein may be induced to be expressed in the lateral root primordia of the root by Abscisic acid (ABA) or auxin.

By using the OsRP3 promoter specifically expressed in the vascular cylinder of the plant root of the present invention, the target gene can be specifically expressed in the root tissue, and using this, the transport of nutrients, ions and water, and the root It can be used to improve the agricultural characteristics of crops through improvement of root traits such as shape control.

1 is a schematic diagram of an OsRP3 promoter-GUS vector.
2 is a genomic PCR analysis of transgenic rice introduced with an OsRP3 promoter-GUS expression vector. DJ (Daejo-gu; Dongjinbyeo); 6-2, 6-3, 177-5 (transgenic rice)
3 shows the results of GUS staining of transgenic rice roots into which the OsRP3 promoter-GUS vector is inserted.
4 shows a comparison of GUS staining by treatment with Abscisic acid (ABA) or naphthalene acetic acid (NAA) in transgenic rice roots into which the OsRP3 promoter-GUS vector is inserted.

Unless defined otherwise, 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. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

As a result of efforts to develop a useful promoter capable of specifically expressing a target gene in the root tissue of a plant, the present invention was completed by isolating the OsRP3 promoter capable of specifically expressing the target gene in the root tissue in rice.

In one aspect, the present invention relates to a promoter comprising the nucleotide sequence of SEQ ID NO: 1 and specifically inducing expression in the root of a plant.

In the present invention, "root" is the root of a plant, which is usually buried in the ground or embedded in other objects to suck up moisture and nutrients and to support the stem.

In one embodiment of the present invention, as a result of transforming rice with the recombinant expression vector containing the OsRP3 promoter of the present invention, the target protein (eg, GUS) operably linked to the promoter is found in rice roots, specifically, the stem (stele). ) or in the vascular cylinder, and in particular, it was confirmed that it was clearly expressed in the metaxylem and protoxylem inside the central column. As a result of a detailed analysis of the GUS staining pattern in the transverse section of the root, GUS expression was not observed in the cells constituting the major tissue layers outside the central column, such as the epidermis and cortex, and in the inner central column region of the root. Clear GUS staining was confirmed (FIG. 3B).

Therefore, it was demonstrated that the OsRP3 promoter can specifically express the target gene in the xylem tissue of the rice root.

Therefore, in the present invention, the root may be a vascular cylinder specifically located at the root.

In the present invention, the vascular cylinder is a vascular bundle tissue located in the stem region of the root, and includes a xylem and a phloem.

In an embodiment of the present invention, it was confirmed that staining was clearly observed in the metaxylem and protoxylem of the vascular bundle center, and the vascular bundle center may specifically be a xylem tissue.

The xylem tissue includes a metaxylem and a protoxylem.

That is, the promoter of the present invention may be one that specifically expresses the target gene in the xylem tissue of the center of the vascular bundle of the root.

In the present invention, the term “promoter” refers to a region of a genome connected to the upper side of a structural gene, and serves to regulate the transcription of the structural gene linked thereto into mRNA. The promoter is activated by binding of several general transcription factors. TATA box, CAAT box region that generally regulates gene expression, region, position and direction that affects gene expression in response to external stimuli It consists of enhancers, etc. that promote the expression of almost all genes regardless of Since proteins necessary for the basic metabolism of the living body must be maintained at a certain concentration in the cell, promoters linked to these genes are always activated only by the action of general transcription factors. On the other hand, proteins that do not normally have a role and are required to function only under special circumstances are linked to a tissue specific promoter or an inducible promoter that induces the expression of the corresponding structural gene. That is, the tissue-specific promoter is activated during the development of an organism, and the inducible promoter is activated by binding of a specific transcription factor activated by an external stimulus from an environmental factor.

The promoter of the present invention is a promoter that specifically induces expression only in a specific region of a plant, particularly in the root tissue of a plant, and has the nucleotide sequence of SEQ ID NO: 1, but a sequence having 80% or more homology with the sequence, preferably 90 A sequence exhibiting at least % homology, more preferably at least 95% homology, and most preferably at least 99% homology, and may include bases having substantially the same or corresponding biological activity. In addition, as a sequence having such homology, as long as it is a nucleotide sequence having substantially the same or corresponding biological activity, it is apparent that promoters having nucleotide sequences in which some sequences are deleted, modified, substituted or added are also included in the scope of the present invention.

In the present invention, "homology" is intended to indicate a degree of similarity to a wild type nucleotide sequence, and includes sequences having the same percentage or more as the nucleotide sequence of the present invention. Comparison of such homology can be performed visually or using an easily purchased comparison program. A commercially available computer program can calculate the homology between two or more sequences as a percentage (%), and the homology (%) can be calculated for adjacent sequences.

The promoter of the present invention can express a target protein or target gene RNA in the root of a plant, and preferably can specifically express the target protein in the root tissue of the plant.

The site-specific promoter of the present invention can act as a promoter for conventional recombinant expression vectors.

In one embodiment of the present invention, the seedlings of transgenic rice, in which the OsRP3 promoter-GUS vector insertion of the present invention has been confirmed, abscisic acid (ABA) or auxin, naphthalene acetic acid (NAA) After treatment with a medium containing plant hormones for 3 days, as a result of analyzing GUS staining in plants, it was confirmed that GUS staining increased in the vascular bundle center line of the roots of plants treated with ABA or NAA. In addition, as a result of analyzing the GUS staining pattern in the transverse section of the root, it was confirmed that GUS staining was observed in the lateral root primordia of the root of the plant treated with ABA or NAA, whereas no staining was observed in the control group. did

It was confirmed that the OsRP3 promoter of the present invention increases the expression of the target gene in the vascular bundle center line of the root by plant hormones of Abscisic acid (ABA) or auxin. (Abscisic acid, ABA) or a plant hormone of auxin can increase the expression of the target gene in the vascular bundle center line of the root.

In addition, it was confirmed that the OsRP3 promoter induces expression by stress hormone (ABA) and growth control hormone (auxin) in the lateral root primordia of the root. The promoter according to the present invention is Abscisic acid (ABA) ) or auxin can induce expression in the lateral root primordia of the root.

In another aspect of the present invention, it relates to a recombinant expression vector comprising a foreign gene encoding a target protein operably linked to the promoter.

As used herein, the term “recombinant expression vector” refers to a vector capable of expressing a target protein or target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert.

The recombinant expression vector of the present invention is a plasmid, virus known in the art, into which the promoter of the present invention and the gene nucleotide sequence encoding the target protein operably linked with the promoter can be inserted or introduced. or any other medium. The tissue-specific expression promoter of the plant according to the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter may be operably linked to the expression control sequence, and the operably linked gene sequence and the expression control sequence may be included in one expression vector including a selection marker and a replication origin.

Said “operably linked” may be a gene and an expression control sequence linked in such a way as to enable gene expression when an appropriate molecule is bound to the expression control sequence. By “expression control sequence” is meant a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a particular host cell. Such regulatory sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation.

The expression vector according to the present invention can be prepared by inserting the promoter of the present invention using an existing vector used for protein expression as a basic backbone and inserting a nucleotide sequence encoding a target protein downstream of the promoter. have. Therefore, as a vector that can be used in the present invention, E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119) having various cloning sites, Bacillus subtilus-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50) ) and Ti plasmids, may be plant viral vectors, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA series may be used. However, those skilled in the art may use any vector as long as it is a vector capable of introducing the promoter of the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter into a host cell.

The target protein that can be used in the present invention may be any target protein derived from an external that is specifically expressed in plant tissue by the promoter of the present invention, that is, an exogenous protein or an endogenous protein and a reporter protein. The exogenous protein refers to a protein not naturally present in a specific tissue or cell, and the endogenous protein refers to a protein expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by a reporter gene, and refers to a label protein that informs its activity in a cell by its presence.

In addition, the expression vector of the present invention may preferably contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic selectable by a conventional chemical method, and may include any gene capable of distinguishing a transformed cell from a non-transformed cell.

Specifically, in one embodiment of the present invention, the OsRP3 promoter of the present invention having the nucleotide sequence of SEQ ID NO: 1 is operated in the upstream part of the GUS gene in a known vector having a reporter system linked to the blue color gene GUS. An OsRP3 promoter-GUS expression vector, which is a possibly linked recombinant expression vector, was prepared.

In another aspect of the present invention, it relates to a transformant transformed by the vector.

In the present invention, “transformation” refers to genetically changing the trait of an individual or cell by DNA, which is a genetic material given from the outside. It means that the gene is specifically expressed in the root of a plant, especially in the vascular cylinder of the root.

Transformation with the recombinant expression vector of the present invention in the present invention can be performed by transformation techniques known to those skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion ( PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method), or a method mediated by Agrobacterium sp., etc. may be used, but is not limited thereto.

In the present invention, the transformant refers to a host cell transformed with a recombinant expression vector comprising a promoter and a nucleotide sequence encoding a target protein operably linked to the promoter.

The transformant is preferably a microorganism, but is not limited thereto, Agrobacterium tumefaciens ), Escherichia coli , Bacillus subtilis ), Streptomyces ( Streptomyces ), Pseudomonas ( Pseudomonas ), Proteus mirabilis ( Proteus mirabilis ), may be Staphylococcus ( Staphylococcus ), preferably Agrobacterium tumefaciens ( Agrobacterium tumefaciens ).

Another aspect of the present invention relates to a plant transformed with the vector or transformant, and the transformant may mean Agrobacterium.

In the present invention, "plant" means a plant or plant cell. Plant includes whole plant, plant part, callus, plant tissue, plant cell and plant seed.

Plants to which the method according to the present invention can be applied include food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat or sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion or carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots or bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies or tulips; And ryegrass, red clover, orchard grass, alpha alpha, may be at least one selected from the group consisting of feed crops including tall fescue or perennial ryegrass, particularly rice ( Oryza sativa ) is preferred.

Specifically, in one embodiment of the present invention, a microbial transformant transformed by a recombinant expression vector including an OsRP3 promoter, Agrobacterium tumefaciens ) LBA4404 Rice ( Oryza sativa ) Transformed rice by inoculation Plants were produced.

Another aspect of the present invention relates to a method for producing a transgenic plant comprising the step of transforming a plant with the vector to express a target protein in the plant.

The production method of the present invention may include the step of transforming a plant with the vector to express the target protein in the plant.

In an embodiment of the present invention, a promoter for specific expression of a target protein comprising the nucleotide sequence represented by SEQ ID NO: 1 and a structural gene encoding the target protein are operatively linked to prepare a recombinant expression vector, After Agrobacterium tumefaciens was transformed with the expression vector, the transformed Agrobacterium tumefaciens was infected with rice to confirm that the target protein was specifically expressed in the root xylem tissue of the rice.

Therefore, the transformation may be specifically infecting rice with Agrobacterium tumefaciens transformed with an expression vector.

In addition, in the present invention, the target protein may be specifically expressed in the root of a plant, specifically, in the vascular cylinder of the root.

The description of the vascular bundle center pole in the present invention is the same as described above.

In the above-described embodiment of the present invention, it was confirmed that the OsRP3 promoter induces the expression of the target protein (GUS) by the stress hormone (ABA) and the growth regulation hormone (auxin) in the lateral root primordia of the root. In the present invention, the target protein may be induced to be expressed in the lateral root primordia of the root by Abscisic acid (ABA) or auxin.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

<Example 1> Root expression gene selection through in silico rice transcript analysis

The root of rice is composed of various tissues. Among the 45,151 gene probes registered in the Rice Root Transcripts Public DB (Rice XPro), the normalized intensity of gene expression in the endodermis, pericycle, and stem of the root was 8 or more, and the In the epidermis, cortex, etc., which are external tissues, 23 types of genes expressed less than 1 were first selected, and among them, rice tissue transcripts Public DB (Rice XPro) was used to compare with other organs such as stems. The gene Os05g0213900 with very high expression specificity in the root was selected.

<Example 2> OsRP3 promoter isolation and GUS fusion carrier production

In order to isolate the promoter region of the Os05g0213900 gene, the base sequence information of 2,000 bp of the translation start upper nucleotide sequence was extracted from the rice (Nipponbarre) genome database, and using the primer pair (Os05g0213900P-F, Os05g0213900P-R) in Table 1 below. PCR was performed using the genomic DNA of Dongjin rice as a template.

primer base sequence Os05g0213900P-F
(SEQ ID NO: 1) 5’-gccgcccccttcacccgaagggataagtccaatg-3’ Os05g0213900P-R
(SEQ ID NO:2) 5’-tcggcgcgcccacccttgtgcacggtgaggtcgcc-3’

The PCR product was cloned into a gateway donor vector and the entire nucleotide sequence was determined (Table 2). As a result, when compared with the promoter sequence of the Os05g0213900 gene extracted from the genome database of Nipponbarre, a promoter of 2,000 bp that is 100% identical was identified, and this was named OsRP3 ( Oryza sativa Root Promoter 3).

OsRP3 promoter base sequence (SEQ ID NO: 3) cgaagggata agtccaatgt tccgccggcc caggcaatta tatcgtttac gtcggcatgt 60
tcaaggctgc atcagtacat tcgacctctt ggattgatct ggtttggatg atatacttgt 120
ctacctattt atcatatatc tctgttaatc tagtcttagc atatcaattt agctctatcg 180
gctgcttctc gttttagggt ttctgcaggc atcggctaaa tcgcgttgct agattagatt 240
agcttagaca tctaccaccc tgaaaactca gtcaacgact tgattgtcta gatattatgt 300
ttcttttcat acttagtgct gcatcagtta agtttgatct actaagtcgt gcttagaacc 360
ataatctcta gcctgcttct tgattgccaa taagggtttc atcggggttt cagctggtga 420
gttatctgga cgttgcatcg gctcataagg attgcatata catataagtt ggatttagcc 480
gatgacaaca aaggcttcag tgtttaatct aatcttgtgg atttcatgac atcagacctc 540
cagccgatgt gtgttttgac cttcgaatcg atgcttattt atcatatcat tatttgctga 600
ttggtttata ctggattata ttgttatttt attacatcgt cgtcagccaa ttgcctttat 660
atcattatct acattggaca tatagccgat tgcttaaacc ctatcgctat cggctggtat 720
cggcatcggc tattatcggc atcggctgga actactccat cggcttgtca gccgatcggc 780
tatttcgatc tactatttgc atatcttgtc agttgcagga tcaaactgac tggtacgctc 840
gaatctcgtc aatatttgga tctgcacagg agctaagcag atctcccagg acggtgtgtt 900
cgattttttt tcgtcaacag tatcacacag ttattagcaa cttgttgatt gatctttgtt 960
gttagtcgtg aacaatcacc gaaacaacat ctcgtgatga acagcataaa caatttcaga 1020
atccagtaat gcatgcattt agtttgacaa aagtttactt gccgcgctag tgcaaaatgg 1080
aggcatatgc cacacacttt gcaacaacgt ttgttgcagt tcgttggctc agcgcactga 1140
ggcactacct aagcaaaaaa aataagcacg acataatata tgtaccggca gatcgactca 1200
gcttccatta gagaaagtca cacacctcga attaagataa tttttgaaat agcacgcctg 1260
ggagaagaag gtaaataata ataataatat gctacaatga tattagatca ctgatgaaga 1320
tggcagcata cacgtcttgg acatggagag gctcgattaa attacattgc attatgggga 1380
agcaccggtg gagtggtgat ggagtcgtgg gtacatgatt aatttcagag ttcaaatcct 1440
ggtgtctacg aatattatgt acacgtaaat gaactgtcaa tcgaaattta gcgagattat 1500
ggatgtaccg ttggtttccg tctttttaaa catgtgagag gggatgcatt cgcaagctgt 1560
ataagtgtgg tgttgcctgt gtaatactcc ctccgtttca aaatgtttga caccgttgac 1620
tttttagtcg tgtttgacca ttcgtcttat tcaaaaattt aagtaattat ttattctttt 1680
catctcattt gattcattgt taaatatact ttcatgtaca catatagttt tacatatttt 1740
acaatttttt tttgaataag acgaatggtc aaatatgtgc taaaagtcaa cggcgtcaaa 1800
cattttgaaa cggagggagt attgatcaat gtgtgtgcgt cccccttcta gtaaagaaat 1860
atattgcatt atgtaaacag atataggcca ggccacccac tataaatacc ttgctctccc 1920
aacattgttc tcacagctca ttacacatag cccgctcggt catcggagcg agaaagctcg 1980
ccggcgacct caccgtgcac 2000

The isolated promoter was cloned into a pBGWFS7 vector containing a GUS reporter gene to prepare a promoter expression analysis vector (pBGWFS7-OsRP3) as shown in FIG. 1 .

<Example 3> Search for motifs in the OsRP3 promoter region

To confirm the expression regulatory factors (cis-acting element) present in the OsRP3 promoter, the base sequence of the OsRP3 promoter was converted to New PLACE (database of plant cis-acting regulatory DNA elements: http://www.dna.affrc.go.jp /PLACE/) was used for analysis.

Some of the expression control factors (cis-acting element) found in the OsRP3 promoter are shown in Table 3. Referring to Table 3, in the OsRP3 promoter, RHERPATEXPA7 motif indicating root hair-specific expression, OSE1ROOTNODULE, OSE2ROOTNODULE, ROOTMOTIFTAPOX1 motif indicating root hair-specific expression, and CANBNNAPA, DPBFCOREDCDC3 indicating seed or embryo-specific expression. A motif was found, suggesting the possibility that the OsRP3 promoter drives root-specific expression.

In addition, DPBFCOREDCDC3, MYB2AT motifs were found, in which the ABRELATERD1 motif involved in drought stress and ABA signaling, a stress hormone, and transcription factors (ABI5, Myb, HD-ZIP, etc.) regulating the ABA response are combined. OsRP3 promoter (promoter) shows the possibility to induce expression by abiotic stress (abiotic stress) signal.

In addition, an ANAERO3CONSENSUS motif that induces expression by anaerobic stimulation was found.

<Example 4> Production of transgenic rice into which OsRP3 promoter-GUS expression vector is inserted

To confirm the function of the OsRP3 promoter, the expression vector of FIG. 1 was introduced into Agrobacterium LBA4404, then Dongjinbyeo callus was infected, and transformants were selected under PPT (6 μg/l) conditions and grown in a greenhouse. By separating genomic DNA from the leaves of transgenic rice, PCR was performed using primers present in the introduction vector (GUSA-anti) and promoter (OsRP3-1957F) to confirm that the introduced vector was inserted ( 2).

<Example 5> GUS staining analysis of transgenic rice for root expression inducibility assay of OsRP3 promoter

5.1. Confirmation of tissue-specific expression induction of the OsRP3 promoter

As a result of GUS staining of seedlings 5 days after germination in transgenic rice in which the OsRP3 promoter-GUS vector insertion was confirmed, it was confirmed that the expression was not found in stems and young leaves, but only in the roots (FIG. 3C).

In the root, GUS staining was confirmed only in the vascular cylinder, and in particular, the staining was clearly observed in the metaxylem and protoxylem inside the central column (FIG. 3A).

As a result of a detailed analysis of the GUS staining pattern in the transverse section of the root, GUS expression was not observed in the cells constituting the major tissue layers outside the central column, such as the epidermis and cortex, and in the inner central column region of the root. Clear GUS staining was confirmed (FIG. 3B). Through the above results, it was demonstrated that the OsRP3 promoter can specifically express the target gene in the xylem tissue of the rice root.

Therefore, it was confirmed that the OsRP3 promoter can specifically express the target gene in the central line of the vascular bundle of rice roots, especially in the xylem tissue of the root.

5.2. Confirmation of induction of expression by plant hormones of the OsRP3 promoter

In Example 4, the OsRP3 promoter contains a motif involved in drought stress and ABA signaling, a stress hormone, and a motif that binds to a transcription factor regulating the ABA response, Abscisic acid (ABA) The possibility of inducing expression by a plant hormone containing was confirmed.

Therefore, the expression control effect of the plant hormone of the OsRP3 promoter in transgenic rice was analyzed. Specifically, the seedlings of transgenic rice, in which the OsRP3 promoter-GUS vector insertion was confirmed, were cultured in a medium containing plant hormones of Abscisic acid (ABA) or naphthalene acetic acid (NAA), an auxin for 3 days. After treatment, GUS staining was analyzed in the plants.

As a result of the analysis, it was confirmed that GUS staining increased in the vascular bundle center of the roots of plants treated with Abscisic acid (ABA) or naphthalene acetic acid (NAA) (FIG. 4A).

In addition, as a result of analyzing the GUS staining pattern in the transverse section of the root in detail, GUS staining was shown in the lateral root primordia of the plant root treated with ABA or NAA ( FIG. 4B ).

On the other hand, no staining was observed in the control group. These results prove that the OsRP3 promoter has a unique property of inducing expression specifically in the lateral root primordia in response to stress hormone (ABA) and growth regulation hormone (auxin) in the root.

These results prove that the OsRP3 promoter has a unique property of inducing expression by stress hormone (ABA) and growth regulation hormone (auxin) in the lateral root primordia of the root, and regulates root stress response and growth. It was confirmed that it can be usefully used for

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> OsRP3 promoter expressed in root vascular cylinder and uses it <130> DP20200245 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Os05g0213900P F primer <400> 1 gccgccccct tcacccgaag ggataagtcc aatg 34 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Os05g0213900P R primer <400> 2 tcggcgcgcc cacccttgtg cacggtgagg tcgcc 35 <210> 3 <211> 2000 <212> DNA <213> Artificial Sequence <220> <223> Oryza sativa Root Promoter 3 <400> 3 cgaagggata agtccaatgt tccgccggcc caggcaatta tatcgtttac gtcggcatgt 60 tcaaggctgc atcagtacat tcgacctctt ggattgatct ggtttggatg atatacttgt 120 ctacctattt atcatatatc tctgttaatc tagtcttagc atatcaattt agctctatcg 180 gctgcttctc gttttagggt ttctgcaggc atcggctaaa tcgcgttgct agattagatt 240 agcttagaca tctaccaccc tgaaaactca gtcaacgact tgattgtcta gatattatgt 300 ttcttttcat acttagtgct gcatcagtta agtttgatct actaagtcgt gcttagaacc 360 ataatctcta gcctgcttct tgattgccaa taagggtttc atcggggttt cagctggtga 420 gttatctgga cgttgcatcg gctcataagg attgcatata catataagtt ggatttagcc 480 gatgacaaca aaggcttcag tgtttaatct aatcttgtgg atttcatgac atcagacctc 540 cagccgatgt gtgttttgac cttcgaatcg atgcttattt atcatatcat tatttgctga 600 ttggtttata ctggattata ttgttatttt attacatcgt cgtcagccaa ttgcctttat 660 atcattatct acattggaca tatagccgat tgcttaaacc ctatcgctat cggctggtat 720 cggcatcggc tattatcggc atcggctgga actactccat cggcttgtca gccgatcggc 780 tatttcgatc tactatttgc atatcttgtc agttgcagga tcaaactgac tggtacgctc 840 gaatctcgtc aatatttgga tctgcacagg agctaagcag atctcccagg acggtgtgtt 900 cgattttttt tcgtcaacag tatcacacag ttattagcaa cttgttgatt gatctttgtt 960 gttagtcgtg aacaatcacc gaaacaacat ctcgtgatga acagcataaa caatttcaga 1020 atccagtaat gcatgcattt agtttgacaa aagtttactt gccgcgctag tgcaaaatgg 1080 aggcatatgc cacacacttt gcaacaacgt ttgttgcagt tcgttggctc agcgcactga 1140 ggcactacct aagcaaaaaa aataagcacg acataatata tgtaccggca gatcgactca 1200 gcttccatta gagaaagtca cacacctcga attaagataa tttttgaaat agcacgcctg 1260 ggagaagaag gtaaataata ataataatat gctacaatga tattagatca ctgatgaaga 1320 tggcagcata cacgtcttgg acatggagag gctcgattaa attacattgc attatgggga 1380 agcaccggtg gagtggtgat ggagtcgtgg gtacatgatt aatttcagag ttcaaatcct 1440 ggtgtctacg aatattatgt acacgtaaat gaactgtcaa tcgaaattta gcgagattat 1500 ggatgtaccg ttggtttccg tctttttaaa catgtgagag gggatgcatt cgcaagctgt 1560 ataagtgtgg tgttgcctgt gtaatactcc ctccgtttca aaatgtttga caccgttgac 1620 tttttagtcg tgtttgacca ttcgtcttat tcaaaaattt aagtaattat ttattctttt 1680 catctcattt gattcattgt taaatatact ttcatgtaca catatagttt tacatatttt 1740 acaatttttt tttgaataag acgaatggtc aaatatgtgc taaaagtcaa cggcgtcaaa 1800 cattttgaaa cggagggagt attgatcaat gtgtgtgcgt cccccttcta gtaaagaaat 1860 atattgcatt atgtaaacag atataggcca ggccacccac tataaatacc ttgctctccc 1920 aacattgttc tcacagctca ttacacatag cccgctcggt catcggagcg agaaagctcg 1980 ccggcgacct caccgtgcac 2000 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GUSA Anti primer <400> 4 cgcgatccag actgaatgcc c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsRP3 1957 F primer <400> 5 cggtcatcgg agcgagaaag ctcg 24

Claims (13)

A promoter that specifically induces expression in the root of a plant, comprising the nucleotide sequence represented by SEQ ID NO: 1. The method of claim 1,
The root is characterized in that the vascular cylinder (vascular cylinder), the promoter. 3. The method of claim 2,
The vascular bundle center column, characterized in that the xylem tissue, the promoter. The method of claim 1,
The promoter is characterized in that for inducing expression in the lateral root primordia of the root by Abscisic acid (ABA) or auxin (auxin), the promoter. A recombinant vector comprising a foreign gene encoding a target protein operably linked to the promoter of any one of claims 1 to 4. Agrobacterium transformed with the vector of claim 5 ( Agrobacterium ). A transgenic plant transformed with the vector of claim 5 or the Agrobacterium of claim 6. 8. The method of claim 7,
The plant is rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats or food crops including sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion or carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots or bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies or tulips; and ryegrass, red clover, orchard grass, alpha alpha, tall fescue or perennial ryegrass, characterized in that at least one selected from the group consisting of feed crops, transgenic plants. A method for producing a transgenic plant comprising the step of transforming the plant with the vector of claim 5 to express the target protein in the plant. 10. The method of claim 9,
The method for producing a transgenic plant, characterized in that the target protein is specifically expressed in the root of the plant. 10. The method of claim 9,
The root is a method for producing a transgenic plant, characterized in that the vascular bundle center (vascular cylinder). 12. The method of claim 11,
The method for producing a transgenic plant, characterized in that the vascular bundle center line is a xylem tissue. 10. The method of claim 9,
The method for producing a transgenic plant, characterized in that the expression of the target protein is induced in the lateral root primordia of the root by Abscisic acid (ABA) or auxin.

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