KR101549126B1 - Vascular tissue specific promoter Br11 - Google Patents

Abstract

The present invention relates to a vascular tissue-specific expression promoter Br11. More specifically, it is confirmed that a promoter Br11 separated from a Chinese cabbage is specifically expressed to vascular tissues. Therefore, the promoter can be helpfully used when improving plants through a biotechnology method by expressing genes useful in agriculture in vascular tissues of plants using the same.

Description

Conduit-specific expression promoter Br11 {Vascular tissue specific promoter Br11}

The present invention relates to a duct-specific expression promoter Br11, an expression vector comprising the same, and a plant transformed with the expression vector.

Breeding It is an agricultural technology that cultivates, propagates and distributes crops whose utility value is higher than that of the past. Methods for breeding include isolated breeding, hybrid breeding, mutation, diploid breeding, and molecular breeding. Molecular breeding techniques have attracted attention in recent years due to advances in molecular biology techniques.

Molecular breeding is a method of introducing a new trait into a living organism by using molecular biology techniques and selecting the same. It is less time and cost than conventional breeding, and transgenic transduction which can not be obtained by conventional breeding is possible. For such molecular breeding, various means such as a means for expressing a useful gene and a transformation technique are required. In particular, the development of a promoter and a vector which exist in the upper part of various genes for expressing a desired gene at a desired site Is required.

Promoters that are specifically expressed in ductal tissues are being developed because they are useful for imparting resistance to diseases infected along conduits or for improving wood quality. Poplar-derived Lmx promoter (US Patent Publication No. 20070180580) and amber-derived PP2-type gene promoter (US Patent No. 5495007) have been developed as conduit-specific promoters and PLRV resistant plants using the conduit-specific promoters RolC and Sh (Michael W et al., Plant Molecular Biology, 33: 729-735, 1997). However, such promoters have a disadvantage in that the expression inducing ability is not sufficient or the versatility is poor, and it is urgent to develop efficient and excellent duct specific promoters.

Thus, the present inventors searched for a promoter specifically expressed in the duct, and confirmed that the Chinese cabbage-derived promoter Br11 was expressed in a duct-specific manner. Therefore, the use of the conduit specific expression promoter Br11 will be useful because it can specifically induce expression of a target gene in a duct.

It is an object of the present invention to provide a vascular tissue-specific expression promoter derived from Chinese cabbage (Brassica rapa) comprising the nucleotide sequence of SEQ ID NO: 1.

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

It is another object of the present invention to provide a microorganism transformed with said recombinant expression vector.

It is still another object of the present invention to provide a transformed dicotyledonous plant transformed with said microorganism.

It is another object of the present invention to provide a method for producing a transgenic plant that expresses a target protein in a duct-specific manner using the recombinant expression vector.

In order to accomplish the above object, the present invention provides a vascular tissue-specific expression promoter derived from Chinese cabbage (Brassica rapa) comprising the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a recombinant expression vector comprising the promoter and a base sequence encoding a target protein operably linked to the promoter.

The present invention also provides a microorganism transformed with said recombinant expression vector.

In addition, the present invention provides a transformed dicotyledonous plant transformed with said microorganism.

In addition,

1) a recombinant expression vector pBGWFS7-PBr11 comprising a vascular tissue-specific expression promoter derived from a cabbage consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a target protein operably linked to the promoter Producing a vector having a cleavage map as shown in Figure 2;

2) introducing the expression vector prepared in step 1) into Agrobacterium; And

And 3) transforming the plant with Agrobacterium of step 2). The present invention also provides a method for producing a transgenic plant that expresses a target protein in a duct-specific manner.

Since the duct-specific expression promoter Br11 derived from Chinese cabbage of the present invention is a novel promoter unique to Korea that induces the expression of the target protein specifically in the ductal tissue of the plant, it is possible to express the agricultural useful gene in the duct of the plant, It is useful when you want to improve by the method.

Brief Description of the Drawings Fig. 1 is a diagram showing the nucleotide sequence of Brassica rapa-derived vascular tissue-specific expression promoter Br11 comprising the nucleotide sequence of SEQ ID NO: 1.
Fig. 2 is a schematic diagram of vectors pBGWFS7-PBr11 and pBGWFS7-P35S (control) for the analysis of promoter activity: (A) Br11 promoter and (B) 35S promoter.
FIG. 3 is a chart showing Br11 promoter activity by growth stage and organization: 7D, 7 days after germination; 21D, 21 days after germination; Nutritional growth; 42D; 42 days after germination Reproductive growth.
FIG. 4 is a diagram showing tissue-specific expression of Br11 promoter by RT-PCR analysis: R, root; St. stem; L, leaf; F, flowers; Si, pod.

Hereinafter, the present invention will be described in detail.

The present invention provides a vascular tissue-specific expression promoter derived from Chinese cabbage (Brassica rapa) comprising the nucleotide sequence of SEQ ID NO: 1.

A conduit is the most differentiated part of a plant tissue, also known as a water tube. It is not found in gymnosperms and ferns, but it appears in most flowering plants. It is presumed that the cell walls of both cells above and below the cell have been lost as the primitive form of the vasculature (or duct) has been differentiated. The conduit consists of vertically elongated water tube cells, ranging in length from cylindrical to dull drum shapes.

The conduit may be, but is not limited to, the leaf, stem, root of the plant, main vein and side vein or whole conduit tissue.

The promoter is a gene site located upstream of the coding site, which generally includes a point at which transcription is initiated. The promoter of the present invention affects gene expression in response to external stimuli, such as a TATA box, a CAAT box site, An enhancer that promotes expression of almost all genes regardless of the site and position and orientation of the gene. The promoter of the present invention is characterized in that it specifically induces expression only in a duct of a plant. Expression of the promoter may be normal and may be temporal depending on the growth degree, night / day, temperature, season, and the like.

The present invention also provides a recombinant expression vector comprising the promoter and a base sequence encoding a target protein operably linked to the promoter.

The recombinant expression vector is preferably pBGWFS7-PBr11 shown in FIG. 2, but is not limited thereto.

The expression vector is a vector capable of expressing a target protein or a target RNA in a host cell, and includes a necessary regulatory element operatively linked to express the gene insert. Expression vectors include expression control sequences and signal sequences or leader sequences for membrane targeting or secretion, and may be prepared variously according to the purpose. An expression control sequence is a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. The expression vector may also include a selection marker for selecting a host cell containing the vector, and may contain a replication origin if it is a replicable expression vector.

The recombinant expression vector of the present invention may include a base sequence encoding various proteins known in the art, and the base sequence encoding the target protein is operably linked to the promoter. Operably linked, it is meant that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments.

The target protein according to the present invention may be any target protein derived from the outside, that is, an exogenous protein or an endogenous protein and a reporter protein, which is intended to be expressed by the promoter of the present invention in a duct-specific manner. The exogenous protein refers to a protein that is not naturally present in a specific tissue or cell of a plant, and the endogenous protein refers to a protein expressed by a gene naturally present in a specific tissue or cell. The reporter protein is a protein expressed by a reporter gene, and refers to a marker protein that indicates its activity in a cell by its presence. The target proteins include antibiotic resistance proteins, herbicide resistance proteins, virus resistance proteins, insect resistance proteins, metabolism related proteins, luminescent proteins, GFP (green fluorescence protein), GUS (β-glucuronidase), GAL But is not limited thereto.

The expression vector according to the present invention can be prepared by inserting the promoter of the present invention using a conventional vector used for protein expression as a basic framework and inserting a nucleotide sequence encoding the target protein downstream of the promoter. The conventional vector used for protein expression may be a known recombinant expression vector, particularly a recombinant expression vector for plant transformation, which can be used for the production of transgenic plants. Examples thereof include a plasmid vector such as an E. coli-derived plasmid, a Bacillus subtilis-derived plasmid, a yeast-derived plasmid and a Ti plasmid, a cosmid vector, a bacteriophage vector and a viral vector. However, common binary vectors such as pCHF3, pPZP, pGA and pCAMBIA a binary vector or a cointegration vector may be used. There are many types of binary vectors that are widely used in plant transformation and almost all binary vectors are available at international centers and university laboratories such as CAMBIA (Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT 2601, Australia) Available.

The recombinant expression vector is preferably pBGWFS7-PBr11, but is not limited thereto.

In one embodiment of the present invention, the Br11 promoter of the present invention having the nucleotide sequence of SEQ ID NO: 1 is inserted into a known pBGWFS7 vector having a reporter system in which a green fluorescent protein gene Egfp and a blue coloring gene Gus are linked, A recombinant expression vector, pBGWFS7-PBr11, operatively linked to the upstream portion of the Gus gene was prepared (see Example 2 and Figure 2).

The present invention also provides a microorganism transformed with said recombinant expression vector.

The recombinant expression vector comprising the promoter of the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter may be introduced into the host using methods known in the art. Examples include, but are not limited to, calcium chloride and heat shock, particle gun bombardment, silicon carbide whiskers, sonication, electroporaion, PEG- A fusion method, a microinjection method, a microinjection method, a liposome mediated method, and a vacuum infiltration method.

The microorganism may be Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus, Agrobacterium, Agrobacterium tumefaciens, and Agrobacterium rhizogene. However, the present invention is not limited thereto.

The recombinant expression vector according to the present invention can be introduced into plants using methods known in the art. For example, but not limited to, Agrobacterium sp. Mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation electroporation, electroporation, PEG-mediated fusion, microinjection, microinjection, liposome mediation, vacuum infiltration, and anoxia method. Preferably, the Agrobacterium sp. Mediated method can be used and the methods exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985; An et al., EMBOJ. 4: 227-288, 1985).

In addition, the present invention provides a transformed dicotyledonous plant transformed with said microorganism.

The dicotyledonous plants may be selected from the group consisting of soybean, potato, red bean, Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, carrot, ginseng, tobacco, cotton, sesame, But are not limited to, those selected from the group consisting of rapeseed, apple tree, pear, jujube tree, peach, sheep grape, grape, citrus, persimmon, plum, apricot, rose, gerbera, carnation, chrysanthemum and red clover.

In addition,

1) a recombinant expression vector pBGWFS7-PBr11 comprising a vascular tissue-specific expression promoter derived from a cabbage consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a target protein operably linked to the promoter Producing a vector having a cleavage map as shown in Figure 2;

2) introducing the expression vector prepared in step 1) into Agrobacterium; And

And 3) transforming the plant with Agrobacterium of step 2). The present invention also provides a method for producing a transgenic plant that expresses a target protein in a duct-specific manner.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Conduit-Specific Promoter Isolation

In order to obtain Br11, a conduit-specific promoter from Chinese cabbage (Brassica rapa), microarray analysis of Chinese cabbage tissues was carried out to identify genes showing tissue-specific expression, and then one of the tissue- A nucleotide sequence of about 2 kb region including TATA box and the like was extracted above the start codon of the unknown gene of the protein of interest. Br11 B1: 5'-AAAAAGCAGGCTTGCTGGTTACACATTTTGTT-3 '(SEQ ID NO: 3) and Br11 B2: 5 (SEQ ID NO: 3) were used to specifically amplify the Br11 promoter using a gateway cloning system from Chinese cabbage BAC clone (KBrH006P22) '-AAAGCTGGGTATGTTTTATGGGGGAAGATT-3' (SEQ ID NO: 4). Then, genomic DNA was extracted from the Chinese cabbage BAC clone and PCR was performed to isolate about 2 Kb of Br11 promoter. At this time, the PCR conditions were denatured at 95 ° C for 10 minutes, followed by 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, and 68 ° C for 2 minutes as one cycle, followed by reaction at 68 ° C for 10 minutes. The nucleotide sequence (SEQ ID NO: 1) of the entire 2000 bp Br11 promoter region of the DNA obtained in the PCR reaction was analyzed and shown in FIG.

In order to use the CaMV 35S promoter, which is known as a systemic spontaneous promoter, as a control for the activity of the promoter activity, 35S B1: 5'-AAAAAGCAGGCTTCGACCTGCAGGCATGCA-3 '(SEQ ID NO: 5) and 35S B2: 5'-AGAAAGCTGGGTGTGCGGCCGCCTGCAGGT-3 '(SEQ ID NO: 6). Using a pK2GW7 (VIB, Belgium) gateway vector containing the 35S promoter as a template, denaturation at 95 ° C for 10 minutes, reaction at 94 ° C for 1 minute, 65 ° C for 30 seconds and 65 ° C for 2 minutes was repeated 30 times The reaction was carried out at 72 ° C for 7 minutes. As a result, the nucleotide sequence (SEQ ID NO: 2) of the entire 357S promoter region of 1077 bp of the DNA obtained in the PCR reaction was analyzed.

< Example  2 > Production of a vector containing a duct-specific promoter

A vector was constructed to test the activity of the Br11 promoter isolated in Example 1 above.

Specifically, in order to test the activity of the Br11 promoter of SEQ ID NO: 1 isolated from Chinese cabbage, pBGWFS7 (VIB, Belgium), in which a reporter system in which the Egfp and GUS genes are linked, was used to test the final plant transformation promoter Carrier pBGWFS7-PBr11 was prepared (Fig. 2A). As a control, a 35S promoter, which is a persistent promoter, was also cloned into pBGWFS7 to construct a vector (Fig. 2B).

The PCR products amplified and isolated in Example 1, namely, the Br11 promoter and the 35S promoter, were amplified by 5'-GGGGACAAGTTTGTACAAAAAAAGCAGGCT-3 '(SEQ ID NO: 7), which is adapter primer B1 containing the entire bacterial-specific recombinant nucleic acid sequence region when the bacterium is infected with bacteriophage ) And B2, 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '(SEQ ID NO: 8). The PCR products were subjected to BP recombination reaction with the pDONR221 vector containing the bacteriophage-specific recombinant nucleic acid sequence region when the bacterium was infected with the bacteriophage, and cloning vectors of pDONR-PBr11 and pDONR-P35S were produced. The two subcloned carriers were transfected with the pBGWFS7 vector containing the reporter system in which the Egfp and Gus genes were linked in order to construct the final transgenic carrier and then subjected to the LR recombination reaction to obtain the final pBGWFS7-PBr11 And The pBGWFS7-P35S carrier was completed. At this time, pBGWFS7, which is used as a binary vector, has a spectinomycin resistance gene for microorganism selection and contains herbicide resistance Bar gene as a plant selection factor.

< Example  3> Production of transgenic plant-specific promoter transgenic plants

PBGWFS7-PBr11 and pBGWFS7-P35S, which are the reporter transporters for plant transformation prepared in Example 2, were transfected into Agrobacterium and infected with Arabidopsis thaliana grown for 4 weeks after sowing. Transgenic T0 transformants were cultivated, and seeds were cultivated and seedlings were seeded. The transformants of T1 generation survived by treating 0.3% basta on the 1st and 17th day of growth on the 10th day of growth, . Subsequently, the T2 posttransformants were grown in the same manner and used for assaying the promoter activity.

< Experimental Example  1> Br11  Analysis of tissue-specific activity of promoter

<1-1> GUS  Tissue specific expression analysis by staining

Conduction - specific promoters were identified in the transgenic Arabidopsis thaliana by tissue - specific activities.

Specifically, a T2 generation Arabidopsis plant transformed with the conduit-specific promoter Br11 produced in Example 3, a Arabidopsis plant transformed with the 35S promoter, and a control non-transformed Arabidopsis plant were germinated, On days 21 and 42, GUS-assay buffer [Sol. I: 5-Bromo-4-chloro-3-indolyl β-Dglucuronide cyclohexylammonium salt (X-gluc CHA salt) 20 mM, Sol. II: NaH2PO4 H2O 100mM, NaEDTA 10mM, Triton-X-100 0.1%, pH 7.5], treated at 37 ° C for 24 hours, and treated with 70% ethanol to remove chlorophyll. The plants were observed with an optical microscope or with naked eyes.

As a result, GUS coloration was confirmed in the ductal tissues of all the organs such as leaves, flowers and roots of the Arabidopsis thaliana transformed with the duct-specific promoter Br11 (Fig. 3).

<1-2> RT - PCR On by Br11  Tissue specific expression analysis of promoter

pBGWFS7-PBr11 Transgenic Arabidopsis RNA was extracted from the tissues of the flowers (F), leaves (L), stems (St), pods (Si) and roots (R) of the T2 generation plants and GUS-specific primers (GUS- (Elongation factor-1a) gene specific for the Arabidopsis thaliana house keeping gene and the 5'-ACCTGCGTCAATGTAATGTTCTGC-3 '(SEQ ID NO: 9); GUS-R, 5'-CTCCCTGCTGCGGTTTTTCA- RT-PCR was performed using primers (EF1a-F, 5'-GTTTCACATTAACATTGTGGTCATT-3 '(SEQ ID NO: 11); EF1a-R, 5'-GAGGTACCAGTAATCATGTTCTTG-3' (SEQ ID NO: 12)).

As a result, the US band and the EF1a-specific band (B) were observed in the tubular tissues of the flowers (F), leaves (L), stems (St), pods (Si) and roots All were detected in all tissues (Fig. 4).

<110> republic of korea <120> Vascular tissue specific promoter Br11 <130> P14R12D0138 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 2000 <212> DNA <213> Brassica rapa <400> 1 tgctggttac acattttgtt tataacacac ctgtcgttta ttcctggatt tttaagggca 60 acatctatca tctggtttac atgtaccctt tctttgttct tttcgaattt aacttttagt 120 cagtattttt attaagtgtg aaacgaactt atttataagt gtaattgcct gcccaaacag 180 gataataatg ggaagcgtga gcagcttcgt gattcagcac gctccttgcc ctgtcaccgt 240 tgtcaaggat aacgactctc actaaataat gaagtctatc tcattatgtt tttaccaaaa 300 caatcatcgc tgcgtatggt tttaattaaa gaataaaaga ccctttctct cgtatttgta 360 tttgaattcg tattcgtatc aatatattat tacatcgcca tagttgttca caccctaaaa 420 acagagagtt tttggaagaa ttctatgttt tcctgcaaat tatttccatt actttcgttg 480 tttttcggcc agaatgactc catgtgattt agggatcaat gtgtggtggg gttatttgaa 540 tagtttgtta tacccaaatg gttttaaaca agtttgcgtg acagaaatca catggatagg 600 tcggttcctt ttcatcggcc actatgaaaa gaaattattag aatatttata aggatagaaa 660 acaaatagta tattaattat tgttattctt tggatacacg atgcgaccaa aaagatatac 720 atgaattact ggtctttttc ttaaaatata ggagtttttt ttaactcaaa attttattaa 780 aactttcata atgaaataca ttatggattg caaaaattcg acaggaaata attcgacagg 840 aaataataat agtttcggaa gcagtaatcc aaaagaaagt aacaataaaa atgagatagg 900 atatatgaaa aataaatatt agagaactaa gtatactaaa gctggaactg atagactgat 960 catgaccata tacactcgtg aaaaatatga gacaaaatat gagaccgaaa acaattaaaa 1020 aaccaataat ttaggtaaat ccaaccctaa agaatacacc accaaaccaa aacatgcaca 1080 acatcgaaac aagcatgtga aaagataaac ttacatcaac acgagagatt gtttgaaact 1140 ggagtcgaaa caaaaccaag tcaaaaccaa acacctaggc tcaaagacac aagaacttgc 1200 aatactaaaa agaaaaatac cgatgaagat ttacagatct tacacacgaa tattcgttag 1260 acataataac aatccgcaga attaaaaatg gccacctctt cgcaaaagaa aatctgcaaa 1320 tcaaacttac tatttcatct cgaatcaacc acaaaaagag atcggaatga cctggagttt 1380 ttttcttctt ctctctcgac tgcttgacct gatcaaaata taggagttaa tagtgtagtt 1440 tatttcagag tttaaaaatt tgtgcgttag taatgtagtt tatttcccac catggtgaga 1500 tgaccatttt gtagagattt gggagtcgag ctggacctga ggatactcaa aagaatgtcg 1560 ggtcaaaatc ttgttaatgg gctagcttaa tgcttgatct aaacaagctc tagaatacca 1620 caacttgtgc aaacttcgaa aagaaaaaaa agctcgttat aataatgtac aactaacact 1680 tactcaaaag ttgcaaggtc ggcgatgaca caaccacaaa tccaccattc gatttgataa 1740 tcgaaattaa gaaataaaat aagtattaga atgtttgttc agtgatctct cagacatcaa 1800 cacctggaaa cttctgttgt tggtaagaac actacctctt tctcactcaa gtgcttactc 1860 cactaccttc agtgccttga acctaaacac aaaattcctg ctatataaat tcacacattc 1920 aagaaccagt ggcaacacac agacaatatc aacacaaaac agaacctcat tattttacca 1980 aatcttcccc cataaaacat 2000 <210> 2 <211> 1077 <212> DNA <213> 35S promoter <400> 2 tcgacctgca ggcatgcaag ctctcccata tggtcganat ctcctttgcc ccggagatca 60 ccatggacga ctttctctat ctctacgatc taggaagaaa gttcgacgga gaaggtgacg 120 ataccatgtt caccaccgat aatgagaaga ttagcctctt caatttcaga aagaatgctg 180 acccacagat ggttagagag gcctacgcgg caggtctcat caagacgatc tacccgagta 240 ataatctcca ggagatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca 300 ggactaactg catcaagaac acagagaaag atatatttct caagatcaga agtactattc 360 cagtatggac gattcaaggc ttgcttcata aaccaaggca agtaatagag attggagtct 420 ctaagaaagt agttcctact gaatcaaagg ccatggagtc aaaaattcag atcgaggatc 480 taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg 540 acaagaagaa aatcttcgtc aacatggtgg agcacgacac tctcgtctac tccaagaata 600 tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa agggtaatat 660 cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa aggacagtag 720 aaaaggaagg tggcacctac aaatgccatc attgcgataa aggaaaggct atcgttcaag 780 atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 840 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg 900 taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 960 catttcattt ggagaggact gcaggacgat ccgtattttt acaacaatta ccacaacaaa 1020 acaaacaaca aacaacatta caatttacta ttctagtcga cctgcaggcg gccgcac 1077 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Br11 amplifying primer <400> 3 aaaaagcagg cttgctggtt acacattttg tt 32 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Br11 amplifying primer <400> 4 aaaaagcagg cttgctggtt acacattttg tt 32 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 35S amplifying primer <400> 5 aaaaagcagg cttcgacctg caggcatgca 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 35S amplifying primer <400> 6 agaaagctgg gtgtgcggcc gcctgcaggt 30 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter primer <400> 7 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter primer <400> 8 ggggaccact ttgtacaaga aagctgggt 29 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GUS primer <400> 9 acctgcgtca atgtaatgtt ctgc 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUS primer <400> 10 ctccctgctg cggtttttca 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EF1a primer <400> 11 gtttcacatt aacattgtgg tcatt 25 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EF1a primer <400> 12 gaggtaccag taatcatgtt cttg 24

Claims (8)

A vascular tissue-specific expression promoter derived from Brassica rapa consisting of the nucleotide sequence of SEQ ID NO: 1.
A recombinant expression vector comprising the promoter of claim 1 and a nucleotide sequence encoding a target protein operably linked to the promoter.
3. The recombinant expression vector according to claim 2, wherein the recombinant expression vector is pBGWFS7-PBr11 shown in Fig.
A microorganism transformed with the recombinant expression vector of claim 2.
The method of claim 4, wherein the microorganism is selected from the group consisting of Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus Wherein the microorganism is selected from the group consisting of Staphylococcus, Agrobacterium tumefaciens and Agrobacterium rhizogene.
A transformed dicotyledonous plant transformed with the microorganism of claim 4.
The method according to claim 6, wherein the dicotyledonous plant is selected from the group consisting of soybean, potato, red bean, Arabidopsis, Characterized in that it is selected from the group consisting of radish, perilla, peanut, rapeseed, apple, pear, jujube, peach, sheep, grape, citrus, persimmon, apricot, rose, gerbera, carnation, chrysanthemum, red clover Dicotyledonous plants.
1) a recombinant expression vector pBGWFS7-PBr11 comprising a vascular tissue-specific expression promoter derived from a cabbage consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a target protein operably linked to the promoter Producing a vector having a cleavage map as shown in Figure 2;
2) introducing the expression vector prepared in step 1) into Agrobacterium; And
3) a step of transforming the plant with Agrobacterium of step 2).

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