KR20090052494A - Leaf-specific gene pto1sx162060 associated with development of leaf - Google Patents

Abstract

The present invention relates to a PTO1SX162060 protein that is specifically expressed in the leaf tissue of tomato, a gene encoding the protein, a recombinant vector comprising the gene, a transformant transformed with the recombinant vector, and an antibody against the protein. will be.

Tomato, Leaf Specific Gene, PTO1SX162060 Protein, Recombinant Vector, Antibody

Description

Leaf-specific gene PTO1SX162060 associated with development of leaf}

The present invention relates to a PTO1SX162060 protein that is specifically expressed in leaf tissue involved in the production of leaves, a gene encoding the protein, a recombinant vector comprising the gene, a transformant transformed with the recombinant vector, and the protein. It relates to an antibody.

Plants act as producers in ecosystems and have biochemical reactions that produce fundamental compounds for living things through photosynthesis, which produces carbohydrates and oxygen. Oxygen is an essential element for life's aerobic respiration, and many animals, including humans, depend on plants for their basic energy.

Tomato is composed of a total of 12 chromosomes, which is being actively studied by being selected as a model for genome research of eggplants and crops. Tomato has a total of 3,500 molecular markers developed and produced with four genetic maps, and since there are about 240,000 ESTs that have been analyzed for sequencing, research on the background of EST is very active.

Therefore, the present inventors are trying to find out whether the PTO1SX162060 gene is specifically expressed in the leaves of tomatoes while studying the genes specifically present in the leaves of tomatoes.

In order to solve the above problems, the present invention provides a PTO1SX162060 protein that is specifically expressed in the leaf tissue.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a transformant transformed with the vector.

The present invention also provides an antibody against the protein.

Leaf tissue specific genes are essential genes involved in the production of leaves, and it is important to reveal information on protein-protein interactions, pathways or regulatory networks involved in development. However, analyzing and generating individual genes to reveal such information takes a very long time to identify genes actually involved in tissue development.

We provide the fact that the PTO1SX162060 gene is specifically expressed in leaf tissues, allowing researchers to easily access information such as protein-protein networks or pathways for the generation of leaf tissues. The fact is that false positives can be reduced in related studies. In addition, the PTO1SX162060 gene can be used as a marker in the diagnosis of the development of leaf-related diseases, making it easier to diagnose. When designing a drug as a therapeutic agent for disease symptoms, the protein target for PTO1SX162060 may be reduced. This allows for faster treatment at the onset of the onset.

In order to achieve the above object, the present invention provides a PTO1SX162060 protein that is specifically expressed in the leaf tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2.

Prediction of tissue-specific genes, such as DNA chips, is not possible to distinguish whether the gene is actually involved in tissue formation mechanisms, housekeeping genes, or experimental errors due to numerous errors in experiments. However, if the candidate group is identified as a tissue-specific gene by performing an Audic-Claverie Test based on the EST (Expressed Sequence Tag) or using an experiment such as RT-PCR, the candidate may be judged as a genetic influence factor. Reliability is increased. The inventors have provided higher reliability for candidate groups of genes involved in tissue development by examining candidate groups of genes involved in tissue development and examining whether they contain tissue specific information.

In order to investigate genes specifically expressed in leaf tissues, the inventors use Expressed Sequence Tag (EST) data to investigate whether EST has a large number of specific genes, and perform Audit-Claverie Test to perform tissue-specific information. After identifying a number of genes that may have a gene, the relevant information is found in the TAIR database, the function is analyzed using the Gene Ontology database, and the protein in the STRING database is used to determine whether the gene is a novel gene. Information about protein interactions was extracted. Among these genes, PTO1SX162060 gene was actually verified by RT-PCR experiment that it is a leaf tissue specific gene. FIG. 1 is a schematic diagram of a system for analyzing genes specific to leaf tissues to determine whether a significant gene is actually a tissue-specific gene and to analyze information about protein-protein interactions.

Accordingly, the present invention provides a PTO1SX162060 protein that is specifically expressed in leaf tissue development. The range of PTO1SX162060 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The PTO1SX162060 protein according to the present invention is preferably derived from tomato, but is not limited thereto.

The PTO1SX162060 protein according to the present invention can be used as a target of therapeutic drugs for diseases related to leaves. Examples of leaf-related diseases include tomato leaf fungus, leaf blight, and powdery mildew. When designing a drug as a treatment for disease symptoms of leaves, the drug target can be reduced with the PTO1SX162060 protein, allowing for faster treatment at the onset of onset. In addition, the promoter of this gene can be used to control the expression of external genes specifically for leaves to accumulate specific useful proteins in the leaves, or to introduce desired traits such as leaf shape and size.

The present invention also provides a gene encoding the PTO1SX162060 protein. Genes of the invention include both genomic DNA and cDNA encoding the PTO1SX162060 protein. Preferably, the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 can do. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the gene according to the present invention. The recombinant vector is preferably a recombinant plant expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the fetuin DNA according to the invention into a plant host are viral vectors such as those that can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

Recombinant plant expression vectors according to one embodiment of the invention will preferably comprise one or more selectable markers. The marker includes dihydrofolate reductase or neomycin resistance to eukaryotic cell culture. The most widely used plant transformation marker is the neomycin phosphotransferase II (nptII) gene isolated from Tn5 and another marker gene is the hygromycin phosphotransferase gene that confers resistance to the antibiotic hygromycin. to be.

In a recombinant plant expression vector according to one embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, and examples thereof include nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline (phaseoline) terminator, Terminators of the octopine gene of agrobacterium tumefaciens, but are not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The present invention also provides a transformant transformed with the recombinant vector according to the present invention. The transformant is preferably a plant. The plant is a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass. Preferably, the plant is Arabidopsis, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, Chinese cabbage, cabbage, mustard, watermelon, melon , Cucumbers, pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, or peas.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The invention also provides antibodies to the proteins of the invention. The antibody may be a monoclonal or polyclonal antibody, and the method for preparing these antibodies may use methods known in the art.

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

Example

Example  One: TISA  Establishment of Leaf-Specific Gene Candidates Using Database

Noh SJ 2006 [DNA Res 13 (5) 229-43] published a TISA (Tissue-specific Alternative Splicing) database containing information on genes, transcript structure, alternative splicing events and tissue specificity. TISA is a database that identifies tissue-specific genes by clustering EST data including the information of the expressed tissue into a genetic map and verifying that the number of ESTs of the corresponding TISA genes is statistically significant. If there was a gene predicted as a leaf tissue specific gene in TISA, it was extracted as a candidate group of leaf tissue specific gene.

Example  2: of leaf tissue specific gene candidates RT - PCR  Experiment

Total RNA was extracted using the Trizol RNA Extraction Manual, all col-0 type. There are six different RNA tissues used, Rosettle Leaf, Coullne Leaf, Stem, Root, Flower and Leaf. Roche Co. CDNA was synthesized according to the Ltd RT-PCR kit manual and PCR was performed. Primers used are as follows. All were designed using eprimer3 primer design software and in-house python wrapping script. PCR conditions were finally extended for 72 min 10 min after 35 cycles of pre-denatured 94 ° C. 3 min, followed by 94 ° C. 30 sec, annealing 58-60 ° C. 30 sec, extension 72 ° C. 1 min. 1% agarose gel electrophoresis was performed to confirm the PCR product.

Primer sequence:

Forward primer: 5'-GCAAGGCAGGTCGAAGAAGT-3 '(SEQ ID NO: 3)

Reverse primer: 5'-TGAGATGACCTCACGCTCAAT-3 '(SEQ ID NO: 4)

Figure 2 shows the RT-PCR results of the PTO1SX162060 gene. As can be seen in Figure 2, it was found that the PTO1SX162060 gene is rarely expressed in other tissues, but specifically expressed in leaf tissues.

Example  3: Gene Ontology Reference of Leaf-Specific Gene Candidates Using

Gene Ontology (http://www.geneontology.org/) is a database of the common functions of genes. It does not infer functions by simple similarity in sequence, but how it integrates the information of known functions. Focusing on. The consortium that created the Gene Ontology database refers to a number of databases to create ontology that can be used to describe biological functions, which aim to establish a controlled vocabulary that can describe the functions. Gene Ontology database classifies gene into Molecular Function, Biological Process, and Cellular Component, and each has hierarchical controlled vocabulary.

Leaf tissue specific gene candidates were searched using the search function provided on the Web to confirm what function the gene candidate group specifically showing in the leaf tissue has on the Gene Ontology database.

Investigations reveal no known information, suggesting that the gene is still unknown.

Example  4: STRING Analysis of Protein Interactions in Leaf-Specific Gene Candidates

STRING (Search Tool for the Retrieval of Interacting Proteins; http://string.embl.de) is a database that provides comprehensive information on protein interactions.In 2000, Snel B 2000 Nucleic Acids Res 28 (18) 3442 Version 7.1 was released in 2007 as a continual upgrade since its release by -4.

In order to confirm the protein interaction information of the leaf tissue specific gene candidate, the sequence of the protein in STRING is determined through our candidate data and BLAST alignment (identity> = 90% and coverage> = 20%). Then, corresponding protein interaction information was extracted.

However, no protein with similarity exists in the corresponding criteria, which is thought to be caused by the unknown genes and proteins.

1 is a schematic of a system for analyzing leaf specific genes.

Figure 2 shows the RT-PCR results of the PTO1SX162060 gene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Leaf-specific gene PTO1SX162060 associated with development of          leaf <130> PN07088 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1804 <212> DNA <213> Tomato <220> <221> gene (222) (1) .. (1804) <223> Tomato PTO1SX162060 gene <400> 1 gagactagtt ccaatttcat atattgtttt taaaaaaaaa aacacaattt tcatgcaaaa 60 tatagtaact aaatagaaag aattagcaat tcatctgcta tttctttcaa aaaaaataaa 120 ataaaaattc atatcctatg gctccaactc ttacttcaaa ttcatttctc ctttcaacta 180 cacctcatcc aagattatca ttcaaaaatc caagattcat tgtttctgct aaaaaatctg 240 gacaaaatca agaaactaca accaattctg gcaacccctt caactttgat ttcggtaagt 300 tacctgatgt gacgtctttg atacctgcta gcacgaacac atcttctgga ttgtcttttg 360 gaaggcaaag ggcaaaggat cctggaacgg tgtttgttgc tggtgcaact gggcaagctg 420 gtatccgcat tgcacagttg ctgttgcgtg aaggttatag tgtcagagct ggagtttctg 480 accttggtgc tgcacaagag ctagctcgtc tagccgttag ctacaaggta atctctaacg 540 atgagtcaaa acgtctgaat gcagttgcat catcatttca agatgcagaa tcgattgcaa 600 aggcaatcgg aaatgctaac aaagttgtgg ttactattag taagggagag gacggtcctg 660 cgactgaagt caccacaaca gatgctgtac aagttataga agcagcacaa ttagctggcg 720 ttggacatgt ggccatagtc tatgatgaat cttctccagt aggctctaca tacaatgtgc 780 tcgacggaat tacatcattc ttcaacaatc tgttctcaaa atctcaaccg ttgacgatag 840 cagagtttct gcagaagata gtagagacag acctaagcta tacactcata aaaaccagat 900 taactgacga tttctcccct gagagctcat acaaaatcgt cgtttcagct gaaggaagtg 960 ctgatgctga cgcctataaa gtggggaagt ctcagatagc taagctcgtt gtggatgttt 1020 tctccaacac agcggtggca gaaaataagg ttgtcgaagt ctttgctgat ccatcagcac 1080 cgccaaagac tgtggatgaa ctttttagcg tcattcctga ggatggtcgg agaaaggcct 1140 atgcagaagc ccttgaaaaa tcgaaagctg aggaagaagc tcgaggagtt gctgaagcat 1200 ccaagaggca ggaacaagaa gcaaagaagc ttgaaaagaa agaagcaaag gctgctgcta 1260 ccatcgcaaa ggaacctgaa gagaaggcgt catcaccttc cgttccttct gttgaaagtc 1320 tactagacaa agcaaaagac tttagtacag gattttcatt tgaaaaactc agctcacagc 1380 tcaaatcagc tgttgaaaag gctaacgagg aatcagacgc tcaagttgct accgttaggg 1440 gacaggccaa ggctaaaaat ttgcctgctc agaaagcagt ggtgaaaact cctccaagga 1500 aaccatttgc ttcgaaaacg aagaaggagg ctccgaaacc agctaaacaa acagaggttg 1560 cttcaacaga gaagaggaag atttttggtg gactgtttca acaagaaacc atctatgttg 1620 atgactaaga tgaatgaata agagaaagcc aaaatattaa ttttctatat atcattttag 1680 cctaattatg aggttacaaa caatggttta actgaggctg agtgagaaag catgtaaatc 1740 cctgcacatt ttctgtggat cttagtgtat atttgcctca aatgcaacat tcaggcactt 1800 gttc 1804 <210> 2 <211> 510 <212> PRT <213> Tomato <220> <221> PEPTIDE (222) (1) .. (510) <223> Tomato PTO1SX162060 protein <400> 2 Met Ala Ser Ser Ser Thr Ser Phe Pro Leu Thr Thr Ala Pro Pro Gln   1 5 10 15 Gly Val Arg Phe Asn Arg Arg Lys Pro Arg Leu Thr Val Trp Ala Lys              20 25 30 Gln Thr Ala Phe Gln Leu Gly Lys Thr Lys Gly Asp Asp Asp Ser Glu          35 40 45 Gly Lys Gln Lys Gly Lys Asn Pro Phe Gln Phe Asp Phe Gly Lys Leu      50 55 60 Pro Asp Met Lys Ser Leu Ile Pro Val Val Thr Asn Pro Ser Thr Gly  65 70 75 80 Leu Val Phe Gly Asn Asn Arg Lys Lys Asp Pro Gly Thr Ile Phe Val                  85 90 95 Ala Gly Ala Thr Gly Gln Ala Gly Ile Arg Ile Ala Gln Thr Leu Leu             100 105 110 Gln Arg Gly Phe Ser Val Arg Ala Gly Val Pro Asp Leu Gly Ala Ala         115 120 125 Gln Asp Leu Ala Arg Val Ala Ala Thr Tyr Lys Ile Leu Ser Asn Asp     130 135 140 Glu Val Lys Arg Leu Asn Ala Val Gln Ser Pro Phe Gln Asp Ala Glu 145 150 155 160 Ser Ile Ala Lys Ala Ile Gly Asn Ala Thr Lys Val Val Val Thr Val                 165 170 175 Gly Ala Thr Glu Asn Gly Pro Asp Ala Gln Val Ser Thr Ser Asp Ala             180 185 190 Leu Leu Val Val Gln Ala Ala Glu Leu Ala Gly Val Ser His Val Ala         195 200 205 Ile Val Tyr Asp Gly Thr Ile Ser Gly Ser Thr Tyr Asn Val Leu Asp     210 215 220 Gly Ile Thr Ser Phe Phe Gly Asn Leu Phe Ala Lys Ser Gln Pro Leu 225 230 235 240 Thr Ile Ser Asp Leu Ile Glu Lys Val Ala Gln Thr Asp Val Ala Tyr                 245 250 255 Thr Leu Ile Lys Thr Ser Leu Thr Glu Asp Phe Ser Pro Glu Lys Ala             260 265 270 Tyr Asn Val Val Val Ala Glu Gly Ser Asn Ser Gly Ser Gly Ser         275 280 285 Ser Ser Ser Glu Ala Tyr Lys Val Pro Lys Leu Lys Ile Ala Ser Leu     290 295 300 Val Ala Asp Ile Phe Ala Asn Thr Ala Val Ala Glu Asn Lys Val Val 305 310 315 320 Glu Val Ser Thr Asp Pro Ser Ala Pro Ser Arg Pro Val Asp Glu Leu                 325 330 335 Phe Ser Val Ile Pro Glu Asp Gly Arg Arg Lys Val Tyr Ala Asp Ala             340 345 350 Ile Ala Arg Glu Arg Ala Glu Glu Glu Ala Lys Val Ala Ala Asp Lys         355 360 365 Ala Arg Glu Ala Ala Glu Ala Ala Lys Glu Phe Glu Lys Gln Met Gln     370 375 380 Lys Leu Ser Glu Lys Glu Ala Glu Ala Ala Ser Leu Ala Glu Asp Ala 385 390 395 400 Gln Gln Lys Ala Asp Ala Val Gly Val Thr Val Asp Gly Leu Phe Asn                 405 410 415 Lys Ala Lys Asp Ile Ser Ser Gly Leu Ser Trp Asn Lys Leu Gly Ser             420 425 430 Gln Phe Ala Thr Ala Ile Gln Asn Ala Ser Glu Thr Pro Lys Val Gln         435 440 445 Val Ala Thr Val Arg Gly Gln Ala Lys Ala Arg Asn Leu Pro Pro Lys     450 455 460 Lys Ala Val Val Lys Gln Arg Pro Ser Ser Pro Phe Ala Ser Lys Pro 465 470 475 480 Lys Glu Glu Arg Pro Lys Lys Pro Glu Lys Glu Val Arg Lys Val Phe                 485 490 495 Gly Gly Leu Phe Lys Gln Glu Thr Ile Tyr Ile Asp Asp Asp             500 505 510 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 gcaaggcagg tcgaagaagt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 tgagatgacc tcacgctcaa t 21  

Claims (8)

PTO1SX162060 protein specifically expressed in the leaf tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2. The protein of claim 1 which is derived from tomato. The protein of claim 1 which is used as a target of therapeutic drug for leaf related diseases. The gene encoding the PTO1SX162060 protein of claim 1. The gene according to claim 4, consisting of a nucleotide sequence represented by SEQ ID NO: 1. Recombinant vector comprising the gene of claim 4. A transformant transformed with the recombinant vector of claim 6. An antibody against the protein of claim 1.

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