KR101972098B1 - U-box domain containing Cla022983 marker gene for diagnosis of gummy stem blight disease of watermelon, and uses thereof - Google Patents

Abstract

The present invention relates to a U-box domain-containing marker Cla022983 gene for diagnosis of gummy stem blight of a watermelon and uses thereof, and more specifically, to a composition and kit for diagnosis of gummy stem blight of a watermelon using a phenomenon that an expression level of a gene encoding a protein represented by amino acid sequence of SEQ ID NO:1 is reduced compared to a normal control group when a watermelon, which is sensitive to gummy stem blight, is infected with gummy stem blight, a diagnosis method, and a method of determining resistance or sensitivity to gummy stem blight of a watermelon. When the composition, kit or diagnosis method of the present invention are used, it is possible to simply, quickly and accurately diagnose a watermelon which is sensitive to gummy stem blight and infected with the same. Particularly, since a watermelon can be diagnosed even when a relatively short period of time has elapsed after the watermelon is exposed to gummy stem blight, it is possible to respond to gummy stem blight more quickly, and diagnosis can be performed by clearly distinguishing gummy stem blight from infection of other pathogens such as anthracnose. Also, when a determination method of the present invention is used, it is possible to efficiently select a watermelon having resistance to gummy stem blight, thereby being very useful for developing a new variety of watermelon.

Description

U-box domain-containing marker Cla022983 gene and its use for diagnosis of vine blight of watermelon {U-box domain containing Cla022983 marker gene for diagnosis of gummy stem blight disease of watermelon, and uses thereof}

The present invention relates to a U-box domain-containing marker Cla022983 gene and its use for diagnosing a vine blight of watermelon. Specifically, when a watermelon susceptible to vine blight disease is infected with a vine blight disease, a protein represented by the amino acid sequence of SEQ ID NO: Kits and diagnostic methods for the diagnosis of vine blight disease using the phenomenon that the expression level of the gene encoding the vine blight of the present invention is reduced as compared with that of the normal control, and a method for determining the resistance or susceptibility of the watermelon to vine blight.

Watermelon is categorized as a vinegared herbaceous plant with a bark and its scientific name is Citrullus vulgaris (or Citrullus lanatus ). Watermelon is very high in water and is mostly composed of saccharides. It has excellent diuretic effect and is known to have effects on frog, cystitis, blood, and skin. It is usually harvested from July to August, and now it is cultivated all year round through facility horticulture due to technological development.

In watermelon cultivation, various diseases such as anthrax, plague, vine blight, Blighting occurs in the stems, leaves, and stomachs. The stalk shows brown spots at the beginning, and develops into pale brown or grayish brown lesions. In leaves, small brown spots appear in the early stages. Pathogens are known as Didymella bryoniae and Mycosphaerella melonis .

Especially in the case of facility cultivation, serial cultivation is inevitable due to its characteristics, and the temperature inside the facility becomes high or humidity becomes high, so that a vine flower pathogenic agent is likely to occur. The disease occurs rapidly after the deposition of watermelon, and the leaves and vines are often killed before the fruit is matured. Therefore, for the stable production of watermelon, it is necessary to diagnose vine blight early and cope with it promptly.

On the other hand, in order to diagnose the vine blight disease, there have been used a method of identifying the diseased parts of diseased plants or analyzing the mycological characteristics by isolating the bacteria. However, this method does not allow for early diagnosis of vine blight disease, making it difficult to quickly respond to vine blight disease.

Accordingly, the present inventors have made efforts to develop a method for quickly and accurately diagnosing vine blight of watermelon early. As a result, a gene expressing a specific expression pattern was detected when a watermelon variety susceptible to vine blight infection was infected with a vine blight disease virus. Using this gene as a marker can not only effectively diagnose vine blight disease, The present invention can be utilized as a marker when developing a watermelon variety having resistance.

Norton, J. D. 1979. Inheritance of resistance to gummy stem blight in watermelon. HortScience 14: 630-632. Skarshaug, A. J. 1981. Centrum development in Didymella bryoniae. Amer. J. Bot. 68: 1096-1103.

Therefore, it is a main object of the present invention to provide a marker for quickly and accurately diagnosing the infection of a vine blight in a watermelon that is susceptible to vine blight and causes serious problems during infection.

Another object of the present invention is to provide a composition and a kit for diagnosing vine blight of watermelon using the marker.

It is still another object of the present invention to provide a diagnostic method capable of efficiently diagnosing vine blight of watermelon using the marker.

It is another object of the present invention to provide a method for efficiently discriminating resistance or susceptibility of watermelon resources to vine blight by using the markers.

According to one aspect of the present invention, there is provided a composition for detecting a vine blight of watermelon comprising a probe for measuring the expression level of a gene encoding a protein represented by the amino acid sequence of SEQ ID NO: 1 from a sample of watermelon.

In the composition of the present invention, the gene may be represented by the nucleotide sequence of SEQ ID NO: 2.

In the composition of the present invention, the probe may be a probe for detecting the mRNA of the gene or the protein encoded by the gene, and the probe is preferably a probe for detecting RNA represented by the nucleotide sequence of SEQ ID NO: 3.

In the composition of the present invention, the probe may be a primer for a polymerase chain reaction (PCR), a probe for hybridization or an antibody, A primer or a probe that specifically binds to the cDNA of RNA is preferable.

In the composition of the present invention, the probe is preferably a primer set consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5.

The composition of the present invention may further include reagents necessary for measuring the expression level of a gene using a probe in addition to the probe. For example, a reagent necessary for performing RT-PCR, a reagent necessary for performing a northern blot, or a reagent necessary for performing a western blot, and the like.

According to another aspect of the present invention, there is provided a kit for the diagnosis of vine blight of watermelons comprising the composition.

The kit of the present invention may further include reagents, apparatuses, or devices necessary for measuring the expression level of a gene using a probe in addition to the above composition. For example, reagents, apparatus, or devices necessary to perform RT-PCR, northern blotting or western blotting.

According to still another aspect of the present invention, there is provided a method for diagnosing a vine blight of a watermelon characterized by measuring the expression level of a gene encoding a protein represented by the amino acid sequence of SEQ ID NO: 1 from a sample of watermelon.

In the diagnostic method of the present invention of the present invention, the gene may be represented by the nucleotide sequence of SEQ ID NO: 2.

In the diagnostic method of the present invention of the present invention, in order to measure the expression level, it is preferable to detect the RNA represented by the nucleotide sequence of SEQ ID NO: 3.

In the diagnostic method of the present invention, when the expression level of the gene is decreased as compared with the normal control group (watermelon not infected with the vine blight disease), the watermelon may be susceptible to vine blight susceptibility and can be judged to be infected with the vine blight disease. In this case, the watermelon which has been found to be resistant to the vine blight or the watermelon which has been found to be susceptible to vine blight may be used as a control group to improve the accuracy of the diagnosis, and the existing vine blight-resistant watermelon or susceptible watermelon And the watermelon inoculated with the vine blight fungus can be used as a control to further improve the accuracy.

According to another aspect of the present invention, the present invention provides a method of isolating RNA from a sample of watermelon, Synthesizing the RNA with cDNA; Performing PCR using the cDNA as a template and a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5; And analyzing the product of the PCR. The present invention also provides a method for diagnosing a vine blight of watermelon. If the concentration of the PCR product (162 bp) is lower than that of the normal control (watermelon not infected with the vine blight disease), the watermelon is susceptible to vine blight and can be judged to be infected with the vine blight disease.

According to another aspect of the present invention, there is provided a method for producing a vinegar, comprising the steps of: And measuring the expression level of a gene encoding the protein represented by the amino acid sequence of SEQ ID NO: 1 from the sample of the watermelon after the inoculation, to thereby determine resistance to or susceptibility to vine blight of watermelon.

In the discrimination method of the present invention, the gene may be represented by the nucleotide sequence of SEQ ID NO: 2.

In the discrimination method of the present invention, in order to measure the expression level of the gene, it is preferable to detect RNA represented by the nucleotide sequence of SEQ ID NO: 3.

In the discrimination method of the present invention, when the expression level of the gene is decreased as compared with the normal control group (watermelon not vaccinated with vine blight disease), the watermelon can be judged as susceptible to vine blight.

According to another aspect of the present invention, there is provided a method for producing a vinegar, comprising the steps of: Separating the RNA from the watermelon sample after the inoculation; Synthesizing the RNA with cDNA; Performing PCR using the cDNA as a template and a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5; And analyzing the product of the PCR. The present invention also provides a method for determining resistance to or susceptibility to vine blight of watermelon. At this time, if the concentration of the PCR product (162 bp) is decreased compared to the normal control (watermelon not vaccinated with vine blight disease), the watermelon can be judged to be susceptible to vine blight disease, otherwise it is judged to be resistant .

The present invention is based on the new fact that when the watermelon susceptible to vine blight infection is infected with a vine blight disease, the expression level of the gene decreases.

In order to detect the genes related to the vine blight, this study was carried out to inoculate a variety of watermelons of various varieties and to carry out RT-PCR for each candidate gene. In contrast to other watermelons, varieties known to be susceptible to vine blight , And that the RT-PCR product of the Cla022983 gene was specifically and significantly reduced when the strain was inoculated.

Such a phenomenon is unusual and does not occur when anthrax is infected, so it is useful for distinguishing the infection from anthrax infection.

The Cla022983 gene encodes the amino acid sequence of SEQ ID NO: 1 and is represented by the nucleotide sequence of SEQ ID NO: 2, and the mRNA sequence for this gene is the nucleotide sequence of SEQ ID NO: 3 Sequence.

According to the present invention, the Cla022983 gene shows 94% homology with the cucumber LOC101210079 gene. Therefore, as the Cla022983 gene in watermelon was used to diagnose vine blight disease or to discriminate resistance or susceptibility, LOC101210079 gene in cucumber could be used for diagnosis of vine blight disease, or resistance and susceptibility. In particular, since the sequence of the primer part of SEQ ID NOS: 4 and 5 differs from the LOC101210079 gene of cucumber by only one nucleotide, this primer can be directly used for the diagnosis of vine blight of cucumber, or for discrimination of resistance and susceptibility.

Using the composition, kit or diagnostic method of the present invention, a watermelon susceptible to vine blight and infected with vine blight disease can be diagnosed easily, quickly and accurately. In particular, since it can be diagnosed at a relatively short time after exposure to vine blight, it is possible to respond more quickly to vine blight disease and to diagnose it distinctly from other pathogenic bacteria such as anthrax have. Also, by using the discrimination method of the present invention, watermelons resistant to vine blight disease can be efficiently screened, and thus it is expected to be very useful for developing a new variety of watermelon.

FIG. 1 is a result of analysis of characteristic domains using the amino acid sequence of eight genes (including Cla022983 gene of the present invention) selected by examining a gene containing a U-box domain among E3 ligase groups in a watermelon genome.
FIG. 2 is a graph showing the results of sampling at a time interval (0, 24, 48, 72 hours) after inoculation of a vine-bearing rot fungus in a vine resistance resistant watermelon (PI189225), a vine wilt middle resistance resistant watermelon (PI482322) and a vine wilt susceptible watermelon (920533) Total RNA was extracted and RT-PCR was performed to analyze the expression pattern of each gene. C: Results for the Cla +21233 gene, 43 + 44: Results for the Cla001333 gene, 45 + 46: Results for the Cla019613 gene, Control group without inoculation of the vine blight fungus, , 47 + 48: results for Cla013298 gene, 49 + 50: results for Cla009870 gene, 51 + 52: results for Cla010378 gene, 53 + 54: results for Cla022983 gene Results for the Cla016973 gene.
FIG. 3 shows the results obtained after inoculation of anthrax with the anthrax-resistant watermelon (Au-Producer-1), anthrax-resistant midwater-resistant watermelon (006-1) and anthrax susceptible watermelon (920533-1) ), And then RT-PCR was performed to analyze the expression pattern of each gene. C: Results for the Cla021233 gene, 43 + 44: Results for the Cla001333 gene, 45 + 46: Results for the Cla019613 gene, 47: +48 Results for Cla013298 gene, 49 + 50: Results for Cla009870 gene, 51 + 52: Results for Cla010378 gene, 53 + 54: Results for Cla022983 gene (gene of the present invention), 55 + 56: Cla016973 Results for genes.
4 and 5 are the results of comparing the mRNA sequence of Cla022983 gene and the mRNA sequence of LOC101210079 gene of cucumber. Query: mRNA sequence of Cla022983 gene, Sbjct: mRNA sequence of LOC101210079 gene of cucumber, red box: forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and reverse primer site represented by the nucleotide sequence of SEQ ID NO:

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

Example 1. Expression pattern of Cla022983 gene on vine blight disease

1-1. Materials and methods

1-1-1. Test material

Watermelon Varieties: Vine Blight Resistant Resources PI189225, Vine Blight Moderate Resistant Resources PI482322, Vine Blight and Anthrax Sensory Resources 920533, Anthrax Resistant Resources Au-Producer-1, Anthrax Moderate Resistant Resources PI560006

1-1-2. Vine blight and anthrax vaccination and sample preparation

Spore suspensions of KACC No. 40937 and KACC No. 40903 were inoculated into a watermelon seedlings grown in the greenhouse for 3 weeks at a concentration of ⁵ × 10 ⁵ , (24, 48, and 72 hours), quenched into liquid nitrogen, and stored in a cryogenic freezer (-80 ° C).

1-1-3. Total RNA extraction

The samples stored in the cryogenic freezer were ground with liquid nitrogen and total RNA was extracted with TRIzol reagent (Ambion).

1-1-4. Complementary DNA (cDNA) synthesis

After confirming that the total RNA extracted from each sample was the same amount (2 μg), it was synthesized with cDNA using QuantiTect Reverse transcription Kit (Qiagen).

1-1-5. Primer design and polymerase chain reaction (PCR)

Of the E3 ligase groups known to be involved in protein degradation, genes containing the U-box domain were examined in a watermelon genome and eight genes were selected. A forward primer and a reverse primer were designed using an open reading frame of these genes. The primer production was performed according to the rule of 'Wallace rule: Tm = 2 ° C (A + T) + 4 ° C (G + C)' and Tm = 50 ~ 58 ° C (see Table 1).

gene primer order Cla021233 Forward 5'-TCCACATGTAGCTGCAGATGG-3 ' Reverse 5'-GATAGCAGAACGAAGAGCACG-3 ' Cla001333 Forward 5'-AACGGTTCAAAGGATGTGTGG-3 ' Reverse 5'-TCTTGCCTCCACGCACACTCG-3 ' Cla019613 Forward 5'-GGAGATTCGATGAAGACGACG-3 ' Reverse 5'-AGCCGCGCTGGGAAATTTTCC-3 ' Cla013298 Forward 5'-ATGGCACAGACCGAGGTAAGC-3 ' Reverse 5'-GCCAATTGCGGAAGGTTGTGG-3 ' Cla009870 Forward 5'-CAGTTCTGTTCTCCTGTTTCC-3 ' Reverse 5'-TGATCCACCGTCGCAAACACG-3 ' Cla010378 Forward 5'-TCTGGCAAAAACCGCCGGAGC-3 ' Reverse 5'-AGTTCGGCTGCCGAAACAACC-3 ' Cla022983 Forward 5'-CCGGGATTGGTTGCGATTACG-3 ' Reverse 5'-TGGCTTAGAATCTGGAGCAGC-3 ' Cla016973 Forward 5'-CTCATCTGACTTCTGACATGC-3 ' Reverse 5'-TCAACATTTCTCCGCTTGTGG-3 '

Using the cDNA of each sample synthesized in 1-1-4 above as a template, the primers and Prime Taq. (GeNet Bio) polymerase at 95 ° C for 5 minutes → (95 ° C for 10 seconds, 50 to 58 ° C for 10 seconds, and 72 ° C for 10 seconds) 28 times → 72 ° C for 5 minutes → 4 ° C Polymerase chain reaction .

1-2. result

Of the E3 ligase groups known to be involved in protein degradation, a gene containing a U-box domain was examined in a watermelon genome to select eight genes, and their characteristic domains were analyzed using their amino acid sequences (see FIG. 1) .

In order to examine the expression pattern of each gene, a primer was prepared based on the nucleotide sequence (see Table 1), and RT-PCR was performed using the primer thus prepared. As a result, (See FIGS. 2 and 3).

Of the 8 genes, the expression pattern of Cla022983 gene was confirmed to be decreased only in the susceptible resource (920533) inoculated with the vine blight, and this expression pattern appeared after 48 hours of inoculation (see FIG. 2). In addition, these patterns did not appear in the resources inoculated with anthrax (see FIG. 3).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> U-box domain containing Cla022983 marker gene for diagnosis of          gummy stem blight disease of watermelon, and uses thereof <130> PA-D17334 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 746 <212> PRT <213> Citrullus lanatus <400> 1 Met Phe Lys Lys Leu Ala Ile Tyr Gly Gln Val Met Ser Ile Phe   1 5 10 15 Pro Ser Leu Glu Ala Ala Arg Pro Arg Ser Ser Lys Thr Gly Ile Arg Ala              20 25 30 Leu Cys Ser Leu His Val Ala Leu Glu Lys Ala Lys Asn Thr Leu Gln          35 40 45 His Cys Ser Glu Ser Ser Lys Leu Tyr Leu Ala Ile Thr Gly Asp Ala      50 55 60 Val Leu Ala Lys Phe Glu Lys Ala Arg Cys Ser Leu Glu Val Ser Leu  65 70 75 80 Ile Cys Val Glu Asp Ile Val Ser Gln Ser Ile Gly Phe Gln Ile Gln                  85 90 95 Gln Ile Val Asn Glu Leu Lys Asn Thr Val Phe Leu Leu Asp Pro Leu             100 105 110 Glu Lys Gln Ile Gly Asp Asp Ile Ile Ala Leu Leu Leu Gln Glu Arg         115 120 125 Lys Phe Asp Asp Ser Asn Gly Tyr Asn Glu Leu Glu His Phe His Gln     130 135 140 Ala Ala Thr Lys Leu Gly Ile Thr Ser Ser Lys Ala Ala Leu Thr Glu 145 150 155 160 Arg Arg Ala Leu Lys Arg Ile Val Glu Arg Ala Arg Leu Glu Glu Asp                 165 170 175 Lys Arg Lys Glu Ser Ile Val Ala Tyr Leu Leu His Leu Met Arg Lys             180 185 190 Tyr Ser Lys Leu Phe Arg Ser Glu Leu Ala Asp Asp Thr Asp Ser Gln         195 200 205 Gly Gly Ser Thr Pro Cys Ser Pro Thr Leu Arg Cys Ser Leu Glu Asp     210 215 220 Asn Gly Leu Ala Ala Asn Gly Lys Val Phe Glu His Gln Leu Ser Lys 225 230 235 240 Leu Ser Ser Phe Asn Phe Lys Pro Asn Tyr Arg Ser Ser Gly Gln Met                 245 250 255 Pro Leu Pro Pro Glu Glu Leu Arg Cys Pro Ile Ser Leu Gln Leu Met             260 265 270 Tyr Asp Pro Val Ile Ile Asp Ser Gly Gln Thr Tyr Glu Arg Ile Cys         275 280 285 Ile Glu Lys Trp Phe Ser Asp Gly His Lys Thr Cys Pro Lys Thr Gln     290 295 300 Gln Lys Leu Ser His Leu Ser Leu Thr Pro Asn Tyr Ser Val Lys Gly 305 310 315 320 Leu Ile Ala Asn Trp Cys Glu His Asn Gly Val Pro Ile Leu Asp Gly                 325 330 335 Pro Pro Lys Ser Leu Asp Leu Asn Tyr Trp Arg Leu Ala Leu Ser Asp             340 345 350 Ser Glu Ser Val Lys Ser Lys Ser Val Asp Asn Val Gly Ser His Thr         355 360 365 Leu Lys Glu Val Lys Val Val Pro Leu Glu Glu Ser Gly Thr Ile Lys     370 375 380 Val Ala Glu Glu Asn Glu Ala Asp Asp Asn Thr Tyr Thr Glu Glu Ser 385 390 395 400 Ala Asp Phe Ile Thr Leu Glu Ser Cys Val Asn Phe Met Ala Val Leu                 405 410 415 Thr Glu Gly Asp Leu Arg Lys Lys Cys Lys Val Val Glu Gln Ile             420 425 430 Arg Leu Ser Leu Lys Asp Asp Asp Glu Ala Arg Ile Leu Met Gly Ala         435 440 445 Asn Gly Phe Ala Glu Ala Leu Met Glu Phe Leu Thr Leu Ala Leu Ile     450 455 460 Glu Glu Asn Ala Asp Ala Gln Glu Thr Gly Ala Met Ala Leu Phe Asn 465 470 475 480 Leu Ser Val Asn Asn Asn Arg Asn Arg Glu Met Met Ile Ala Ala Gly                 485 490 495 Val Ile Ser Leu Leu Glu Asn Met Ile Leu Lys Ser Asn Leu His Gly             500 505 510 Pro Ala Thr Ala Leu Tyr Leu Asn Leu Ser Cys Leu Glu Asp Ala Lys         515 520 525 Pro Ile Ile Ser Ser Ser Thr Ala Val Pro Phe Leu Ile Gln Leu Leu     530 535 540 Thr Cys Asn Gly Glu Ser Gln Thr Lys Leu Asp Ala Leu His Thr Leu 545 550 555 560 Tyr Asn Leu Ser Thr Thr Pro Ser Ile Pro Val Leu Le Ser Ala                 565 570 575 Gly Ile Val Gly Gly Leu Gln Ser Phe Ile Ala Ala Pro Ser Asp Ser             580 585 590 Met Trp Thr Glu Thr Ser Leu Ala Ile Leu Met Asn Leu Ala Ser Ser         595 600 605 Gln Leu Gly Ile Glu Glu Ile Thr Ser Ala Pro Glu Leu Ile Ser Gly     610 615 620 Leu Ala Ala Ile Val Asp Ala Gly Glu Arg Ala Glu Gln Glu Gln Ala 625 630 635 640 Val Ser Cys Leu Leu Ile Leu Cys Arg Gly Ser Glu Lys Cys Ser Gln                 645 650 655 Met Val Leu Gln Glu Gly Val Ile Pro Gly Leu Val Ala Ile Thr Val             660 665 670 Asn Gly Thr Ser Arg Gly Lys Val Lys Ala Gln Lys Leu Leu Met Leu         675 680 685 Phe Arg Glu Gln Arg Gln Lys Asp Thr Asp Ile Ile Gln Gln Arg Asp     690 695 700 Gly Asn Ser Asp Thr Ala Met Ala Ala Pro Asp Ser Lys Pro Leu Cys 705 710 715 720 Lys Ser Met Ser Lys Lys Lys Met Gly Lys Ala Leu Ser Phe Phe Ala                 725 730 735 Arg Ser Lys Arg Phe Ser Leu Tyr Gln Cys             740 745 <210> 2 <211> 3165 <212> DNA <213> Citrullus lanatus <400> 2 atgttcaaga aactcgctgc aatttatggc caagttatgt caattttccc ttccttggaa 60 gcagcacgac ctagaagcaa aactggtatc cgggctttat gttcattaca tgtagctctt 120 gagaaggcta agaatactct tcagcattgc tcagaatcca gtaaactcta cttggtatga 180 cataacaatt atcattattt caacaattct cttatacaac gtagatttaa tcacgtggaa 240 taaccaacca atgtataaaa ctattatgct ctacttcctt tttcttcttc taaaagtcac 300 tatgaatgtc atgcttatgg gcttgcattt gtatgacagg ctataactgg agatgctgtt 360 ctagccaaat ttgaaaaggc aagatgttct cttgaagtta gtcttatatg cgtcgaagat 420 attgtttcac aatcaattgg atttcaggtg aaaattgctt tgttttattt ctacttttcc 480 tttcccttgt catgctttta acttgacctg tatcctgaca tgatgattag aatgctgttc 540 atatccattt tcattgggca gagttttgga agcagcatgg tgaaagttca tgattttgat 600 tttgaaagca acttaatctc gattagttat tcattcttcc cttattagat cacataatgg 660 actcaatcat gtctagaatc ttggacactt tgatcacttg catttggaat atgctgatgg 720 attttagttt gttcataact gtgtatcttg tgtttcgttt tgtttgagtt tatatgtgcg 780 gtatgatcaa tggcttatgt aaatgatact ttttccagat tcagcagata gtgaacgaac 840 tcaagaacac tgttttctta cttgatccac ttgagaaaca aattggtgat gatatcattg 900 cattactctt gcaagaaaga aaatttgatg attccaatgg ctataatgag ctggagcatt 960 ttcatcaggc tgccacgaaa cttggaatta cctcctccaa agcagccctc acagagagga 1020 gagctctcaa gagaattgta gaacgagctc gcttagaaga agacaagcga aaggaatcaa 1080 ttgtagctta ccttttgcat ctgatgagga agtattctaa gttatttaga agtgagttgg 1140 cagatgacac cgattcacag ggtgggtcaa ctccttgttc tcctactctt cggtgttctc 1200 ttgaggataa tggacttgca gcgaatggta aagtttttga acatcagctt tcaaagctta 1260 gttcttttaa tttcaagcca aattatagga tatcagggca gatgcctctt ccacctgagg 1320 aattaaggtg tccaatatca ttgcagctta tgtacgaccc tgttataatt gattctgggc 1380 aaacatatga aaggatctgt atagagaagt ggttcagtga tggtcataaa acctgcccaa 1440 agactcaaca gaagctctcg cacctgtcct tgacacctaa ttactctgtc aagggtctca 1500 ttgctaattg gtgtgaacat aacggagttc ctatcctgga tggtccacca aaatcacttg 1560 atctgaatta ttggagactt gcattgtctg attctgagtc tgtaaaatca aaatctgtgg 1620 acaatgttgg ctctcacacg ttgaaggaag ttaaggtagt tcctttggag gaaagtggaa 1680 caattaaggt tgctgaagaa aatgaagcag atgataatac atacacggag gagtcagctg 1740 attttattac actggagag tgtgtaaatt ttatggcagt attgactgaa gagggagact 1800 tgagaaaaaa atgtaaggtt gtggagcaga taagactttc attgaaggat gatgatgaag 1860 ctcgaatttt gatgggagcc aatggctttg ctgaagcact tatggaattc ctaactttag 1920 ctcttattga agaaaatgct gatgctcagg aaactggagc catggctctc ttcaatcttt 1980 ctgtcaacaa caacaggtct gtactattta tccaagccta cgtttgaaaa tgaaagtaca 2040 tttcagctac gaggtagact gaatagtggc caatctagca tcttctatga ataattgtct 2100 gcatatttca tgtttgtgac gaggaaatca tggagtcgca tgtcattgaa ctgcttgtga 2160 cttgcattat acatatcaat tattatcatc tcatatttct cgcttctagt gatggagtct 2220 ggtaatagtt tttctttttc aaatttagtc atttggaaaa taaaattggt agaatctatt 2280 ggatggttgg aggctggttg aacccctggc cctctctctc tctctcttga tattgatggc 2340 ttaaactgcc caatgacata gttgtgattt atgtatttct tcagaaatag ggaaatgatg 2400 atagcggcag gtgtaatctc gttgttggag aacatgattt tgaaatcgaa tttacatgga 2460 cctgcaacgg ccctatattt gaacctttcc tgcctggaag atgccaaacc tatcattagt 2520 tcgagtacag ccgttccttt cctgatccag ctgcttactt gtaatggtga atcccaaacc 2580 aagctcgatg cgcttcatac cctttataac ctttcaacca cgccgtccat cattcctgtt 2640 cttctttccg ctggtattgt cggtggactt caatccttta ttgcagctcc tagcgacagc 2700 atgtggacag agacttcttt agctatctta atgaacttag cttcaagcca gttagggata 2760 gaagaaataa cttcagctcc agagcttatc agtgggttgg cagcaatcgt ggatgctggc 2820 gaacgtgccg agcaggagca agctgtatca tgtttgttga ttttgtgtag agggagtgaa 2880 aaatgcagtc aaatggtctt acaggaagga gtgattccgg gattggttgc gattacggta 2940 aatggaactt caagagggaa agtaaaagct caaaaacttc tgatgttgtt tagggagcag 3000 aggcaaaaag atactgatat cattcagcag cgagacggaa atagcgacac ggccatggct 3060 gctccagatt ctaagccact ttgcaaatca atgtcaaaga aaaagatggg gaaagcacta 3120 agcttttttg cgaggagcaa acgattttca ctataccaat gttga 3165 <210> 3 <211> 2241 <212> RNA <213> Citrullus lanatus <400> 3 auguucaaga aacucgcugc aauuuauggc caaguuaugu caauuuuccc uuccuuggaa 60 gcagcacgac cuagaagcaa aacugguauc cgggcuuuau guucauuaca uguagcucuu 120 gagaaggcua agaauacucu ucagcauugc ucagaaucca guaaacucua cuuggcuaua 180 acuggagaug cuguucuagc caaauuugaa aaggcaagau guucucuuga aguuagucuu 240 auaugcgucg aagauauugu uucacaauca auuggauuuc agauucagca gauagugaac 300 gaacucaaga acacuguuuu cuuacuugau ccacuugaga aacaaauugg ugaugauauc 360 auugcauuac ucuugcaaga aagaaaauuu gaugauucca auggcuauaa ugagcuggag 420 cauuuucauc aggcugccac gaaacuugga auuaccuccu ccaaagcagc ccucacagag 480 aggagagcuc ucaagagaau uguagaacga gcucgcuuag aagaagacaa gcgaaaggaa 540 ucaauuguag cuuaccuuuu gcaucugaug aggaaguauu cuaaguuauu uagaagugag 600 uuggcagaug acaccgauuc acaggguggg ucaacuccuu guucuccuac ucuucggugu 660 ucucuugagg auaauggacu ugcagcgaau gguaaaguuu uugaacauca gcuuucaaag 720 cuuaguucuu uuaauuucaa gccaaauuau aggauaucag ggcagaugcc ucuuccaccu 780 gaggaauuaa gguguccaau aucauugcag cuuauguacg acccuguuau aauugauucu 840 gggcaaacau augaaaggau cuguauagag aagugguuca gugaugguca uaaaaccugc 900 ccaaagacuc aacagaagcu cucgcaccug uccuugacac cuaauuacuc ugucaagggu 960 cucauugcua auugguguga acauaacgga guuccuaucc uggauggucc accaaaauca 1020 cuugaucuga auuauuggag acuugcauug ucugauucug agucuguaaa aucaaaaucu 1080 guggacaaug uuggcucuca cacguugaag gaaguuaagg uaguuccuuu ggaggaaagu 1140 ggaacaauua agguugcuga agaaaaugaa gcagaugaua auacauacac ggaggaguca 1200 gcugauuuua uuacacugga gaguugugua aauuuuaugg caguauugac ugaagaggga 1260 gacuugagaa aaaaauguaa gguuguggag cagauaagac uuucauugaa ggaugaugau 1320 gaagcucgaa uuuugauggg agccaauggc uuugcugaag cacuuaugga auuccuaacu 1380 uuagcucuua uugaagaaaa ugcugaugcu caggaaacug gagccauggc ucucuucaau 1440 cuuucuguca acaacaacag aaauagggaa augaugauag cggcaggugu aaucucguug 1500 uuggagaaca ugauuuugaa aucgaauuua cauggaccug caacggcccu auauuugaac 1560 cuuuccugcc uggaagaugc caaaccuauc auuaguucga guacagccgu uccuuuccug 1620 auccagcugc uuacuuguaa uggugaaucc caaaccaagc ucgaugcgcu ucauacccuu 1680 uauaaccuuu caaccacgcc guccaucauu ccuguucuuc uuuccgcugg uauugucggu 1740 ggacuucaau ccuuuauugc agcuccuagc gacagcaugu ggacagagac uucuuuagcu 1800 aucuaauga acuuagcuuc aagccaguua gggauagaag aaauaacuuc agcuccagag 1860 cuuaucagug gguuggcagc aaucguggau gcuggcgaac gugccgagca ggagcaagcu 1920 guaucauguu uguugauuuu guguagaggg agugaaaaau gcagucaaau ggucuuacag 1980 gaaggaguga uuccgggauu gguugcgauu acgguaaaug gaacuucaag agggaaagua 2040 aaagcucaaa aacuucugau guuguuuagg gagcagaggc aaaaagauac ugauaucauu 2100 cagcagcgag acggaaauag cgacacggcc auggcugcuc cagauucuaa gccacuuugc 2160 aaaucaaugu caaagaaaaa gauggggaaa gcacuaagcu uuuuugcgag gagcaaacga 2220 uuuucacuau accaauguug a 2241 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Cla022983 <400> 4 ccgggattgg ttgcgattac g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Cla022983 <400> 5 tggcttagaa tctggagcag c 21

Claims (14)

1. A composition for diagnosing vine blight disease of watermelon comprising a probe for measuring the expression level of a gene encoding a protein represented by the amino acid sequence of SEQ ID NO: 1 from a sample of watermelon. The method according to claim 1,
Wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1,
Wherein the probe is a probe for detecting RNA represented by the nucleotide sequence of SEQ ID NO: 3. The method of claim 3,
Wherein the probe is a primer or a probe that specifically binds to the RNA or the cDNA of the RNA. 5. The method of claim 4,
Wherein the probe is a primer set consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5. A kit for diagnosing a vine blight of watermelon comprising the composition of any one of claims 1 to 5. Wherein the expression level of a gene encoding a protein represented by the amino acid sequence of SEQ ID NO: 1 is measured from a sample of watermelon. 8. The method of claim 7,
Wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2. 8. The method of claim 7,
To determine the level of expression,
A method for diagnosing a vine blight of a watermelon characterized by detecting an RNA represented by the nucleotide sequence of SEQ ID NO: 3. Separating RNA from a sample of watermelon;
Synthesizing the RNA with cDNA;
Performing PCR using the cDNA as a template and a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5; And
And analyzing the product of the PCR. Inoculating a watermelon with a vine-blotted strain; And
Measuring the expression level of a gene encoding the protein represented by the amino acid sequence of SEQ ID NO: 1 from the sample of the watermelon after 48 hours of inoculation; and determining the resistance or susceptibility of the watermelon to vine blight. 12. The method of claim 11,
Wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2. 12. The method of claim 11,
To determine the level of expression of the gene,
And detecting the RNA represented by the nucleotide sequence of SEQ ID NO: 3. Inoculating a watermelon with a vine-blotted strain;
Separating the RNA from the watermelon sample after 48 hours of inoculation;
Synthesizing the RNA with cDNA;
Performing PCR using the cDNA as a template and a forward primer represented by the nucleotide sequence of SEQ ID NO: 4 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 5; And
And analyzing the product of the PCR.

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