CN113528538B - Cucumber CsSTK gene, protein, expression vector and application - Google Patents

Abstract

The invention discloses cucumber CsSTK gene, protein, expression vector and application, and belongs to the technical field of molecular biology. The present application discloses a cucumber CsSTK gene, the nucleotide sequence of which is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the CsSTK gene is shown in SEQ ID NO: 2; gene expression vector. Also disclosed is the application of the CsSTK gene or the protein or the expression vector in improving the resistance of cucumber corynebacterium leaf spot. The present invention verifies the transgenic plants obtained by overexpressing CsSTK through genetic engineering, and through inoculation identification, the onset symptoms of cucumber corynebacterium leaf spot are significantly alleviated, indicating that overexpression of CsSTK can significantly enhance the resistance to cucumber corynebacterium leaf spot.

Description

Cucumber CsSTK gene, protein, expression vector and application

technical field

The invention relates to the technical field of molecular biology, in particular to a cucumber CsSTK gene, protein, expression vector and application.

Background technique

Cucumber (scientific name: Cucumis sativus L.) is an annual trailing or climbing herb of the Cucurbitaceae family, widely cultivated throughout China. In production, cucumber is susceptible to infection by a variety of pathogenic bacteria, which seriously affects its yield and quality. Corynespora cassiicola is a common fungal disease caused by the pathogenic fungus Corynespora cassiicola. Existing control methods mainly use chemical methods for control, and long-term use of chemical methods for control can play a role in the control of cucumber corynebacterium leaf spot, but long-term use is likely to cause pathogenic bacteria resistance. At the same time, chemical pesticide residues have adverse effects on soil and human health. In view of this situation, the main method is to cultivate new resistant varieties or to find safer biological preparations. However, it takes a long time to cultivate resistant varieties and there is a shortage of varieties resistant to Corynebacterium leaf spot. Although biological preparations are relatively chemical The preparation is safer, but the cost is high and the effect is slow. In addition, there are very few studies on cucumber Corynebacterium leaf spot, and most of them focus on the physiology, biochemistry, drug resistance, and genetic diversity of the pathogen. The research on the mechanism of disease resistance is still in its infancy.

With the continuous progress of molecular technology and the completion of the cucumber genome sequencing, a variety of transcription factors in the cucumber genome have been systematically analyzed, such as ERF, MLO, etc., but little research has been done on the function of cucumber STK gene, especially the relationship between cucumber STK gene and cucumber Correlation studies on corynebacterium leaf spot resistance have not been reported.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of cucumber CsSTK gene, protein, expression vector and application, in order to solve the problems existing in the above-mentioned prior art, by overexpressing CsSTK in susceptible varieties, the resistance to cucumber corynebacterium leaf spot is obviously enhanced .

For achieving the above object, the present invention provides the following scheme:

The present invention provides a cucumber CsSTK gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

SEQ ID NO: 1:

atgtcaaaggttcttgcagcaatattaggaggttctgcaggagccgtggcattggtgggattgattattatacttttaagattcttagcacgctcgagaaacactgcaagaacttccgagactggctcttctgatccatctgttcaagtgggaaggcatgttggtattgaattgactctacgagatgctaggcgttttgagatggcagagttggtgttggccactaatgatttcagcgacaagaacttgattggagaagggaaatttggggaggtctataagggtatgcttcaggatggaatgttcgtcgctataaaaaagcggcatggagcgcctagtcaggatttcgtagatgaggtacactacctctcgtctattcagcatcgaaatctcgtgactcttttgggctactgccaggaaaataatctacagtttctcatctttgattatatacccaacggaagtgtttctagccacatatatggcactgagcagcgttcggctgagaagctggagttcaagatcagactctcaatagctctgggggcagctaaaggtctgtcacatcttcactccatgagccctcgtttgacacacagaaacttcaagacatccaacgttcttgtagatgagaattttatagctaaagtggcagatgcaggacttcacaatgtcatgcgaagatttgacgtttcagaatcatcgtcccgagcaacagcagatgagatatttcttgcacctgaggtaaaagagtttcgacaattttccgagaaaagtgacgtatatagtttcggcgtattccttttggagttggtaagcggtcaaaaagcgactgatgcacctgtttccaatcccaattatactctggtggactggatacaaaacaatcagagaaagagcgatattggtagcattacggatccaaggttagggaagagcttcactgaggaaggtatgggtgaactaatggatttgatacttcaatgcgtcgaatattcaa gcgagaggcgtccagtgatgagctacgtggtgacagagctggaaaggatactggagaaagagatgaacttaacaacagtgatgggagaaggaagcccaactgttactcttgggagtcagttgttcaaaaccacaaagtaa.

The present invention also provides the protein encoded by the CsSTK gene.

Preferably, its amino acid sequence is shown in SEQ ID NO:2.

SEQ ID NO: 2:

MSKVLAAILGGSAGAVALVGLIIILLRFLARSRNTARTSETGSSDPSVQVGRHVGIELTLRDARRFEMAELVLATNDFSDKNLIGEGKFGEVYKGMLQDGMFVAIKKRHGAPSQDFVDEVHYLSSIQHRNLVTLLGYCQENNLQFLIFDYIPNGSVSSHIYGTEQRSAEKLEFKIRLSIALGAAKGLSHLHSMSPRLTHRNFKTSNVLVDENFIAKVADAGLHNVMRRFDVSESSSRATADEIFLAPEVKEFRQFSEKSDVYSFGVFLLELVSGQKATDAPVSNPNYTLVDWIQNNQRKSDIGSITDPRLGKSFTEEGMGELMDLILQCVEYSSERRPVMSYVVTELERILEKEMNLTTVMGEGSPTVTLGSQLFKTTK。

The present invention also provides an expression vector containing the CsSTK gene.

The present invention also provides a transformant constructed by using the expression vector.

The present invention also provides the application of the CsSTK gene or the protein or the expression vector in improving the resistance of cucumber corynebacterium leaf spot.

The present invention discloses the following technical effects:

1. The present invention analyzes cucumber-related gene sequence information by using genetic engineering technology, gene expression analysis, gene cloning and sequence analysis technology.

2. The present invention constructs an overexpression vector containing the cucumber CsSTK gene, which can be directly used for the genetic transformation of cucumber.

3. The cucumber CsSTK gene provided by the present invention is a new coding gene of cucumber responding to Corynebacterium leaf spot, and the CsSTK gene is overexpressed after the overexpression vector pCXSN-CsSTK is transferred into the cucumber cotyledon nodes by the transformation method mediated by Agrobacterium, After inoculation with Corynebacterium leaf spot, the symptoms of transgenic plants were significantly lighter than those of the susceptible line D0401, indicating that the susceptible varieties overexpressing CsSTK had significantly enhanced resistance to Corynebacterium leaf spot of cucumber.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Figure 1 shows the CsSTK PCR product and digestion identification; M: DNA marker 2K; 1: PCR product; 2: Plasmid EcoRI digestion identification;

Figure 2 shows the PCR detection of pCXSN-CsSTK overexpression vector; M: DNA marker 2K; 1-5: PCR product;

Figure 3 is PCR detection of bacterial liquid; M: DNA marker 2K; 1-5: PCR product;

Figure 4 shows the process of obtaining transgenic positive plants; a: sowing; b: co-culture of cotyledon nodes; c-d: differentiation of cotyledon nodes; e: differentiated shoots and rooted; f: domestication;

Figure 5 shows the PCR detection of D0401-resistant plants transformed with CsSTK; M: DNA marker 2K; +: positive control; -: sterile water control; C: negative control; K: pCXSN-1250; 1-13: transgenic CsSTK gene resistant plants;

Figure 6 shows the identification of CsSTK transgenic plants by inoculation; OE2, OE5, OE7: 3 strains with the highest expression of CsSTK overexpressing lines in the T0 generation; D9320: resistant line; D0401: susceptible line;

Figure 7 is the gene expression analysis of overexpressed plants; Col: control; OE-STK: overexpressed CsSTK plants; OE-ERF: overexpressed CsERF004 plants;

Figure 8 is the analysis of hormone levels in plants overexpressing CsSTK;

Figure 9 shows the PCR detection of CsSTK open reading frame without stop codon; M: DNA marker 2K; 1: PCR product;

Figure 10 shows the PCR detection and double digestion identification of 35S-CsSTK-eGFP expression vector; M: DNA marker 2K; 1: PCR detection; 2: plasmid double digestion identification;

Figure 11 shows the PCR detection of 35S-CsSTK-eGFP fusion expression vector; M: DNA marker 2K; 1-7: PCR product;

Figure 12 shows the localization analysis of 35S-CsSTK-eGFP fusion protein in Arabidopsis protoplasts.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.

It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which the documents are referred. In the event of conflict with any incorporated document, the content of this specification controls.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present application are only exemplary.

As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.

The cucumber varieties D9320 and D0401 involved in the following examples are provided by the cucumber research group of the School of Horticulture and Landscape Architecture, Northeast Agricultural University.

Example 1 Cloning of cucumber CsSTK gene

Using the fresh leaf cDNA of D9320 after inoculation with the pathogen Corynebacterium leaf spot 48h as the cloning template, the specific primers for cloning the full-length CsSTK gene were designed and amplified by PCR using Primerpremier 6.0.

Specific primers are as follows:

CsSTK-F: ATGTCAAAGGTTCTTGCAGC

CsSTK-R: TTACTTTGTGGTTTTGAACAACTG

A 20 μL amplification reaction system specifically includes the following components: 10 μL of 2×Taq PCR MasterMix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of cDNA template, and 8 μL of ddH ₂ O.

PCR amplification program: 94°C for 2 min; 30 cycles of 94°C for 30s, 52°C for 30s, and 72°C for 1 min; 72°C for 10 min; 4°C Hold.

The amplified target fragment was ligated with pEASY-T3 vector to obtain pEASY-T3-CsSTK Escherichia coli bacteria liquid, which was identified by PCR and sent to Harbin Qingke Biological Company for sequencing. The electrophoresis results showed that the target band appeared at the 1140bp position. DNAMAN 8 was used for sequence alignment analysis, and the results were 100% correct. The plasmid pEASY-T3-CsSTK was identified by single restriction enzyme digestion with EcoRI (Fig. 1), indicating that CsSTK was successfully cloned. After determination, the nucleotide sequence of CsSTK gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

Example 2 Construction and transformation of plant overexpression vector

The cloned gene CsSTK sequence full-length primers were used for cloning and gel recovery. The pCXSN-1250 vector is a plant expression vector containing a 35s promoter. The pCXSN-1250 empty vector was digested with restriction endonuclease XcmⅠ. The cut empty vector and the target fragment were mixed according to the proportion, and ligated with T4 ligase at 16°C for 16 h to obtain the pCXSN-CsSTK overexpression vector, which was transferred into DH-5α competent cells by freeze-thaw method. The cells were screened and cultured on the medium containing Kan, and colony PCR identification was carried out after 12 h.

pCXSN-CsSTK sequencing primers:

pCXSN-1250-F:CGGCAACAGGATTCAATCTTA;

pCXSN-1250-R: CAAGCATTCTACTTCTATTGCAGC.

The amplification system and reaction procedure were the same as those in Example 1.

The electrophoresis results of PCR products showed that the target band appeared at the position of 1140bp (Fig. 2). After the bacterial solution was sent to the company for sequencing, the sequencing result was completely correct, indicating that CsSTK has been successfully constructed on the pCXSN-1250 vector, and subsequent experiments can be carried out.

The recombinant plasmid was introduced into Agrobacterium tumefaciens competent cells according to the instructions, cultivated in YEB solid medium (containing Kan and rifampicin), to grow a single colony, use gene primers for identification (primers used, reaction system and reaction The procedure is the same as that of Example 1), and the target band exists at the position of 1140bp (Fig. 3), indicating that the pCXSN-CsSTK overexpression vector was successfully introduced into Agrobacterium tumefaciens.

Example 3 Obtaining of transgenic positive plants

After soaking the seeds in warm water for 30 minutes, remove the seed coat, disinfect with 75% ethanol for 1 minute and remove the surface tension of the seeds, then disinfect with 2-3% sodium hypochlorite for 10 minutes, rinse with sterilized water for several times to remove the disinfectant on the surface of the seeds, Use sterile filter paper to absorb water, and evenly distribute it on the germination medium (MS solid powder 4.33g·L ^-1 +30g·L ^-1 sucrose + 2g·L ^-1 vegetative gel, pH=5.8), 28 Incubate in the dark for 48h.

Use 1/2MS medium (1/2MS solid powder 4.33g·L ^-1 +30g·L ^-1 sucrose, pH=5.8) to resuspend the above-mentioned pCXSN-CsSTK overexpression vector into A. Under the bacterial condition, cut off the protective film on the seed surface of the above-mentioned culture, peel off the cotyledons, remove the growth point and the hypocotyl, and use the bacterial liquid to carry out shock infection, after the sterile filter paper absorbs the bacterial liquid on the surface of the cotyledon nodes, transfer to a total of The culture medium (germination medium+0.5mg·L ^-1 6-BA+1.0mg·L ^-1 ABA, pH=5.8) was co-cultured under dark conditions, and the cotyledon nodes were slightly curled and Agrobacterium grew around them after 2 days At this time, the cotyledon nodes were transferred to differentiation medium (co-culture medium+400mg·L ^-1 cefotaxime sodium (Cef) + 1.0mg·L ^-1 glyphosate, pH=5.8), placed in tissue culture The room was cultivated, and the cotyledon nodes grew up gradually and turned green, and after 10d, differentiated buds were grown at the position of removing the hypocotyl, and about 28d, the differentiated buds grew into obvious cucumber plants, and the differentiated buds were completely cut off and put into rooting medium ( Germination medium + 400mg·L ^-1 Cef, pH=5.8), the main root and lateral root obviously grow at the bottom of the differentiated buds on 7d, transfer them to a nutrient bowl where vermiculite and soil are evenly mixed at 1:1, and placed in an artificial climate The acclimatization was carried out in the box, and the plastic film was used for moisturizing. As the acclimation progressed, the plastic film was continuously removed (Figure 4).

Example 4 Molecular biological detection of transgenic positive plants

The DNA of the plant to be identified was used as a template, the pCXSN-CsSTK bacterial solution was used as a positive control, and sterile water, normal plant DNA and pCXSN-1250 empty vector plant DNA were used as negative controls respectively. PCR amplification was performed using pCXSN-1250-F/R.

The amplification primers are as follows:

pCXSN-1250-F:CGGCAACAGGATTCAATCTTA;

pCXSN-1250-R: CAAGCATTCTACTTCTATTGCAGC.

Amplification conditions and reaction procedures were the same as in Example 1.

As shown in Figure 5, no target band appeared in the lane with sterile water and the total DNA of non-transgenic cucumber plants as negative control, and there was a band at 750bp with the total DNA of leaves of plants transfected with pCXSN-1250 empty vector as negative control, Both the positive control and the successful transgenic plants showed the target band at the target position and were different from the negative control, indicating that pCXSN-CsSTK had been successfully transformed into cucumber plants.

Example 5 Identification of CsSTK transgenic plants inoculation

Corynespora cassiicola (Berk&Curt) Wei.], donated by Tianjin Kerun Cucumber Research Institute. 10 μL·drop ^- 1 of the prepared spore suspension of 1×10 ⁵ spore·mL ^-1 was inoculated on the underside of the leaf in vitro, and 30 drops were inoculated. The culture conditions were 26/18°C day and night temperature, 16/8h photoperiod, and continuous moisturizing. Disease investigation was carried out 7 days after inoculation.

The leaves of 3 T ₀ generation plants with high relative expression of CsSTK obtained in the above Example 4 were selected for inoculation identification, and the specific steps were as follows: 1×10 5 prepared 1×10 ⁵ Corynespora cassiicola (Berk & Curt) Wei ., donated by Tianjin Kerun Cucumber Research Institute) 10 μL/drop of spore suspension of spore·mL-1 was inoculated on the back of the leaf in vitro, and 30 drops were inoculated. The culture conditions were 26°C/18°C day and night temperature, 16h/8h photoperiod, and continuous moisturizing. Disease investigation was carried out 7 days after inoculation. D9320 and D0401 were used as disease-resistant and susceptible controls. The investigation was carried out 7 days after inoculation with pathogenic bacteria in vitro.

The results showed that the leaves of the susceptible line D0401 turned yellow obviously after inoculation with Corynebacterium cucumber, the whole leaves were water-soaked, and the disease symptoms were obvious. The disease symptoms of the susceptible line D0401 were mild, indicating that the susceptible varieties overexpressing CsSTK had significantly enhanced resistance to cucumber corynebacterium leaf spot (Fig. 6).

Example 6 Gene expression analysis of transgenic plants

Gene expression analysis of overexpressed CsSTK was carried out by qRT-PCR technology. At the same time, the overexpression of CsERF004, a downstream gene of CsSTK gene, can also enhance cucumber resistance. Therefore, the CsSTK gene and CsERF004 gene were compared and analyzed.

As shown in Figure 7, the results showed that CsERF004, CsPR1 and CsPR4 were significantly up-regulated in plants overexpressing CsERF004; CsSTK, CsERF004, CsPR1 and CsPR4 were significantly up-regulated in plants overexpressing CsSTK. According to the gene expression results, it can be seen that both overexpression of CsSTK and overexpression of CsERF004 can cause the up-regulated expression of CsPR1 and CsPR4; after overexpression of CsSTK, the expression of CsERF004 is significantly up-regulated, but the expression of CsSTK has no significant change after overexpression of CsERF004.

Example 7 Level analysis of different substance contents in CsSTK transgenic plants

Referring to gas chromatography (Liu Dong. Study on the molecular mechanism of double resistance to cucumber downy mildew and corynebacterium leaf spot [D]. Harbin: Northeast Agricultural University Ph.D. dissertation, 2017.) and enzyme-linked immunosorbent assay (Zhao J, Li G, Yi G X, et al. Comparison between conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA for small molecules[J]. Analytica Chimica Acta, 2006, 571(1): 79-85.) respectively on transgenic plants Determination of ethylene release, salicylic acid and methyl jasmonate content was carried out.

The results are shown in Figure 8. The results show that the content of salicylic acid and the amount of ethylene released in the plants overexpressing CsSTK were significantly higher than those of the control, and the content of methyl jasmonate was significantly lower than that of the control.

Example 8 Analysis of subcellular localization of CsSTK gene

1. Construction of 35S-CsSTK-eGFP transient expression vector

By extracting and inoculating the disease-resistant variety D9320 leaf RNA of Corynebacterium leaf spot 48h, carry out the synthesis of the first strand of cDNA with reverse transcription (5 × Integrated RT MasterMix of Beijing DiNing Company. According to the product manual, the specific operation process is as follows:

To a 200 μL sterilized and pre-cooled EP tube, add 2 pg to 2 μg of RNA sample and 4 μL of 4×DIRT Reaction Mix, add DEPC-ddH ₂ O to make up to 16 μL, incubate at 42°C for 2 min to remove gDNA, and place the EP tube immediately. Put on ice and collect all the liquid in the tube to the bottom of the tube, then add 4 μL of 5×Integrated RT MasterMix, incubate at 42°C for 20min in the PCR machine, and then incubate at 85°C for 5min to stop the reaction, the synthesis of the first strand of cDNA is completed, set aside Store at -20°C. After sequence analysis, GFP-CsSTK-F/R was used for cloning to obtain a CsSTK open reading frame without a stop codon (Fig. 9).

CsSTK transient expression vector construction primers:

GFP-CsSTK-F CCAAGCTTATGTCAAAGGTTCTTGCAGCAATA

GFP-CsSTK-RGGACCCGGGTCTTTGTGGTTTTGAACA

The cloning reaction system and reaction procedure were the same as in Example 1.

The cloned gene sequence was sequenced, and the sequence alignment was 100% correct using DNAMAN 8, indicating that the CsSTK open reading frame was cloned successfully, and subsequent experiments could be carried out.

The pGII-eGFP empty vector and the PCR recovery product were subjected to double enzyme digestion (BamHI and SmalI) respectively. After gel recovery, the two samples were mixed according to the proportion, and then T4 ligase was used to connect the two recovered products to obtain 35S-CsSTK-eGFP transient expression The results of electrophoresis of the PCR products and double-enzyme digestion products showed that the target band appeared at the 1140bp position (Figure 10), indicating that CsSTK was successfully constructed on the pGII-eGFP vector, and the 35S-CsSTK-eGFP transient expression vector was constructed. , which can be used for subsequent experiments.

2. The 35S-CsSTK-eGFP transient expression vector was introduced into Agrobacterium tumefaciens

The 35S-CsSTK-eGFP fusion expression vector and the pGII-eGFP empty vector plasmid were extracted respectively, and the two vectors were respectively transformed into GV3101 Agrobacterium competent cells containing the helper plasmid pSoup, and cultured upside down in the dark for 48 hours. After a single colony grew, PCR identification was carried out. , PCR product electrophoresis results showed that the target band appeared at the 1140bp position (Figure 11), indicating that the 35S-CsSTK-eGFP fusion expression vector was successfully transferred into Agrobacterium tumefaciens.

3. Extract the protoplast cells of Arabidopsis thaliana, and then introduce the 35S-CsSTK-eGFP fusion expression vector and pGII-eGFP empty vector into Arabidopsis thaliana protoplasts respectively, and observe the 35S-CsSTK-eGFP fusion under a laser confocal microscope Subcellular localization of expressed proteins.

The results showed that the pGII-eGFP protein showed fluorescent signals in all parts of the cell, while the 35S-CsSTK-eGFP fusion protein only showed green fluorescent signals on the cell membrane (Fig. 12). It shows that CsSTK is a cell membrane localized protein, and it performs the main function on the cell membrane.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, but not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can Variations and improvements should fall within the protection scope determined by the claims of the present invention.

sequence listing

<110> Northeast Agricultural University

<120> Cucumber CsSTK gene, protein, expression vector and application

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atgtcaaagg ttcttgcagc aatattagga ggttctgcag gagccgtggc attggtggga 60

ttgattatta tacttttaag attcttagca cgctcgagaa acactgcaag aacttccgag 120

actggctctt ctgatccatc tgttcaagtg ggaaggcatg ttggtattga attgactcta 180

cgagatgcta ggcgttttga gatggcagag ttggtgttgg ccactaatga tttcagcgac 240

aagaacttga ttggagaagg gaaatttggg gaggtctata agggtatgct tcaggatgga 300

atgttcgtcg ctataaaaaa gcggcatgga gcgcctagtc aggatttcgt agatgaggta 360

cactacctct cgtctattca gcatcgaaat ctcgtgactc ttttgggcta ctgccaggaa 420

aataatctac agtttctcat ctttgattat atacccaacg gaagtgtttc tagccacata 480

tatggcactg agcagcgttc ggctgagaag ctggagttca agatcagact ctcaatagct 540

ctggggggcag ctaaaggtct gtcacatctt cactccatga gccctcgttt gacacacaga 600

aacttcaaga catccaacgt tcttgtagat gagaatttta tagctaaagt ggcagatgca 660

ggacttcaca atgtcatgcg aagatttgac gtttcagaat catcgtcccg agcaacagca 720

gatgagatat ttcttgcacc tgaggtaaaa gagtttcgac aattttccga gaaaagtgac 780

gtatatagtt tcggcgtatt ccttttggag ttggtaagcg gtcaaaaagc gactgatgca 840

cctgtttcca atcccaatta tactctggtg gactggatac aaaacaatca gagaaagagc 900

gatattggta gcattacgga tccaaggtta gggaagagct tcactgagga aggtatgggt 960

gaactaatgg atttgatact tcaatgcgtc gaatattcaa gcgagaggcg tccagtgatg 1020

agctacgtgg tgacagagct ggaaaggata ctggagaaag agatgaactt aacaacagtg 1080

atgggagaag gaagcccaac tgttactctt gggagtcagt tgttcaaaac cacaaagtaa 1140

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Met Ser Lys Val Leu Ala Ala Ile Leu Gly Gly Ser Ala Gly Ala Val

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Ala Leu Val Gly Leu Ile Ile Ile Leu Leu Arg Phe Leu Ala Arg Ser

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Arg Asn Thr Ala Arg Thr Ser Glu Thr Gly Ser Ser Asp Pro Ser Val

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Gln Val Gly Arg His Val Gly Ile Glu Leu Thr Leu Arg Asp Ala Arg

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Arg Phe Glu Met Ala Glu Leu Val Leu Ala Thr Asn Asp Phe Ser Asp

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Lys Asn Leu Ile Gly Glu Gly Lys Phe Gly Glu Val Tyr Lys Gly Met

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Leu Gln Asp Gly Met Phe Val Ala Ile Lys Lys Arg His Gly Ala Pro

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Ser Gln Asp Phe Val Asp Glu Val His Tyr Leu Ser Ser Ile Gln His

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Arg Asn Leu Val Thr Leu Leu Gly Tyr Cys Gln Glu Asn Asn Leu Gln

130 135 140

Phe Leu Ile Phe Asp Tyr Ile Pro Asn Gly Ser Val Ser Ser His Ile

145 150 155 160

Tyr Gly Thr Glu Gln Arg Ser Ala Glu Lys Leu Glu Phe Lys Ile Arg

165 170 175

Leu Ser Ile Ala Leu Gly Ala Ala Lys Gly Leu Ser His Leu His Ser

180 185 190

Met Ser Pro Arg Leu Thr His Arg Asn Phe Lys Thr Ser Asn Val Leu

195 200 205

Val Asp Glu Asn Phe Ile Ala Lys Val Ala Asp Ala Gly Leu His Asn

210 215 220

Val Met Arg Arg Phe Asp Val Ser Glu Ser Ser Ser Arg Ala Thr Ala

225 230 235 240

Asp Glu Ile Phe Leu Ala Pro Glu Val Lys Glu Phe Arg Gln Phe Ser

245 250 255

Glu Lys Ser Asp Val Tyr Ser Phe Gly Val Phe Leu Leu Glu Leu Val

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Ser Gly Gln Lys Ala Thr Asp Ala Pro Val Ser Asn Pro Asn Tyr Thr

275 280 285

Leu Val Asp Trp Ile Gln Asn Asn Gln Arg Lys Ser Asp Ile Gly Ser

290 295 300

Ile Thr Asp Pro Arg Leu Gly Lys Ser Phe Thr Glu Glu Gly Met Gly

305 310 315 320

Glu Leu Met Asp Leu Ile Leu Gln Cys Val Glu Tyr Ser Ser Glu Arg

325 330 335

Arg Pro Val Met Ser Tyr Val Val Thr Glu Leu Glu Arg Ile Leu Glu

340 345 350

Lys Glu Met Asn Leu Thr Thr Val Met Gly Glu Gly Ser Pro Thr Val

355 360 365

Thr Leu Gly Ser Gln Leu Phe Lys Thr Thr Lys

370 375

Claims (1)

1. the application of CsSTK gene or the protein encoded by CsSTK gene or the expression vector containing CsSTK gene in improving the resistance of cucumber corynebacterium leaf spot, it is characterized in that, the nucleotide sequence of described CsSTK gene is such as SEQ ID NO : 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

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