Resistance of CaCP450 gene to cucumber blight and its application

Abstract

The present invention belongs to the technical field of plant molecular biology and plant genetic engineering, discloses the resistance and application of CaCP450 gene to cucumber blight, and specifically discloses the application of cytochrome gene CaCP450 in resistance to cucumber blight. The present invention first discovered that the CaCP450 gene derived from pepper CM334 has obvious resistance to cucumber blight. The resistance of cucumber cotyledons with heterologous overexpression of CaCP450 gene to cucumber blight is significantly enhanced, and the area of ​​lesions after inoculation is significantly reduced compared with the control. The CaCP450 gene can be used for cucumber products resistant to cucumber blight and for cultivating cucumber transgenic plants resistant to blight, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to the resistance of CaCP450 gene to cucumber blight and its application. Background Art

[0002] Cucumber blight is a worldwide oomycete disease. The pathogen is Phytophthora melonis or Phytophthora capsici, which causes cucumber leaves, stems, and fruits to turn brown and cause death or seedling death. The disease occurs in all areas of my country where cucumbers are grown. In the north, cucumbers are more seriously affected in open-field cultivation in summer and autumn and protected cultivation in spring, while in the south, they are more seriously affected in the hot and humid seasons of spring and summer and autumn. The entire growth period and all parts of cucumbers can be affected. The growth point and the base of the tender stems of the seedlings are water-soaked and constricted, and they wilt and die. In the adult stage, the stems are affected, and the bases and nodes are water-soaked and dark brown spots, which gradually soften and constrict, the leaves droop, the plants wilt, and die; the leaves often have large dark green spots with unclear edges near the leaf margins; the fruits and vegetables show water-soaked, nearly circular sunken spots at the affected parts, which rot quickly, and a sparse white or grayish-white mold layer grows on the surface. Once the disease occurs, the control effect of chemical agents is not ideal, resulting in economic losses. Cultivating disease-resistant varieties is one of the most economical, environmentally friendly and effective ways to control cucumber blight, and discovering disease-resistant genes (or proteins) is the basis of disease-resistant breeding. Summary of the invention

[0003] The first aspect of the present invention aims to provide the application of CaCP450.

[0004] The second aspect of the present invention aims to provide the use of nucleic acid molecules encoding CaCP450 in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight.

[0005] The third aspect of the present invention aims to provide a vector comprising a nucleic acid molecule encoding CaCP450 for use in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight.

[0006] The fourth aspect of the present invention aims to provide an expression cassette comprising a nucleic acid molecule encoding CaCP450 for use in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight.

[0007] The fifth aspect of the present invention aims to provide a transgenic cell line comprising a nucleic acid molecule encoding CaCP450 for use in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight.

[0008] The sixth aspect of the present invention aims to provide a recombinant bacterium or recombinant virus containing a nucleic acid molecule encoding CaCP450 for use in resisting cucumber blight, preparing products resistant to cucumber blight, or cultivating transgenic plants resistant to cucumber blight.

[0009] The seventh aspect of the present invention aims to provide a reagent for targeted upregulation of the expression of CaCP450 for use in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight.

[0010] The eighth aspect of the present invention aims to provide a method for cultivating transgenic plants resistant to cucumber blight.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of CaCP450 as described in any one of (1) to (3):

[0013] (1) Resistance to cucumber blight;

[0014] (2) Preparation of products resistant to cucumber blight;

[0015] (3) Cultivating transgenic plants resistant to cucumber blight;

[0016] The amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0017] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0018] In some embodiments of the present invention, the plant is cucumber.

[0019] The second aspect of the present invention provides the use of a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0020] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0021] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0022] In some embodiments of the present invention, the plant is cucumber.

[0023] The third aspect of the present invention provides the use of a vector comprising a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0024] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0025] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0026] In some embodiments of the present invention, the plant is cucumber.

[0027] A fourth aspect of the present invention provides the use of an expression cassette comprising a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0028] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0029] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0030] In some embodiments of the present invention, the plant is cucumber.

[0031] A fifth aspect of the present invention provides a transgenic cell line comprising a nucleic acid molecule encoding CaCP450 for use in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0032] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0033] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0034] In some embodiments of the present invention, the plant is cucumber.

[0035] A sixth aspect of the present invention is the use of a recombinant bacterium or recombinant virus comprising a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight, or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0036] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0037] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0038] In some embodiments of the present invention, the plant is cucumber.

[0039] The seventh aspect of the present invention provides the use of a reagent for targeted upregulation of the expression of CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight, or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:2.

[0040] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0041] In some embodiments of the present invention, the plant is cucumber.

[0042] The eighth aspect of the present invention provides a method for cultivating cucumber blight-resistant transgenic plants, comprising the following steps: introducing a nucleic acid molecule encoding CaCP450 into a target plant to obtain a cucumber blight-resistant transgenic plant; the amino acid sequence of the CaCP450 is shown in SEQ ID NO:2.

[0043] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

[0044] In some embodiments of the present invention, the cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

[0045] In some embodiments of the present invention, the plant is cucumber.

[0046] The beneficial effects of the present invention are:

[0047] The present invention first discovered that the CaCP450 gene from pepper CM334 has obvious resistance to cucumber blight. The resistance of cucumber cotyledons that heterologously overexpress the cytochrome gene CaCP450 to cucumber blight is significantly enhanced, and the area of ​​lesions after inoculation is significantly reduced compared with the control. The CaCP450 gene can be used for cucumber products resistant to cucumber blight and for cultivating cucumber transgenic plants resistant to blight, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is the transcriptional expression of CaCP450 gene in resistant and susceptible pepper materials.

[0049] Figure 2 This is the plasmid map of the overexpression vector pGWB5.

[0050] Figure 3 Detection of the expression level of CaCP450 genes in cucumber leaves.

[0051] Figure 4 Resistance detection in cucumber leaves heterologously expressing CaCP450 genes. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below through specific examples.

[0053] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0054] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0055] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products. This experiment used cucumber B80 (Tingquan Wu, Rui Wang, Xiaomei Xu, Xiaoming He, Baojuan Sun, Yujuan Zhong, Zhaojuan Liang, Shaobo Luo* and Yu′e Lin*. Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthoramelonis, Phytophthora capsici and water immersion in disease resistant andsusceptible cucumber cultivars.Gene, 2014,549(2):214-222), pepper CM334 (XiaomeiXu, Juan Chao, Xueli Cheng, Rui Wang, Baojuan Sun, Hengming Wang, Shaobo Luo, Xiaowan Xu, Tingquan Wu, Ying Li*. Mapping of a novel race specific resistancegene to phytophthora root rot of pepper(Capsicum annuum)using bulkedsegregant analysis combined with specific length amplified fragmentsequencing strategy.PLoS One,2016,11(3):e0151401) Materials were provided by the New Technology Research Laboratory of Facility Agriculture of our unit.The cucumber blight fungus originated from diseased cucumber plants in the Zhongluotan experimental field in Baiyun District, Guangzhou (Wang Rui, Jin Qingmin, Wu Tingquan, Lin Yu'e*. Inhibitory effect of leek crude extract on cucurbit blight fungus and pepper blight fungus. Abstracts of the 2015 Annual Academic Conference of the Chinese Society of Horticulture, 2015, 94; Wang Rui, Lin Yu'e, Liang Zhaojun, Jin Qingmin, Wu Tingquan*. Isolation and identification of the pathogen of cucumber blight in Guangdong. Chinese Agricultural Science Bulletin, 2016, 32(01): 76-80).

[0056] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0057] Example 1 Transcriptional expression of CaCP450 genes in resistant and susceptible pepper materials

[0058] The cytochrome gene CaCP450 is a cytochrome family gene that was discovered during the transcriptome analysis of pepper resistant materials (CM334) and susceptible materials (10399). 24 hours after the inoculation of pepper phytophthora, the transcriptional expression of the CaCP450 gene in pepper resistant materials increased rapidly, while the transcription in susceptible materials was severely inhibited ( Figure 1 ).

[0059] Example 2 Construction of CaCP450 gene heterologous overexpression vector

[0060] The full-length CDS of CaCP450 gene (SEQ ID NO: 1) was constructed into pGWB5 (pGWB5 plasmid map as shown in Figure 2 The specific position is between attR1 and attR2 after the 35S promoter (the ccdB gene is replaced by gene recombination exchange), and an overexpression vector (pGWB5-CaCP450) is obtained.

[0061] The CaCP450 gene CDS sequence is as follows:

[0062] ATGGCTTTTCTTTGGCTAGCTCTTGCCGTAGTTGCCATAGTTTTTCTTCTTCTTC

[0063] AACAACTACAGAAGAAGTTCAGTAAGAAGAAGAAGATACTTCCTCCAGGTCCAAG

[0064] AGGGCTCCCACTTCTTGGGAATCTTCACATGTTTGGAGATAATCTTCACCAAGACC

[0065] TTAACAAAATAGCCAAGAAACATGGCCCTATAATGTCCATACATTTTGGTCTTGTTC

[0066] CTGTAATAGTTGCTTCCACTCCTTATGCTGCTGAACAATTCTTGAAAACACATGATT

[0067] TAACCTTTTCAAGTAGACCCTCTAATCAAGTATCAGAAAACATTTTTTACAAGCGG

[0068] AAAAATCTTGTCTCGGCCCGTTACGGACCATATTGGAGGAACATGCGCAAGTTGTG

[0069] CACTTTGCAGCTGCTAAGCAGTGCCAAGATCCTTGCTTTCCAGCCTATGAGGAAAC

[0070] AAGAACTTGGGATTCTTATAAATTTCGTAAAACAGGCAGCTATGGAACACAAAGTA

[0071] GTTGATCTTACTGCAAAAGTTGGTTCTCTCAGTGCAAACATGTCATGTTTGATGATA

[0072] TTTGGAAAGAAGTACATGGACGAAGATCTTGGCGAAAAGGGGTTCAAAGCTATAA

[0073] TTCAAGATATGCTGCATATAGCAGCATTGCCAAATTTTTCTGATTTTTTCCCTTTTCT

[0074] TCGCATATTTGATCTCCAGGGATATAATCGTCGTGTGAAAGAATTAGCTAAACTGTT

[0075] TGACGAGTTCTTGGAGAGAGTAATCGACGAACATGTTGGTGAATCCAATGAGCAG

[0076] AAACAAGCAAAGGATATGGTGGATACACTGATGGAGATTCTGCAGTCCGGTGCGG

[0077] CTGAATTCGAGTTCGATCGTCGCCATGTCAAAGCTATTATGCTGGACTTGCTGATAG

[0078] CATCTATGGACACTTCATCAACAACAATTGATTGGATCCTAACAGAACTTTTAAGGC

[0079] ACCCCAATGTGATGAAAAAAGTGCAGAATGAGTTGGAACAAGTCATTGGTAAAAA

[0080] CAGAATGGTGGAAGAGTCAGACTTAGAAAGCCTAGAGTATTTAGATATGGTCATAA

[0081] AAGAAAGTAACAGACTCCATCCTGCTGCACCCTTATTGCTTCCTCACGAGTCAATT

[0082] GAAGACTGCACGGTTGATGGCTTTCACATACCTAAGGGATCCCGAGTATTAATCAAT

[0083] ATTTGGGCAATTGGCAGAGATCCAGACACTTGGGTCGAGCCTGAAAAGTTCAAAC

[0084] CTGAGAGATTTCAAGGAAGCAATATAGATCTGCGCGGACGACATTTTCAACTTCTA

[0085] CCATTTGGTTCCGGTAGAAGAAGTTGTCCTGGTTTGCAGCTAGGGCTCACCGTTGT

[0086] TCGCTTGATGGTCGCTCAATTGGTTCACTGCTTTAACTGGGAGTTGCCGAATGGCA

[0087] TGATGCCGAAGGATATTGACATCACTGAGAAATTTGGTTTAGTAACGGCCAGAGCA

[0088] CAACATCTAATGGCGATTCCTAATTATCGACTACACATATGA(SEQ ID NO:1)。

[0089] The amino acid sequence of the CaCP450 protein is as follows:

[0090] MAFLWLALAVVAIVFLLLQQLQKKFSKKKKILPPPGPRGLPLLGNLHMFGDNLHQ

[0091] DLNKIAKKHGPIMSIHFGLVPVIVASTPYAAEQFLKTHDLTFSSRPSNQVSENIFYKRKN

[0092] LVSARYGPYWRNMRKLCTLQLLSSAKILAFQPMRKQELGILINFVKQAAMEHKVVDL

[0093] TAKVGSLSANMSCLMIFGKKYMDEDLGEKGFKAIIQDMLHIAALPNFSDFFPFLRIFD

[0094] LQGYNRRVKELAKLFDEFLERVIDEHVGESNEQKQAKDMVDTLMEILQSGAAEFEFD

[0095] RRHVKAIMLDLLIASMDTSSTTIDWILTELLRHPNVMKKVQNELEQVIGKNRMVEES

[0096] DLESLEYLDMVIKESNRLHPAAPLLLPHESIEDCTVDGFHIPKGSRVLINIWAIGRDPDT

[0097] WVEPEKFKPERFQGSNIDLRGRHFQLLPFGSGRRSCPGLQLGLTVVRLMVAQLVHCF

[0098] NWELPNGMMPKDIDITEKFGLVTARAQHLMAIPNYRLHI(SEQ ID NO:2).

[0099] Example 3 Construction of a cucumber leaf gene expression model for transient heterologous expression

[0100] (1) The overexpression vector pGWB5-CaCP450 in Example 2 was transformed into Agrobacterium GV3101 and cultured in an inverted manner on a medium with corresponding resistance for 48 h.

[0101] (2) Pick a single clone and add it to 4 mL of LB medium containing the corresponding antibiotics and rifampicin, and shake the culture at 28°C and 180 rpm for 24 h.

[0102] (3) Add the culture medium containing the corresponding antibiotics and rifampicin at a ratio of 1:100 and shake at 28°C and 180 rpm until the OD reaches 600 The value is about 3.0.

[0103] (4) Centrifuge at 3000 rpm for 5 min to collect the cells, resuspend them in suspension (10 mM MES, 10 mM MgCl2), and adjust the OD 600 When the pH value reaches about 0.4, add 200 mM acetosyringone.

[0104] (5) Let stand at room temperature for 3 hours.

[0105] (6) Use a syringe needle to poke a hole on each side of the main vein of the cucumber leaf.

[0106] (7) Use a 1 mL syringe to draw up an equal amount of the bacterial solution after it has been allowed to stand. Aim the syringe at the needle hole on the back, block the front with your hand, and inject the bacterial solution.

[0107] (8) The injected cucumber seedlings were cultured in the dark for 12 h and then in the light at 22°C for 3 to 4 days.

[0108] (9) The cucumber cotyledons were separated from the culture medium and inoculated with fungus blocks of cucumber phytophthora. The cotyledons were then cultured in a petri dish at 28°C in the dark for 24 h.

[0109] (10) Collect treated and control leaves and detect gene expression levels (for specific methods, refer to Rui Wang, Yu'eLin, Qingmin Jin, Chunpeng Yao, Yujuan Zhong, Tingquan Wu*. RNA-Seq analysis of gynoecious and weak female cucumber revealing the cell cycle pathway may regulate sex determination in cucumber. Gene, 2019, 687: 289-297).

[0110] The results showed that CaCP450 gene was expressed at a higher level in cucumber leaves with transient heterologous CaCP450 gene expression, while the wild type control (CK) had no expression ( Figure 3 ). This indicates that the model of transient heterologous expression of CaCP450 gene in cucumber leaves was successfully constructed.

[0111] Example 4 Disease resistance experiment of cucumber leaves

[0112] The cucumber leaf model of transient heterologous expression of CaCP450 gene successfully constructed in Example 3 and the wild-type cucumber leaves were inoculated with Phytophthora cucumeris for 24 hours, and the resistance function of the target gene to Phytophthora cucumeris and Phytophthora capsici was determined based on the size of the lesions on the cucumber leaves treated with different methods.

[0113] The lesion area of ​​30 leaves in each treatment was averaged, and then the leaf areas of heterologous expression and control were used for significance analysis and comparison.

[0114] The experimental results showed that transient heterologous expression of the CaCP450 gene (pGWB5-CaCP450) in the leaves of the susceptible cucumber material B80 could significantly enhance the resistance of cucumber leaves to the blight caused by Phytophthora cucurbitae compared with the wild type (WT) ( Figure 4 ).

[0115] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of CaCP450 in any one of (1) to (3): (1) Resistance to cucumber blight; (2) Preparation of products resistant to cucumber blight; (3) Cultivating transgenic plants resistant to cucumber blight; The amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

2. Use of a nucleic acid molecule encoding CaCP450 in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

3. Use of a vector comprising a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

4. Use of an expression cassette comprising a nucleic acid molecule encoding CaCP450 in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

5. Use of a transgenic cell line comprising a nucleic acid molecule encoding CaCP450 in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

6. Use of a recombinant bacterium or recombinant virus comprising a nucleic acid molecule encoding CaCP450 in resisting cucumber blight, preparing products resistant to cucumber blight or cultivating transgenic plants resistant to cucumber blight, wherein the amino acid sequence of the CaCP450 is shown in SEQ ID NO:

2.

7. Use of a reagent for targeted upregulation of the expression of CaCP450 in resistance to cucumber blight, preparation of products resistant to cucumber blight or cultivation of transgenic plants resistant to cucumber blight, wherein the amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

8. The use according to any one of claims 1 to 7, characterized in that: The cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

9. The use according to any one of claims 2 to 6, characterized in that: The sequence of the nucleic acid molecule is shown in SEQ ID NO:

1.

10. A method for cultivating a transgenic plant resistant to cucumber blight, comprising the following steps: introducing a nucleic acid molecule encoding CaCP450 into a target plant to obtain a transgenic plant resistant to cucumber blight; The amino acid sequence of CaCP450 is shown in SEQ ID NO:

2.

11. The method according to claim 10, characterized in that The cucumber blight is a disease caused by Phytophthora cucumeris and / or Phytophthora capsici.

12. The method according to claim 10, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO:1.

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