Application of HCR1 gene in improving Phytophthora resistance of plants

Abstract

The present invention relates to the technical field of genetic engineering, and particularly to the application of the HCR1 gene in improving the resistance of plants to Phytophthora. The present invention provides the application of the HCR1 gene in improving the resistance of plants to Phytophthora or cultivating transgenic plants resistant to Phytophthora, and the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1. The present invention introduces the mechanism of "accumulation on the membrane - formation of the death saw - membrane lysis - bacterial cell necrosis" of the phage HCR1 gene into plants, and discovers that the HCR1 gene can improve the resistance of plants to Phytophthora. By domesticating exogenous genes to achieve the purpose of plant disease resistance, it can not only expand the source of disease resistance gene mining, but also the disease resistance traits can be stably inherited.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of the HCR1 gene in improving the resistance of plants to Phytophthora blight. Background Art

[0002] There are two types of disease resistance bodies in plant cells. One binds to specific effectors, forms polymers, forms pores in the cell membrane, triggers calcium ion flow, and then triggers plant immune responses. The other needs the help of helper genes to polymerize and form pores in the cell membrane to cause plant disease resistance responses after binding to effectors. However, there are still great inconveniences in the modification and design of current plant disease resistance bodies, and at the same time, when infected by various pathogens such as fungi, bacteria, and viruses, the loss of race resistance occurs. Meanwhile, the excavation of current disease resistance genes is limited. To clone and discover more disease resistance bodies from plants, it requires a large amount of manpower, material resources, and financial resources. Moreover, traditional plant disease-resistant varieties are prone to the loss of race resistance after continuous planting for several years, making some plant endogenous disease resistance genes unable to play a lasting disease resistance role. Summary of the Invention

[0003] To solve the above problems, the present invention provides the application of the HCR1 gene in improving the resistance of plants to Phytophthora blight. The present invention discovers that the HCR1 gene can improve the resistance of plants to Phytophthora blight, and the disease resistance traits can be stably inherited.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides the application of the HCR1 gene in improving the resistance of plants to Phytophthora blight or cultivating transgenic plants resistant to Phytophthora blight, and the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1.

[0006] Preferably, the plant includes tobacco.

[0007] Preferably, the pathogen of Phytophthora blight includes Phytophthora capsici.

[0008] Preferably, the nucleotide sequence of the HCR1 gene is as shown in SEQ ID NO.2.

[0009] The present invention provides a recombinant vector for improving the resistance of plants to Phytophthora blight, and the recombinant vector includes an original vector, an RBOH promoter, and the HCR1 gene located downstream of the RBOH promoter; the nucleotide sequence of the RBOH promoter is as shown in SEQ ID NO.22; the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1.

[0010] Preferably, the original vector includes the pCAMBIA1381 vector.

[0011] The present invention provides a method for constructing the recombinant vector described in the above technical solution, comprising the following steps:

[0012] Using Arabidopsis thaliana genomic DNA as a template, PCR amplification is carried out with primers NbRBOHpro-F and NbRBOHpro-R to obtain the RBOH promoter containing restriction enzyme sites; the nucleotide sequences of the primers NbRBOHpro-F and NbRBOHpro-R are shown in SEQ ID NO.18 and SEQ ID NO.19;

[0013] Using the HCR1 gene as a template, PCR amplification is carried out with primers p1381-HCR1-F and p1381-HCR1-R to obtain the HCR1 gene sequence containing restriction enzyme sites; the nucleotide sequences of the primers p1381-HCR1-F and p1381-HCR1-R are shown in SEQ ID NO.20 and SEQ ID NO.21;

[0014] Connect the RBOH promoter containing restriction enzyme sites with the linearized pCAMBIA1381 vector digested by EcoR I to obtain the recombinant vector pCAMBIA1381-RBOH;

[0015] After digesting the recombinant vector pCAMBIA1381-RBOH with the restriction endonuclease Hind III, connect it with the HCR1 gene sequence containing restriction enzyme sites to obtain the recombinant vector.

[0016] The present invention provides an engineered bacterium for improving the resistance of plants to Phytophthora diseases, characterized by comprising the recombinant vector described in the above technical solution or the recombinant vector constructed by using the construction method described in the above technical solution.

[0017] Preferably, the initial strain for constructing the engineered bacterium includes Agrobacterium tumefaciens GV3101.

[0018] The present invention provides a method for improving the resistance of plants to Phytophthora diseases, by introducing the recombinant vector or the engineered bacterium into plants; the recombinant vector is the recombinant vector described in the above technical solution or the recombinant vector constructed by using the construction method described in the above technical solution; the engineered bacterium is the engineered bacterium described in the above technical solution.

[0019] Beneficial effects:

[0020] The present invention provides the application of the HCR1 gene in improving the resistance of plants to Phytophthora blight or cultivating transgenic plants resistant to Phytophthora blight. The amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1. The present invention introduces the mechanism of "accumulation on the membrane - formation of the death saw - membrane lysis - necrosis of bacterial cells" of the phage HCR1 gene into plants, and finds that the HCR1 gene can improve the resistance of plants to Phytophthora blight. By domesticating exogenous genes to achieve the purpose of plant disease resistance, not only can the source of disease resistance gene mining be expanded, but also the disease resistance traits can be stably inherited. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0022] Figure 1 Results of inducing the hypersensitive necrosis reaction of plants by transient expression of exogenous genes in Nicotiana benthamiana; Bright light and UV light represent white light and ultraviolet light respectively; the scale bar is 1 cm;

[0023] Figure 2 Results of Phytophthora capsici infection after 18 h of transient expression of the HCR1 gene in Nicotiana benthamiana, as well as statistics of lesion area and relative pathogen biomass;

[0024] Figure 3 Results of Phytophthora capsici infection and statistics of diseased area of different transgenic T0 lines;

[0025] Figure 4 Statistics of the infection effect of Phytophthora capsici, diseased area and relative pathogen biomass of different transgenic T1 lines. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides the application of the HCR1 gene in improving the resistance of plants to Phytophthora blight or cultivating transgenic plants resistant to Phytophthora blight. In the present invention, the plants preferably include tobacco; the pathogen of Phytophthora blight preferably includes Phytophthora capsici; the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1; the nucleotide sequence of the HCR1 gene is preferably as shown in SEQ ID NO.2, and the specific sequence information is as follows:

[0027] SEQ ID NO.1:

[0028] MKMPEKHDLLAAILAAKEQGIGAILAFAMAYLRGRYNGGAFTKTVIDAT MCAIIAWFIRDLLDFAGLSSNLAYITSVFIGYIGTDSIGSLIKRFAAKKAGVEDG RNQ*;

[0029] SEQ ID NO.2:

[0030] 5'-ATGAAGATGCCGGAGAAGCACGACCTCCTCGCCGCTATTCTCGCCG CTAAGGAGCAAGGCATCGGCGCTATCCTCGCCTTCGCTATGGCCTACCTCCGCGGCAGATACAACGGCGGCGCTTTCACAAAGACAGTGATCGACGCCACCATGTGCGCCATCATCGCCTGGTTCATCAGAGACCTCCTCGACTTCGCGGGCCTCTCTTCTAACCTCGCCTACATCACCTCCGTGTTCATCGGCTACATCGGCACCGACTCCATCGGCTCTCTCATCAAGCGCTTCGCCGCCAAGAAGGCCGGCGTTGAGGATGGCAGAAACCAGTGA-3'。

[0031] The present invention introduces the mechanism of "accumulation on the membrane - formation of the death scythe - membrane lysis - bacterial cell necrosis" of the phage HCR1 gene (that is, after the phage injects DNA into the bacterial cytoplasm, the HCR1 gene can be continuously expressed on the cell membrane, a large amount of aggregation forms the "death scythe" within 2 minutes, and then rapidly releases lytic factors to cause lysis and death of bacterial cells) into plants. By introducing the HCR1 gene into plant disease resistance, its role in plant disease resistance is demonstrated. After the HCR1 gene is introduced into plants, it can rapidly cause an immune response in plants, timely inhibit the invasion of pathogenic bacteria, and has the advantages of significant disease resistance effect and stable inheritance of disease resistance traits. The present invention domesticates plant exogenous genes to enhance the disease resistance ability of plants, and becomes a broad-spectrum resistance gene against a variety of pathogenic bacteria.

[0032] Based on the above advantages, the present invention provides a recombinant vector for improving the resistance of plants to Phytophthora, and the recombinant vector includes an original vector, an RBOH promoter, and an HCR1 gene located downstream of the RBOH promoter; the nucleotide sequence of the RBOH promoter is as shown in SEQ ID NO.24; the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1. In the present invention, the original vector preferably includes the pCAMBIA1381 vector.

[0033] The present invention provides a method for constructing the recombinant vector described in the above technical solution, comprising the following steps:

[0034] Using Arabidopsis thaliana genomic DNA as a template, PCR amplification is carried out with primers NbRBOHpro-F and NbRBOHpro-R to obtain an RBOH promoter containing restriction enzyme sites; the nucleotide sequences of the primers NbRBOHpro-F and NbRBOHpro-R are shown in SEQ ID NO.18 and SEQ ID NO.19;

[0035] Using the HCR1 gene as a template, PCR amplification is carried out with primers p1381-HCR1-F and p1381-HCR1-R to obtain an HCR1 gene sequence containing restriction enzyme sites; the nucleotide sequences of the primers p1381-HCR1-F and p1381-HCR1-R are shown in SEQ ID NO.20 and SEQ ID NO.21;

[0036] Connect the RBOH promoter containing restriction enzyme sites with the linearized pCAMBIA1381 vector digested with EcoR I to obtain the recombinant vector pCAMBIA1381-RBOH;

[0037] After digesting the recombinant vector pCAMBIA1381-RBOH with the restriction endonuclease Hind III, connect it with the HCR1 gene sequence containing restriction enzyme sites to obtain the recombinant vector.

[0038] Based on the above advantages, the present invention provides an engineered bacterium for improving the resistance of plants to Phytophthora blight, comprising the recombinant vector described in the above technical solution or a recombinant vector constructed by using the construction method described in the above technical solution. In the present invention, the initial strain for constructing the engineered bacterium preferably includes Agrobacterium tumefaciens GV3101.

[0039] Based on the above advantages, the present invention provides a method for improving the resistance of plants to Phytophthora blight, by introducing the recombinant vector or the engineered bacterium into plants; the recombinant vector is the recombinant vector described in the above technical solution or a recombinant vector constructed by using the construction method described in the above technical solution; the engineered bacterium is the engineered bacterium described in the above technical solution.

[0040] To further illustrate the present invention, the application of the HCR1 gene provided by the present invention in improving the resistance of plants to Phytophthora blight will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] S1. Vector construction

[0043] 1. Total RNA of rice was extracted using the Trizol method (R401-01-AA, Novoprotein, Nanjing, China) according to the instruction manual, and then cDNA was obtained using reverse transcriptase (R312-01-AB, Novoprotein, Nanjing, China) according to the instruction manual.

[0044] 2. Using the synthesized HCR1 gene (shown in SEQ ID NO.2) as a template, three full-length fragments of the HCR1 gene were amplified respectively. Hind III restriction sites and 14bp vector homologous sequences were added to the 5' ends of the upstream and downstream primers, and the downstream primer contained the stop codon TAA sequence for the independent expression of HCR1. Hind III restriction sites and 14bp vector homologous sequences were added to the 5' ends of the upstream and downstream primers, but the downstream primer did not contain the stop codon TAA sequence for the fusion expression of HCR1-eGFP. SmaI restriction sites and 14bp vector homologous sequences were added to the 5' ends of the upstream and downstream primers, and the downstream primer contained the stop codon TAA sequence for the fusion expression of eGFP-HCR1. The full-length Xa23 gene was amplified by PCR using the extracted rice cDNA as a template to obtain the Xa23 gene fragment. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 15 sec, for 35 cycles; final extension at 72°C for 5 min. The PCR products were recovered using an agarose gel recovery kit (DC301-01, Novoprotein, Nanjing, China). The primer sequences are as follows:

[0045] HCR1-F: 5'-AAATCGACTCTAGAAAGCTTATGAAGATGCCGGAGAAGCA-3', SEQ ID NO.3;

[0046] HCR1-R: 5'-CTCACCATGGTCTCAAGCTTTCACTGGTTTCTGCCATCCTC-3', SEQ ID NO.4;

[0047] HCR1-eGFP-F: as shown in SEQ ID NO.3;

[0048] HCR1-eGFP-R: 5'-CTCACCATGGTCTCAAGCTTCTGGTTTCTGCCATCCT CAAC-3', SEQ IDNO.5;

[0049] eGFP-HCR1-F: 5'-ACGAGCTGTACAAGCCCGGGATGAAGATGCCGGAG AAGCA-3', SEQ IDNO.6;

[0050] eGFP-HCR1-R: 5'-TAGTCCATGTCGACCCCGGGTCACTGGTTTCTGCCAT CCTC-3', SEQ ID NO.7;

[0051] Xa23-F: 5'-AAATCGACTCTAGAAAGCTTATGTTGCATCATCTCAAGGAG CT-3', SEQ ID NO.8;

[0052] Xa23-R: 5'-CTCACCATGGTCTCAAGCTTTTAAACAGGGAGAATAACCAT CTTGTCG-3', SEQ ID NO.9;

[0053] 3. Digest the pCAMBIA1300 vector with the restriction endonucleases Hind III and Sma I respectively, and recover and purify the digested products using an agarose gel recovery kit to obtain the Hind III-linearized pCAMBIA1300 vector and the Sma I-linearized pCAMBIA1300 vector;

[0054] Ligate the Hind III-linearized pCAMBIA1300 vector with the above two full-length HCR1 gene fragments and Xa23 gene fragments with Hind III restriction sites respectively under the action of a homologous recombination enzyme (#RDA01, Boyuan YANXUAN, Wuhan, China), and then transform the ligation products into Escherichia coli DH5α to obtain the recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, and pCAMBIA1300-Xa23;

[0055] Ligate the Sma I-linearized pCAMBIA1300 vector with the above HCR1 gene fragment with Sma I restriction sites under the action of a homologous recombination enzyme (#RDA01, Boyuan YANXUAN, Wuhan, China), and then transform the ligation products into Escherichia coli DH5α to obtain the recombinant vector pCAMBIA1300-eGFP-HCR1.

[0056] 4. Using the combined length of the full-length sequence of the HCR1 gene or Xa23 gene and a partial empty vector sequence as the positive clone target band, detect the positive clones of the recombinant vectors in three PCR amplification systems.

[0057] The detection primers for detecting the recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, and pCAMBIA1300-Xa23 are:

[0058] p1300-Hind III-F: 5'-CGCCATTTCGCCTTTTCA-3', SEQ ID NO.10;

[0059] p1300-Hind III-R: 5'-TGCAGATGAACTTCAGGGTCAG-3', SEQ ID NO.11;

[0060] The detection primers for detecting the recombinant vector pCAMBIA1300-eGFP-HCR1 are:

[0061] p1300-Sma I-F: 5'-GCAAAGACCCCAACGAGAAG-3', SEQ ID NO.12;

[0062] p1300-Sma I-R: 5'-CCGGCAACAGGATTCAATCT-3', SEQ ID NO.13;

[0063] S2. Detection of the cell lethality of the HCR1 gene

[0064] Transient expression of foreign genes in Nicotiana benthamiana induces the hypersensitive necrosis reaction (HR) in plants. The recombinant vectors containing foreign genes are CAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, pCAMBIA1300-eGFP-HCR1, pCAMBIA1300-Xa23 constructed by S1, and the empty vector pCAMBIA1300. The pCAMBIA1300 empty vector is used as a negative control, and pCAMBIA1300-Xa23 containing the eGFP gene and the Xa23 gene can induce the HR reaction as a positive control. The specific steps are as follows:

[0065] The four recombinant vectors pCAMBIA1300-HCR1, pCAMBIA1300-HCR1-eGFP, pCAMBIA1300-eGFP-HCR1, pCAMBIA1300-Xa23 and the empty vector pCAMBIA1300-eGFP were respectively transferred into Agrobacterium tumefaciens GV3101 by the freeze-thaw method. After culturing at 28 °C and 200 rpm for 18 h, the cells were centrifuged at 1500 g for 5 min using a centrifuge, and then the cells were resuspended with the infection solution (10 mM MgCl2, 10 mM MES and 100 μM acetosyringone). The concentration value OD was measured using a visible light spectrophotometer. 600nm Then, the infection solution was used again to adjust the OD of each component. 600nmThe value is 1.0. Agrobacterium resuspensions containing HCR1 gene, eGFP-HCR1 gene or HCR1-eGFP gene, empty vector negative control eGFP gene and positive control Xa23 gene were injected into four different parts of the same leaf of Nicotiana benthamiana at the 6-leaf stage. 48 hours after injection, the tobacco leaves were observed and photographed under white light and ultraviolet light. The results are shown in Figure 1 .

[0066] Figure 1 The results showed that HCR1 gene, eGFP-HCR1 gene and positive control Xa23 gene could induce HR response in tobacco cells, while HCR1-eGFP gene and empty vector negative control eGFP gene could not induce HR response. Figure 1 It can be seen that the HCR1 gene has the basic characteristics of plant disease resistance protein.

[0067] Example 2 HCR1 gene can inhibit the infection of pepper phytophthora in tobacco leaves

[0068] Transient expression of HCR1 gene in Nicotiana benthamiana can inhibit infection by Phytophthora capsici: The experiment was divided into two groups. Agrobacterium GV3101 containing vectors pCAMBIA1300-HCR1, pCAMBIA1300-eGFP and pCAMBIA1300-Xa23 were cultured at 28°C, 200 rpm and shaken for 18 h. The cells were centrifuged at 1500 g for 5 min, resuspended in infection solution (10 mM MgCl2, 10 mM MES and 100 μM acetosyringone), and the OD was measured using a visible light spectrophotometer. 600nm Adjust the OD of each component 600nm The value is 1.0. With the Xa23 gene as the positive control, the Agrobacterium resuspension containing the HCR1 gene and the empty vector negative control eGFP gene were injected into two symmetrical parts of the same leaf of Nicotiana benthamiana at the 6-leaf stage. After culturing at 24°C for 18 hours, the fungus block of Phytophthora capsici LT263 was inoculated at the injection site of Agrobacterium. After culturing at 24°C in the dark for 18 hours, the tobacco leaves were observed and photographed under ultraviolet light, the infection area of Phytophthora capsici was counted, and the biomass of the pathogen was detected by PCR. The results are shown in Figure 2It is deposited in the Rice Disease Resistance Laboratory of the College of Plant Science and Technology, Huazhong Agricultural University, and is disclosed in the literature [Sha G, Sun P, Kong XJ, Han XY, Sun QP, Fouillen L, et al. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance. Nature, 2023, 618(7967): 1017-1023. https: / / doi.org / 10.1038 / s41586-023-06205-2].

[0069] The primers for PCR detection of pathogen biomass are as follows:

[0070] Reference primers:

[0071] NbEF1a-F: 5'-AGAGGCCCTCAGACAAAC-3', SEQ ID NO.14;

[0072] NbEF1a-R: 5'-TAGGTCCAAAGGTCACAA-3', SEQ ID NO.15;

[0073] Primers for detecting Phytophthora capsici:

[0074] Pc-tubulin-F: 5'-CAGAGGGTGCTGAGCTTATTGACT-3', SEQ ID NO.16;

[0075] Pc-tubulin-R: 5'-GAGAGTGGGTGATCTGGAAACCC-3', SEQ ID NO.17;

[0076] Figure 2 The results showed that transient expression of the HCR1 gene in Nicotiana benthamiana could significantly inhibit the infection of Phytophthora capsici, and the lesion area was significantly smaller than that of the empty vector negative control eGFP gene (p < 0.01); the biomass contained in the infection site of Phytophthora capsici after expressing the HCR1 gene was significantly less than that of the negative control eGFP gene (p < 0.01).

[0077] From Figure 2 it can be seen that the HCR1 gene has plant disease resistance.

[0078] Example 3 Obtaining of HCR1 transgenic tobacco

[0079] S1. Vector construction

[0080] The full-length fragments of the RBOH promoter and the HCR1 gene were amplified by PCR and respectively constructed into the EcoR I and Hind III restriction sites of the plant stable expression vector pCAMBIA1381 through homologous recombination.

[0081] 1. Using Arabidopsis genomic DNA as a template, the RBOH promoter was amplified with primers NbRBOHpro-F and NbRBOHpro-R; using the synthesized HCR1 gene (shown in SEQ ID NO.2) as a template, the full-length HCR1 gene sequence was amplified with primers p1381-HCR1-F and p1381-HCR1-R. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C according to the amplification efficiency of 30 sec / 1 kb for 35 amplification cycles; final extension at 72°C for 5 min. The primer sequences are as follows:

[0082] NbRBOH pro-F: 5'-ATCCAGATCCAGTGGGATCCATAATAATGTAATAGCGAGAATACCTCTATTG-3', SEQ ID NO.18;

[0083] NbRBOH pro-R: 5'-CCGAGCTCACCCGGGGATCCCGGATGATGATTTTC AGAATTTTGCA-3', SEQ ID NO.19;

[0084] p1381-HCR1-F: 5'-GTCGACCTGCAGCCAAGCTTATGAAGATGCCGGAG AAGCA-3', SEQ IDNO.20;

[0085] p1381-HCR1-R: 5'-GTGGACTCCTCTTAAAGCTTTCACTGGTTTCTGCCA TCCTC-3', SEQ IDNO.21;

[0086] The nucleotide sequence of the RBOH promoter is shown in SEQ ID NO.22, and the specific sequence information is as follows:

[0087] SEQ ID NO.22:

[0088]

[0089] 2. Digest the pCAMBIA1381 vector with the restriction endonuclease EcoR I. After recovering and purifying the digested product using an agarose gel recovery kit, ligate it with the RBOH promoter fragment amplified in step 1 under the action of a homologous recombinase. Then transform the ligation product into Escherichia coli DH5α to obtain a recombinant vector pCAMBIA1381-RBOH containing the RBOH promoter.

[0090] 3. Digest the recombinant vector pCAMBIA1381-RBOH with the restriction endonuclease Hind III. After recovering and purifying the digested product using an agarose gel recovery kit, ligate it with the HCR1 gene fragment amplified in step 1 under the action of a homologous recombinase. Then transform the ligation product into Escherichia coli DH5α to obtain a recombinant vector pCAMBIA1381-RBOH-HCR1 containing the RBOH promoter and the HCR1 gene.

[0091] 4. Using the combined length of the HCR1 gene sequence and the empty vector sequence as the positive clone target band, the recombinant vector PCR detection primers are as follows:

[0092] p1381-F: 5'-GCCCTTTGGTCTTCTGAGACTG-3', SEQ ID NO.23;

[0093] p1381-R: 5'-CTGCATCGGCGAACTGATC-3', SEQ ID NO.24;

[0094] S2. Preparation of transgenic tobacco materials with the HCR1 gene

[0095] Prepare transgenic Nicotiana benthamiana using the recombinant vector pCAMBIA1381-RBOH-HCR1. The transgenic method is as follows:

[0096] (1) Agrobacterium-mediated infection of tobacco: Cut Nicotiana benthamiana leaves into 2 cm 2 square leaves, and then place the explants into the Agrobacterium GV3101 bacterial solution (OD 600nm value is 0.4 - 0.6) containing the recombinant vector pCAMBIA1381-RBOH-HCR1 and soak for 10 min. Subsequently, transfer the explants to sterile filter paper to absorb the moisture, and place them in the differentiation medium for dark culture for 3 d; the basal medium of the differentiation medium is MS medium, and it only contains 0.5 mg / L NAA and 2 mg / L 6-BA.

[0097] (2) Differentiation and screening of transgenic tobacco: Transfer the explants to a differentiation medium containing 300 mg / L cefamycin and 50 mg / L, and culture them under light at 25°C. Subculture once every 3 - 4 weeks. When the clustered buds grow, cut the resistant buds with a length of 1 - 1.5 cm from the base and transfer them to a seedling strengthening medium for culture; the basal medium of the seedling strengthening medium is MS medium, and it also only contains 0.1 mg / L 6 - BA, 0.01 mg / L NAA, 30 mg / L hygromycin and 100 mg / L cefamycin.

[0098] (3) Rooting of transgenic tobacco: Select the tobacco seedlings with roots and transfer them to a rooting medium for culture and detection; the basal medium of the rooting medium is MS medium, and it also only contains 30 mg / L hygromycin and 100 mg / L cefamycin.

[0099] (4) Extract a small sample of genomic DNA by CTAB method.

[0100] (5) PCR identification of positive transgenic plants: Perform PCR detection with specific primers. The size of the obtained target fragment is 350 bp, and the HCR1 transgenic tobacco is obtained, named NbRBOH - HCR1.

[0101] The PCR detection primers are shown as SEQ ID NO.20 and SEQ ID NO.21.

[0102] After culturing the obtained NbRBOH - HCR1 transgenic tobacco for one month, perform disease resistance detection.

[0103] Example 4 Resistance analysis of HCR1 transgenic tobacco to Phytophthora capsici

[0104] S1. Resistance detection of To - generation NbRBOH - HCR1 transgenic tobacco

[0105] After culturing the NbRBOH - HCR1 transgenic tobacco for one month, using wild - type tobacco (denoted as WT) as a control, take leaves at the same position, then gently make cross - shaped wound spots at the inoculation site with a needle, place the Phytophthora capsici LT263 fungal block in the center of the wound spot, observe the Phytophthora infection situation under ultraviolet light 28 h after culturing in the dark and moisturized at 24°C, and count the diseased area. Five tobacco lines with significant resistance (p < 0.05) are screened, denoted as RBOH:HCR1 - 3, RBOH:HCR1 - 6, RBOH:HCR1 - 9, RBOH:HCR1 - 14 and RBOH:HCR1 - 15 (see Figure 3 ).

[0106] S2. Resistance detection of T1 - generation NbRBOH - HCR1 transgenic tobacco

[0107] Harvest the T0 generation of NbRBOH-HCR1 transgenic tobacco seeds with significant resistance to Phytophthora capsici, and conduct resistance verification according to the method described in S1. Observe the infection situation of Phytophthora and count the disease area and relative pathogen biomass. The results are shown in Figure 4 , where the leaves with the same number are different leaves of the same plant.

[0108] Figure 3 and Figure 4 The results show that the disease severity of the NbRBOH-HCR1 transgenic tobacco lines 3, 6, 9, 14, and 15 decreased after inoculation with Phytophthora capsici, and their Phytophthora disease area and relative pathogen biomass were significantly smaller than those of the control, indicating that the HCR1 transgenic tobacco has significant resistance to Phytophthora capsici infection.

[0109] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of the HCR1 gene in enhancing the resistance of plants to Phytophthora blight or cultivating transgenic plants resistant to Phytophthora blight, wherein the amino acid sequence encoded by the HCR1 gene is as shown in SEQ ID NO.1; the plant is tobacco; the pathogen of Phytophthora blight includes Phytophthora capsici.

2. The application according to claim 1, wherein The nucleotide sequence of the HCR1 gene is as shown in SEQ ID NO.2.

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