NbCYP707A4 gene and application thereof in hydrangea leaf spot indicating plant
By knocking out the NbCYP707A4 gene in tobacco, we created tobacco that is highly susceptible to leaf spot disease. This serves as an indicator plant for hydrangea leaf spot disease, solving the problems of predicting and preventing hydrangea leaf spot disease and achieving dual reductions in economic benefits and environmental protection.
Patent Information
- Application Number
- CN202510879516.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-12
AI Technical Summary
There is no effective method in the existing technology to predict and prevent leaf spot disease of Hydrangea macrophylla, which leads to high morbidity, large economic losses and serious environmental pollution.
CRISPR/Cas9 gene knockout technology was used to knock out the NbCYP707A4 gene in tobacco, creating tobacco that is highly susceptible to leaf spot disease. This plant serves as an indicator plant for hydrangea leaf spot disease, and the disease can be predicted and prevented by observing the incidence of tobacco disease.
It can timely predict and prevent hydrangea leaf spot disease, reduce economic losses, lower production costs, and alleviate environmental pollution. It is suitable for hydrangea and other horticultural crops.
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Figure CN120624480A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical fields of genetic engineering and plant cultivation, and particularly relates to an NbCYP707A4 gene and an application thereof in hydrangea leaf spot disease indicator plants. Background Art
[0002] Bigleaf hydrangea (Hydrangea macrophylla) is one of the world's three major garden plants. It can be used for landscaping, potted plants, and cut flowers, and has high ornamental and economic value. Leaf spot is one of the main diseases of bigleaf hydrangea. The hot and humid climate, where the plant's peak growth period overlaps with the plum rain season in Jiangsu, Zhejiang, and Shanghai, is particularly susceptible to leaf spot, with an incidence rate exceeding 80%. This is a major factor restricting the high-quality and high-yield nature of hydrangeas.
[0003] Corynespora leaf spot, caused by Corynespora cassiicola, is one of the main types of leaf spot diseases in hydrangeas. Once the lesions are formed, they are irreversible, causing great economic losses every year. There is still no specific medicine for leaf spot caused by Corynespora cassiicola. After the onset of the disease, a large number of chemical agents need to be applied in rotation, leading to serious problems such as increased production costs and increased environmental pollution. C. cassiicola is a typical fungus with a high risk of drug resistance. Therefore, it is necessary to predict and prevent hydrangea leaf spot before the onset of the disease, control the large-scale damage of pathogens, and thus achieve scientific management. The use of plant indicators is a simple, practical and easy-to-promote prediction method for predicting the occurrence of diseases and insect pests in production. At present, there are no reports on the use of indicator plants for disease prediction and prevention of leaf spot diseases in ornamental plants such as hydrangeas.
[0004] Therefore, it is necessary to develop a leaf spot disease indicator plant and use it to predict and control leaf spot disease in hydrangea and other horticultural crops. Summary of the Invention
[0005] In response to the deficiencies in the prior art, the present invention provides the NbCYP707A4 gene and its application in hydrangea leaf spot indicator plants. The present invention has found that the NbCYP707A4 gene is a key gene for tobacco leaf spot resistance, and that tobacco highly susceptible to Corynespora leaf spot can be prepared by knocking out the gene using CRISPR / Cas9 gene knockout technology. The highly susceptible tobacco to Corynespora leaf spot can serve as an indicator plant for hydrangea leaf spot, can timely predict and prevent the occurrence of hydrangea leaf spot, reduce economic losses, achieve a reduction in hydrangea production costs, and alleviate environmental pollution caused by excessive application of pesticides. The invention is of great significance to hydrangea and other horticultural crops and has good practicality.
[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:
[0007] The present invention first provides a NbCYP707A4 gene, the nucleotide sequence of the NbCYP707A4 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the NbCYP707A4 gene is shown in SEQ ID NO: 2.
[0008] The present invention also provides the use of the above-mentioned NbCYP707A4 gene in resisting plant leaf spot disease; overexpression of the NbCYP707A4 gene can improve the plant's resistance to leaf spot disease, and silencing / inhibiting the expression of the NbCYP707A4 gene can reduce the plant's resistance to leaf spot disease.
[0009] Preferably, the plant includes Nicotiana benthamiana; and the leaf spot disease includes Corynespora leaf spot disease caused by Corynespora multifungi.
[0010] The present invention also provides a method for obtaining tobacco highly susceptible to leaf spot disease, the method comprising:
[0011] The NbCYP707A4 gene in tobacco was knocked out using CRISPR / Cas9 gene knockout technology to obtain tobacco highly susceptible to leaf spot disease.
[0012] The present invention also provides the use of the highly susceptible tobacco to leaf spot disease obtained by the method as an indicator plant for leaf spot disease.
[0013] Preferably, the application includes: predicting and / or preventing and controlling the occurrence of hydrangea leaf spot disease.
[0014] The present invention also provides a method for predicting and / or preventing and controlling hydrangea leaf spot disease, the method comprising:
[0015] Place tobacco plants that are highly susceptible to leaf spot disease in the hydrangea greenhouse near varieties that are susceptible to leaf spot disease, and observe the incidence of leaf spot disease in the tobacco plants. When spots appear on the leaves of tobacco plants that are highly susceptible to leaf spot disease and hydrangeas do not develop leaf spot disease, spray the hydrangeas with fungal agents to prevent the occurrence of leaf spot disease.
[0016] Preferably, the leaf spot disease includes Corynespora leaf spot caused by Corynespora multifungi.
[0017] Preferably, the suitable seedling age of the indicator plant is 30-60 days after transplanting and planting;
[0018] The indicator plant is used in the rainy season when hydrangea leaf spot disease is prone to occur.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention uses tobacco, which is susceptible to Corynespora leaf spot, as an indicator crop for horticultural crops, particularly hydrangea leaf spot. Tobacco has the advantages of low planting cost, short growth cycle, and ease of management. Furthermore, as a model plant for molecular biology research, it has a complete genome and a relatively mature gene editing technology system, making it an ideal material for use as an indicator plant for hydrangea leaf spot. The leaf spot pathogen (Corynespora cassiicola) isolated from hydrangea leaf spot-infected strains can cause leaf spot symptoms in Nicotiana benthamiana leaves, and the symptoms are more severe when infected simultaneously than on hydrangea leaves.
[0021] Since the time difference between the onset of leaf spot symptoms in tobacco and hydrangea after infection with leaf spot pathogens is very short under natural conditions, the present invention discovered that NbCYP707A4 is a key gene for tobacco's resistance to leaf spot disease. By knocking out NbCYP707A4 using CRISPR / Cas9 gene knockout technology, non-transgenic tobacco with high susceptibility to leaf spot disease can be created. The time difference between the onset of leaf spot symptoms in this highly susceptible tobacco and hydrangea is increased, and this tobacco can serve as an indicator plant for hydrangea leaf spot disease.
[0022] The present invention places highly susceptible tobacco plants, transplanted 30-60 days after planting, at both ends and in the middle of a greenhouse hydrangea bed during the rainy season, when hydrangea leaf spot is prone to occur, near susceptible varieties. The tobacco plants are observed for leaf spot disease. When lesions appear on the leaves of the highly susceptible tobacco plants and the hydrangeas are not affected by leaf spot disease, a fungicide is sprayed on the hydrangeas to prevent the occurrence of leaf spot disease. The method of the present invention can timely predict and prevent hydrangea leaf spot disease, thereby reducing economic losses, lowering hydrangea production costs, and alleviating environmental pollution caused by excessive pesticide application. This method has important implications for hydrangeas and other horticultural crops. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a diagram showing the tissue situation of Nicotiana benthamiana leaves infected by the hydrangea leaf spot pathogen Corynespora cassiicola.
[0024] Figure 2 This figure shows the changes in endogenous hormones in plants before and after infection of resistant and susceptible cultivars by Corynespora cassiicola. In the figure, a: salicylic acid SA; b: jasmonic acid JA; c: abscisic acid ABA; d: ethylene ET.
[0025] Figure 3Figure 4: Disease incidence and lesion area of resistant and susceptible varieties 48 hours after inoculation with Corynespora cassiicola on detached leaves of resistant and susceptible varieties after exogenous ABA spraying. In the figure, a and b: disease incidence and lesion area of susceptible varieties inoculated with leaf spot pathogen after ABA treatment; c and d: disease incidence and lesion area of resistant varieties inoculated with leaf spot pathogen.
[0026] Figure 4 The expression levels of HmCYP707A4 in the ABA decomposition pathway of hydrangea in the resistant and susceptible varieties inoculated with Corynespora cassiicola at 0 h and 24 h; in the figure, a: position of HmCYP707A4 in the ABA decomposition pathway of hydrangea; b: expression levels of the resistant and susceptible varieties inoculated with Corynespora cassiicola at 0 h and 24 h, among which, RCK: resistant variety was infected with Corynespora cassiicola for 0 h; RT: resistant variety was infected with Corynespora cassiicola for 24 h; SCK: susceptible variety was infected with Corynespora cassiicola for 0 h; ST: susceptible variety was infected with Corynespora cassiicola for 24 h.
[0027] Figure 5 This is a diagram showing the functional verification of NbCYP707A4's resistance to leaf spot disease.
[0028] Figure 6 This is a diagram showing the verification of the leaf spot resistance function of tobacco that is highly susceptible to leaf spot disease. DETAILED DESCRIPTION
[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the scope of the present invention is not limited thereto. In the following examples, various processes and methods not described in detail are conventional methods well known in the art. The sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.
[0030] The seamless cloning kit, high-fidelity DNA polymerase, BamHI, and SalI used in Example 2 of the present invention were all purchased from Yisheng Biotechnology Co., Ltd.
[0031] The leaf spot pathogen C.cassiicola used comes from the Flower Innovation Team of the Leisure Agriculture Research Institute of Jiangsu Academy of Agriculture.
[0032] Agrobacterium strain EHA105 was purchased from Sangon Biotechnology Co., Ltd.
[0033] The decarboxylation culture medium consists of: MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 500 mg / L Cef + 50 mg / L K; the basal culture medium was purchased from Soleb Biotechnology Co., Ltd.
[0034] Example 1:
[0035] Tobacco, a member of the Solanaceae family, is a crop susceptible to Corynespora leaf spot. In this example, the fungus Corynespora cassiicola was inoculated into the lower mature leaves of Nicotiana benthamiana to investigate whether the fungus could cause disease in Nicotiana benthamiana and the severity of the disease. The specific steps are as follows:
[0036] The fungus cakes (5mm in diameter) of Corynespora cassiicola were inoculated onto the lower mature leaves of Nicotiana benthamiana and hydrangea leaves, with the mycelium surface attached to the leaves. After inoculation, Tween 80 was used to moisturize the inoculation site. The lower mature leaves of Nicotiana benthamiana and hydrangea leaves were observed 48 hours after inoculation. The results are as follows: Figure 1 shown.
[0037] from Figure 1 It can be seen that 48 hours after inoculation, the inoculated part of Nicotiana benthamiana wilts and turns yellow, and symptoms appear ( Figure 1 A), indicating that Nicotiana benthamiana can be infected by Corynespora leaf spot pathogen; and when the same infection method was used to inoculate hydrangea leaves for 48 hours, the symptoms and lesion area of tobacco leaves were significantly more severe than those of hydrangea ( Figure 1 B) This suggests that N. benthamiana has the potential to be used as an indicator plant for hydrangea leaf spot. However, the onset of disease in N. benthamiana and hydrangea is too close, so a more susceptible N. benthamiana strain would need to be created to develop disease more quickly.
[0038] Example 2:
[0039] In the early stage of this invention, the molecular mechanism of hydrangea leaf spot disease resistance was studied by identifying the leaf spot disease resistance of different varieties, and the typical leaf spot disease-resistant variety "White Angel" and the susceptible variety "Ocean Heart" were screened. By studying the disease-resistant variety "White Angel" and the disease-susceptible variety "Ocean Heart", the key genes affecting hydrangea leaf spot disease resistance were screened.
[0040] The leaf spot pathogen C. cassiicola (isolated and preserved by the flower innovation team of the Leisure Agriculture Research Institute of Jiangsu Academy of Agricultural Sciences) was used to infect the leaf spot disease-resistant variety 'White Angel' and the susceptible variety 'Ocean Heart' at 0h and 24h, and the changes in the endogenous hormones ABA, SA, JA and ET in the leaf tissues of the disease-resistant variety 'White Angel' and the susceptible variety 'Ocean Heart' were observed. The results are as follows Figure 2 As shown in the figure, ABA showed the greatest difference in content between resistant and susceptible varieties before and after infection, preliminarily indicating that ABA plays an important role in regulating hydrangea's resistance to leaf spot disease.
[0041] To verify that ABA does play a key role in regulating hydrangea leaf spot resistance, we further sprayed one-gallon potted seedlings of the resistant variety 'White Angel' and the susceptible variety 'Heart of the Ocean' with 100 μM ABA. 24 hours after spraying, we took detached leaves from the 4th leaf position from the top to the bottom of the resistant and susceptible varieties. The results showed that spraying ABA significantly increased the area of lesions in both resistant and susceptible varieties of hydrangea, confirming that ABA has a significant negative effect on hydrangea leaf spot resistance ( Figure 3 ), which clearly shows that ABA plays a key regulatory role in the disease resistance response of hydrangea to C. cassiicola infection.
[0042] RNA-seq analysis of leaf tissues of the disease-resistant variety 'White Angel' and the susceptible variety 'Ocean Heart' at 0h and 24h after C. cassiicola infection revealed that HmCYP707A4, a key enzyme gene in the ABA decomposition pathway, was a differentially expressed gene, and its expression level in the disease-resistant variety 'White Angel' was 15 times higher than that in the susceptible variety 'Ocean Heart' ( Figure 4 ), indicating that HmCYP707A4 (SEQ ID NO: 16) is a key disease resistance candidate gene.
[0043] SEQ ID NO:16
[0044]
[0045] In this example, a homologous gene sequence of HmCYP707A4 of Hydrangea chinensis was found in Nicotiana benthamiana and named NbCYP707A4. The nucleotide sequence of the NbCYP707A4 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the NbCYP707A4 gene is shown in SEQ ID NO: 2.
[0046] SEQ ID NO: 1
[0047]
[0048] SEQ ID NO:2
[0049] MLAFCYISSVAKRKMTNFDLIFYIFIFFLTIFLYFCFVTTRNKKLSFQRKAYKLPPGSMGWPYIGETLQLYSQDPNIFFINRQRRYGEIFKTKILGCPCVMLASPEAARFVLVNQANLFKPTY PKSKETLIGQSALFFHQGNYHIHIRKLVQTSLNPEAICNKIPHIEELAISALNSWAGGHVVDTYIEMKKYSFEVGILAIFGHLDAHLKDQLKKNYSIVDKGYNSFPTNLPGSPYRKAIQARKR LGKILGEVISGRKEKKLIEKGLLSCFLNAKLDEKGLVLNEDQIADNIIGVLFAAQDTTASVLTWILKYLHDNAKLLQSVKAEQKAVCQSNEQENHGLTWTQTRKMPMTNKVVLETRLLASIIS FTFREAVADVEYKGYLIPKGWKVMPLFRNIHHNPDFFPGPQKFDPSRFENVQKPNTFMPFGIGIHACPGNELAKLEILIMMHHVVTKFRWEVEGSNSGIEYGPFPVPVGGLPARFWKESTTST*
[0050] Gene cloning primers F: ATGTTAGCATTCTGCTATATAAGCT (SEQ ID NO: 3) and R: TTAGGTTGAAGTGGTAGATTCTTTC (SEQ ID NO: 4) were designed, and NbCYP707A4 was cloned using a seamless cloning kit using Nicotiana benthamiana RNA as a template.
[0051] NbCYP707A4 was amplified by PCR using a high-fidelity DNA polymerase. A BamHI restriction enzyme site (SEQ ID NO:6) was introduced at the 5' end of the forward primer (SEQ ID NO:5), and a SalI restriction enzyme site (SEQ ID NO:8) was introduced at the 5' end of the reverse primer (SEQ ID NO:7). The amplified product was purified to obtain the target gene fragment NbCYP707A4 containing restriction enzyme sites at both ends.
[0052] The pORE-R4 empty vector was used as the backbone and double-digested with BamHI and SalI. The reaction system was as follows: 2 μg of vector DNA, 5 μL of 10× buffer, 1 U of BamHI / μg DNA, and 1 U of SalI / μg DNA. ddH2O was added to 50 μL of the system. The reaction was incubated at 37°C for 3 hours, and the linearized vector was purified using a gel recovery kit.
[0053] Among them, the forward primer is: ATGTTAGCATTCTGCTATATAAGCT (SEQ ID NO: 5);
[0054] BamHI restriction enzyme site: GGTTGAAGTGGTAGATTCTTTC (SEQ ID NO: 6);
[0055] Reverse primer: GGTTGAAGTGGTAGATTCTTTC (SEQ ID NO: 7);
[0056] SalI restriction enzyme site: 5'-AGTATCGATGCGGCCGCAAAGTCGACG-3' (SEQ ID NO: 8).
[0057] The target gene fragment and the linearized vector were connected using a seamless cloning kit (purchased from Shanghai Yisheng Biological Co., Ltd.) and incubated at 50°C for 30 minutes to complete the connection, obtaining the overexpression vector pORE-R4-NbCYP707A4, and transformed into Agrobacterium GV3101 competent cells (purchased from Shanghai Yisheng Biological Co., Ltd.) by the freeze-thaw method; transient transformation was performed on the lower epidermal cells of Nicotiana benthamiana (Flower Innovation Team, Institute of Leisure Agriculture, Jiangsu Academy of Agricultural Sciences). Young leaves with strong growth, smooth surface, and plump flesh at the 4-6 leaf stage were selected, and the transient expression infection solution was injected into the tobacco from the back of the leaf using a 1 mL syringe. The same volume of empty vector was injected as a control, and the leaves were cultured in the dark at 24°C for 1 day and in the light for 2 days. The leaf spot pathogen C. cassiicola was inoculated at the injection site for 48 hours, and the disease progression of the strains carrying the empty vector pORE-R4 (donated by the Chrysanthemum Laboratory of Nanjing Agricultural University) and the overexpression vector pORE-R4-NbCYP707A4 was observed. The results are as follows Figure 5 shown.
[0058] As can be seen from the figure, the symptoms of the overexpression strain were significantly alleviated compared with the control, which confirmed that NbCYP707A4 has the function of resisting Corynespora leaf spot.
[0059] An online tool (https: / / chopchop.cbu.uib.no) was used for target prediction and selection, off-target risk prediction, and target specificity analysis. Two target sites were identified and target primers were designed:
[0060] Target 1: AGGTTGTCCTTGTGTCATGCTGG (SEQ ID NO: 9);
[0061] Primer: AtU3dT1F:gtcaGGTTGTCCTTGTGTCATGCTGG (SEQ ID NO: 10);
[0062] AtU3dT1R:aaacCCAGCATGACACAAGGACA (SEQ ID NO: 11);
[0063] Target 2: CAATTTCTGCATTGAATTCATGG (SEQ ID NO: 12);
[0064] AtU3bT2F:gtcaCAATTTCTGCATTGAATTCATGG (SEQ ID NO: 13);
[0065] AtU3bT2R:aaacCCATGAATTCAATGCAGAAATTG (SEQ ID NO: 14).
[0066] The target fragment pYLgRNAU3d-U3b (SEQ ID NO: 15) was synthesized by General Biotechnology Co., Ltd., and then the target fragment was constructed into a CRISPR / gRNA vector. The CRISPR / gRNA vector was transformed into Agrobacterium strain EHA105 by electroporation. Positive clones were selected and expanded, and the leaf discs of sterile tobacco seedlings of about 40 days old were infected for 8-10 minutes. The cells were co-cultured in the dark for 3 days, transferred to decarboxylation medium for 7 days, and then transferred to selection medium for subculture every two weeks. When the differentiated resistant buds grew to 1-2 cm, the resistant buds were transferred to rooting screening medium for screening. Preliminary resistant strains were obtained, i.e., non-transgenic tobacco tissue culture seedlings with NbCYP707A4 knockout that were highly susceptible to corynespora leaf spot disease, which were hardened and transplanted.
[0067] pYLgRNAU3d-U3b (SEQ ID NO: 15):
[0068] AGGTTGTCCTTGTGTCATGCTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGG ACCCTGACACTGGAATCGGCAGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACG AAACAAGTGAACTTTTAGGATCAACTTCAGTTTATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCACAATTTCTGCATTGAATTCATGG.
[0069] After hardening and transplanting, take the leaf discs of mature leaves and place them on a 1 / 1000 agar plate and inoculate the spore suspension of leaf spot pathogen C. cassiicola (1*10 6 8 μL of the NbCYP707A4 knockout non-transgenic tobacco with high susceptibility to Corynespora leaf spot was placed on the leaf disc 24 hours after inoculation. Figure 6 shown.
[0070] As can be seen from the figure, the leaf disc symptoms of non-transgenic tobacco with NbCYP707A4 knockout and highly susceptible to Corynespora leaf spot disease are significantly more severe than those of the control, indicating that the tobacco created by gene editing is significantly more susceptible to the disease and develops the disease faster, and can be used as an indicator plant for hydrangea leaf spot disease.
[0071] Example: 3:
[0072] During the rainy season, the non-transgenic tobacco plants with NbCYP707A4 knockout obtained in Example 2 were hardened and transplanted for about 30 days before being planted. Then, 30 potted tobacco plants with the same growth potential and seedling age were placed in a hydrangea greenhouse. Ten plants were placed at each end and in the middle of each seedling bed, close to the hydrangea varieties susceptible to the corynespora leaf spot.
[0073] In a greenhouse, two seedling beds with the same number of varieties were randomly selected. One bed, free of highly susceptible tobacco, was sprayed with the fungicide pyraclostrobin diluted 1000 times in water after hydrangea leaf spot developed. The other bed, free of hydrangea leaf spot, was sprayed with the fungicide pyraclostrobin diluted 1000 times in water within 1-3 days of the appearance of lesions on the leaves of highly susceptible tobacco. The results showed that the hydrangeas in the bed without highly susceptible tobacco as an indicator plant still had an 85.2% incidence of leaf spot, even after being sprayed with pyraclostrobin, while the hydrangeas in the bed with highly susceptible tobacco as an indicator plant had an incidence of only 12.4%. This indicates that using indicator plants and timely application of fungicides can significantly reduce the incidence of leaf spot in greenhouse hydrangeas.
[0074] In summary, the present invention found that the NbCYP707A4 gene is a key gene for tobacco leaf spot resistance. After knocking out the gene through CRISPR / Cas9 gene knockout technology, tobacco highly susceptible to Corynespora leaf spot can be prepared; the highly susceptible tobacco to Corynespora leaf spot can be used as an indicator plant for leaf spot disease, especially hydrangea leaf spot disease, and can timely predict and prevent the occurrence of hydrangea leaf spot disease, reduce economic losses, achieve a reduction in hydrangea production costs, and alleviate environmental pollution caused by excessive agricultural application. It is of great significance to hydrangea and other horticultural crops and has good practicality.
[0075] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.
Claims
1. NbCYP707A4 gene, characterized in that The nucleotide sequence of the NbCYP707A4 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:
2.
2. Use of the NbCYP707A4 gene according to claim 1 in resisting plant leaf spot disease.
3. The use according to claim 2, characterized in that Overexpression of the NbCYP707A4 gene can improve the plant's resistance to leaf spot disease, while silencing / inhibiting the expression of the NbCYP707A4 gene reduces the plant's resistance to leaf spot disease.
4. The use according to claim 2 or 3, characterized in that The plant includes Nicotiana benthamiana; and the leaf spot disease includes Corynespora leaf spot disease caused by Corynespora multifungi.
5. A method for obtaining tobacco highly susceptible to leaf spot disease, characterized in that: The method comprises: The NbCYP707A4 gene in tobacco was knocked out using CRISPR / Cas9 gene knockout technology to obtain tobacco highly susceptible to leaf spot disease.
6. Use of the highly susceptible tobacco to leaf spot disease obtained by the method according to claim 5 as an indicator plant for leaf spot disease.
7. The use according to claim 6, characterized in that The application includes: predicting and / or preventing and controlling the occurrence of hydrangea leaf spot disease.
8. A method for predicting and / or preventing the occurrence of hydrangea leaf spot disease, characterized in that: The method comprises: The highly susceptible tobacco to leaf spot disease obtained by the method of claim 5 is placed in a hydrangea greenhouse near a variety susceptible to leaf spot disease, and the incidence of leaf spot disease in the highly susceptible tobacco is observed. When spots appear on the leaves of the highly susceptible tobacco to leaf spot disease and the hydrangea does not develop leaf spot disease, the hydrangea is sprayed with a fungicide to prevent the occurrence of leaf spot disease.
9. The method according to claim 8, characterized in that The leaf spot disease includes Corynespora leaf spot disease caused by Corynespora multifungi.
10. The method according to claim 8, characterized in that The suitable seedling age of the indicator plant is 30-60 days after transplanting and planting; The indicator plant is used in the rainy season when hydrangea leaf spot disease is prone to occur.