Application of nta11g04230 gene as tobacco immune negative regulator in improving tobacco disease resistance
By regulating the expression of the Nta11g04230 gene in tobacco through genetic engineering and modulating the SA signaling pathway, the problem of lack of disease resistance genes in tobacco black shank disease was solved, achieving high-efficiency resistance of tobacco to Phytophthora infestans and providing a new prevention and control approach.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HENAN TOBACCO CO NANYANG CO
- Filing Date
- 2025-12-17
- Publication Date
- 2026-06-30
AI Technical Summary
Existing tobacco varieties lack effective resistance gene resources against tobacco black shank disease. Traditional fungicide control methods have problems with drug resistance and environmental pollution, making it difficult to effectively control black shank disease caused by Phytophthora indica.
By using genetic engineering techniques, the Nta11g04230 gene in tobacco was silenced or overexpressed to regulate the SA signaling pathway and improve the resistance of tobacco to Phytophthora indica. Plants with silenced or overexpressed Nta11g04230 gene were constructed using Agrobacterium-mediated transformation.
It significantly enhances tobacco's resistance to Phytophthora nicotineis, provides new genetic resources and control pathways, and strengthens tobacco's resistance to black shank disease.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering, specifically involving the application of the Nta11g04230 gene and its encoded protein in improving tobacco resistance to black shank disease. Background Technology
[0002] The tobacco industry is an important component of my country's large-scale agriculture and a significant economic crop. Tobacco black shank is one of the major epidemic pests affecting tobacco production in my country, covering almost all producing areas. The pathogen is *Phytophthora nicotianae*, a semi-living, heterothallic oomycete. During its asexual reproduction stage, it produces numerous sporangia on the surface of infected tissues via sporangiophores. These sporangia germinate at 20-25°C and release zoospores at 10-15°C. The zoospores can directly penetrate leaf or stem tissues, causing lesions after 3-4 days and spreading by wind to neighboring fields, leading to large-scale infection. Under suitable conditions, *Phytophthora nicotianae* can rapidly complete its life cycle, forming a large number of zoospores that cause systemic damage to the roots, stems, and leaves of tobacco plants.
[0003] Phytophthora tobaccoii exhibits high genetic variability, with dynamic regions of its genome containing numerous effector proteins that may tolerate greater non-allelic recombination and other forms of structural variation. These characteristics promote increased virulence and other changes favorable to the pathogen, enabling Phytophthora tobaccoii to rapidly adapt to the selective pressures of modern agriculture, thus making tobacco black shank difficult to control. Currently, major tobacco cultivars are generally susceptible to tobacco black shank, and there is a lack of effective and usable resistance gene resources.
[0004] Currently, the main control strategy for tobacco black shank is the use of fungicides. However, oomycete diseases differ significantly from fungal diseases in their physiological and biochemical characteristics, limiting the number of fungicides available for controlling tobacco black shank. Excessive or prolonged use of single chemical agents can easily lead to drug resistance, pesticide residues, and environmental pollution. Therefore, identifying and utilizing genes related to resistance to tobacco black shank from the tobacco genome is of great significance for achieving green control of tobacco black shank.
[0005] Plants contain several genes that negatively regulate immunity (also known as susceptibility genes). These genes are frequently utilized by pathogens and contribute to their infection and colonization. During the infection of host plants by *Phytophthora infestans*, many of its effector proteins target and utilize susceptibility factors within the plant, aiding in the successful colonization of the fungus. Meanwhile, research has shown that plants lacking susceptibility genes are more likely to acquire durable resistance. For example, a natural mutant of the barley gene Mlo (encoding a calmodulin-binding protein) exhibits durable resistance to powdery mildew. Barley varieties carrying this gene mutation have been successfully used in Europe for over 36 years and still demonstrate good field resistance. Summary of the Invention
[0006] To address the lack of effective genes in molecular resistance breeding for tobacco black shank, this invention initially designed and constructed a plant cDNA expression library. Through transient expression in *Nicotiana benthamiana* followed by inoculation with *Phytophthora indicum*, a previously unstudied and unreported tobacco gene, Nta11g04230, was screened. The Nta11g04230 gene was obtained using genetic engineering techniques. Further functional studies revealed that the Nta11g04230 gene participates in the immune response against *Phytophthora indicum*, the pathogen of tobacco black shank. Overexpression of the Nta11g04230 gene was constructed using genetic methods and genetic engineering techniques. These plants showed reduced resistance to *Phytophthora indicum* and weakened SA-mediated resistance signals. Furthermore, Nta11g04230 gene silencing was constructed using Agrobacterium-mediated transformation. Silent Nta11g04230 gene plants showed enhanced resistance to *Phytophthora indicum* and strengthened SA-mediated resistance signals. This invention provides a new gene resource for improving tobacco resistance to black shank.
[0007] On the one hand, the present invention provides the application of the Nta11g04230 gene in improving tobacco resistance to black shank, silencing the Nta11g04230 gene to improve tobacco resistance to black shank, and the CDS sequence of the Nta11g04230 gene is shown in SEQ ID NO:1.
[0008] Furthermore, in the aforementioned application, the Nta11g04230 gene can enhance tobacco black shank resistance by regulating the SA signaling pathway.
[0009] Furthermore, in the aforementioned application, the regulation of the SA signaling pathway involves upregulating the amount of SA synthesized in tobacco and the expression of the SA signaling pathway marker gene NtPR1.
[0010] Furthermore, in the aforementioned application, the tobacco black shank disease is caused by infection with Phytophthora indica.
[0011] In another aspect, the present invention also provides the application of protein Nta11g04230 in improving tobacco resistance to black shank, reducing the expression of protein Nta11g04230 or weakening the activity of protein Nta11g04230 to improve tobacco resistance to black shank, wherein protein Nta11g04230 is encoded by the Nta11g04230 gene, and the amino acid sequence of protein Nta11g04230 is shown in SEQ ID NO:2.
[0012] In another aspect, the present invention provides a method for improving tobacco resistance to black shank disease by silencing the Nta11g04230 gene or silencing a specific silenced fragment of the Nta11g04230 gene in tobacco, wherein the CDS sequence of the Nta11g04230 gene is shown in SEQ ID NO:1; and the nucleotide sequence of the specific silenced fragment of the Nta11g04230 gene is shown in SEQ ID NO:3.
[0013] Finally, the present invention also provides a method for breeding tobacco varieties resistant to black shank, comprising silencing the Nta11g04230 gene in the plant or expressing a specific silenced fragment of the Nta11g04230 gene.
[0014] Furthermore, in the method, the black shank disease is caused by infection with Phytophthora indica.
[0015] Furthermore, in the method, a gene silencing vector is constructed and transformed into a plant using Agrobacterium-mediated transformation to obtain Nta11g04230 gene-silenced plants.
[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:
[0017] This invention reveals for the first time that the Nta11g04230 gene acts as a negative regulator in the interaction between tobacco and Phytophthora indica. Overexpression of the Nta11g04230 gene in tobacco reduces tobacco resistance to Phytophthora indica, while silencing the Nta11g04230 gene in tobacco significantly enhances tobacco resistance to black shank. This invention provides a new gene resource for the prevention and control of tobacco black shank.
[0018] This invention explores the negative regulatory mechanism of the Nta11g04230 gene in tobacco and Phytophthora infestans immunity. It finds that the Nta11g04230 gene regulates tobacco resistance to Phytophthora infestans by inhibiting the SA signaling pathway. In tobacco plants with the Nta11g04230 gene silenced, the SA signaling pathway-related gene NtPR1 is upregulated, thus negatively regulating the immune response.
[0019] In summary, this invention provides a new technical approach for the prevention and treatment of tobacco-induced black shank disease and for the improvement of tobacco-induced black shank disease resistance. Attached Figure Description
[0020] Figure 1 This is a diagram illustrating the expression pattern of the Nta11g04230 gene when infected by Phytophthora indicum.
[0021] Figure 2 The results show the expression of the Nta11g04230 gene in plants overexpressing the Nta11g04230 gene.
[0022] Figure 3 The results show the expression of the Nta11g04230 gene in plants with the Nta11g04230 gene silenced.
[0023] Figure 4 This is a diagram showing the growth status of plants overexpressing the Nta11g04230 gene.
[0024] Figure 5 This is a diagram showing the growth status of plants with the Nta11g04230 gene silenced.
[0025] Figure 6 Figure 1 shows the resistance test results of plants overexpressing the Nta11g04230 gene to Phytophthora nicotinae. Figure 2a shows the phenotypic pattern of tobacco leaves after Phytophthora nicotinae infection; Figure 3b shows the statistical results of the diameter of lesions on tobacco leaves infected with Phytophthora nicotinae. "***" indicates P<0.001.
[0026] Figure 7 Figure 1 shows the resistance test results of Nta11g04230 gene-silenced plants to Phytophthora nicotineis. Figure 2a shows the leaf phenotype after Phytophthora nicotineis infection; Figure 3b shows the statistical results of the diameter of Phytophthora nicotineis-infected leaf lesions. "***" indicates P<0.001.
[0027] Figure 8 Figure 1 shows the SA content and NtPR1 expression results in Nta11g04230 gene-silenced and Nta11g04230 gene-overexpressing plants. Detailed Implementation
[0028] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.
[0029] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.
[0030] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0031] Example 1
[0032] This example demonstrates the acquisition of the CDS fragment of the Nta11g04230 gene.
[0033] The nucleotide sequence of the CDS of the tobacco Nta11g04230 gene in tobacco genome sequencing was obtained through the Nicomics website (http: / / lifenglab.hzau.edu.cn / Nicomics / ). The nucleotide sequence of the CDS of the tobacco Nta11g04230 gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein Nta11g04230 is shown in SEQ ID NO:2. Using cDNA from tobacco Yunyan 87 as a template for PCR amplification, the CDS sequence of the Nta11g04230 gene was amplified using the Phanta Flash Super-Fidelity DNA Polymerase rapid high-fidelity DNA polymerase reaction system. The Nta11g04230 gene fragment was ligated into the pEASY-Blunt Zero vector using the pEASY-Blunt Zero Cloning Kit (Catalog No. CB501) and sequenced. The primers used to amplify the CDS sequence of the Nta11g04230 gene were Nta11g04230-F and Nta11g04230-R, and the sequences of the primers Nta11g04230-F and Nta11g04230-R are shown in Table 1. The pEASY-Nta11g04230 recombinant vector was sent to a biotechnology company for sequencing, and the sequencing results of the CDS fragment of the Nta11g04230 gene are shown in SEQ ID NO:1.
[0034] SEQ ID NO:1
[0035] ATGGAGAAGATCGTCTTTTCATTCTCAGCTCAGGCCAAGAAGATAGCTGATGGACTCGATCATTATAATTCCACCATCTTAAATTGTATGATTCTCGTCTTAGTAATCACATTTCAGGAATTTCGTTCTTGCTTTTCTTCTTTAATTCGAGTCACTCTTTCTTTCGTTTTTACTCCATGTAAGAAGTCTGATAATGCTGAAACAACTTCTTCAACGGCGAAAAATAACTGTCGATCTGAAATTGTTGCTACTAATATTAAGGCACTATTGGATGAAGATGATGTTGAGATTGTACTA GATACACTATTGACTTTTTGTAACTCAAACGGGGATGATTTTGATAAGGTAGGATTGGCTGAGGTTTTTTGTTCGTTCGACGAAACAGAGCCTAGTTTAGAAGAAGTTAAAGAAGCTTTTAATATGTTTGATGAGAATGGAGATGGCTATATTGATGCAAATGAGCTAAAGAAAGTCATTTACAAGATGGGTTTCTTGGAATTTTCATTGGAAGATTGCCAGAGGATGATTGTGCCATTTGATGATAACACAGATGGAAAAATTGAATTTGCTGAATTTCTGAAGCTCCTGGAGTAA
[0036] SEQ ID NO:2
[0037] MEKIVFSFSAQAKKIADGLDHYNSTILNCMILVLVITFQEFRSCFSSLIRVTLSFVFTPCKKSDNAETTSSTAKNNCRSEIVATNIKALLDEDDVEIVLDTLLTFCNSNGDDFDKVGLAEVFCSFDETEPSLEEVKEAFNMFDENGDGYIDANELKKVIYKMGFLEFSLEDCQRMIVPFDDNTDGKIEFAEFLKLLE
[0038] SEQ ID NO:3
[0039] TTTCAGGAATTTCGTTCTTGCTTTTCTTCTTTAATTCGAGTCACTCTTTTCTTTCGTTTTTACTCCATGTAAGAAGTCTGATAATGCTGAAACAACTTCTTCAACGGCGAAAAATAACTGTCGATCTGAAATTGTTGCTACTAATATT AAGGCACTATTGGATGAAGATGATGTTGAGATTGTACTAGATACACTATTGACTTTTTGTAACTCAAACGGGGATGATTTTGATAAGGTAGGATTGGCTGAGGTTTTTTGTTCGTTCGACGAAACAGAGCCTAGTTTAGAAGAAGTTA
[0040] Table 1 Primer sequence listing
[0041]
[0042] Example 2
[0043] This embodiment is to test whether the Nta11g04230 gene is induced by Phytophthora infestans infection.
[0044] Fresh mycelial blocks of *Phytophthora tobaccoeris* were placed on V8 solid medium and incubated at 22°C for 10–14 days. The growth status and sporangia of *Phytophthora tobaccoeris* were then examined under a microscope. On plates with well-grown *Phytophthora tobaccoeris*, pre-cooled dH₂O at 4°C was added to submerge the mycelium. The plates were then incubated at 4°C for 1–2 hours, and the release and number of zoospores were observed under a microscope. The zoospore suspension was diluted to the usable concentration and placed on ice for inoculation of tobacco leaves.
[0045] Leaves of the wild-type tobacco variety Yunyan 87 were inoculated with *Phytophthora nicotineae*. Healthy leaves from the middle section of Yunyan 87 plants that had grown in the substrate for five weeks were cut, and the petioles were wrapped with moistened absorbent cotton. The detached leaves were placed in a plastic dish with the underside facing upwards. Approximately 1000 zoospores were inoculated into the veins on the underside of the tobacco leaves. Leaf samples of Yunyan 87 plants inoculated with *Phytophthora nicotineae* zoospores were selected at 0, 6, 12, 26, and 48 hours. RNA was extracted, and the expression level of the Nta11g04230 gene was detected using real-time quantitative PCR. The results are as follows: Figure 1 As shown, the results indicate that after inoculation with Phytophthora indica, the expression level of the Nta11g04230 gene was slightly upregulated at 6 hours, downregulated at 48 hours, and relatively stable at other time points, indicating that the expression level of this gene was affected to some extent by Phytophthora indica infection.
[0046] Example 3
[0047] This example demonstrates the construction of a plant overexpressing the Nta11g04230 gene.
[0048] Using cDNA from tobacco Yunyan 87 as a template, the CDS sequence of Nta11g04230 was amplified using Phanta Flash Super-Fidelity DNA Polymerase (brand: Novizan, catalog number: P521). Nta11g04230 was cloned into a linearized backbone Super1300 digested with restriction endonucleases Sma1 / Sac1 to obtain a recombinant plasmid for constructing Nta11g04230 overexpression transformation materials and for inoculation testing.
[0049] Following the Agrobacterium electroporation competent cell transformation method, the recombinant plasmid Super1300-Nta11g04230 was transformed into GV3101 competent cells. The validated GV3101 strain was inoculated into liquid LB medium containing Kan and Rif, and cultured in shake flasks until orange-yellow. The bacterial suspension was collected (2500 rpm, 10 min), gently resuspended in LB liquid medium, and the resuspended medium concentration was adjusted to OD. 600 =0.4 was used for tobacco leaf disc transformation. Leaf disc preparation: Several leaves, each 5 mm in length, were cut from one-month-old tobacco tissue culture seedlings and placed on R3B solid plates. Two sheets of filter paper were placed on top of the stem segments, and 2 mL of PACM (Plant Artificial Conditioning Medium) was added to moisten the filter paper. The plates were then placed in a 23°C light incubator. After 24 hours of PACM treatment, the leaves were transferred to the infection solution and soaked for 5 minutes. The infected stem segments were then transferred to sterile filter paper to dry and placed on R3B solid plates for 48 hours. The stem segments were then transferred to MS solid medium plates containing zeatin, cephalosporins, vancomycin, and kanamycin and incubated at 23°C light. Every 20 days, the plates were transferred to fresh MS solid medium plates to induce callus and bud differentiation until buds were induced. The transformed plants were then transferred to MS medium flasks containing kanamycin resistance for growth. After one month, the transformed plants were identified. DNA was extracted from the leaves of the tobacco transformed plants grown on antibiotic-containing MS medium for one month. PCR amplification was performed using vector primers, and vector insertion was detected by electrophoresis. RNA was extracted from leaves of stable transformed tobacco materials and wild-type tobacco Yunyan 87 grown on MS medium. Using NtNADH as an internal reference gene, the expression level of the Nta11g04230 gene in tobacco transformants and wild-type Yunyan 87 was quantitatively detected. The results are as follows: Figure 2 As shown, two tobacco plants overexpressing the Nta11g04230 gene were obtained and labeled as OE-Nta11g04230-1 and OE-Nta11g04230-3.
[0050] The above-mentioned tobacco plants that overexpressed the Nta11g04230 gene were cultured in pots with wild-type tobacco (Yunyan 87) for five weeks. The plant phenotypes were then observed, and the results are as follows: Figure 4 As shown, the Nta11g04230 gene overexpressing plants OE-Nta11g04230-2 and OE-Nta11g04230-3 showed no significant differences in growth phenotype compared to wild-type tobacco (Yunyan 87).
[0051] Example 4
[0052] This example demonstrates the construction of a Nta11g04230 gene-silenced plant.
[0053] A specific region in the Nta11g04230 gene sequence was selected as the RNAi silencing fragment of the Nta11g04230 gene. The nucleotide sequence of the RNAi silencing fragment of the Nta11g04230 gene is shown in SEQ ID NO:3. Using cDNA of wild-type tobacco Yunyan 87 as a PCR amplification template, the RNAi silencing fragment of the Nta11g04230 gene was amplified using Phanta Flash Super-Fidelity DNA Polymerase (brand: Novizan, catalog number: P521) for rapid high-fidelity DNA polymerase. The RNAi silencing fragment of the Nta11g04230 gene was cloned into the linearized pKANNIBAL vector, which was double-digested with restriction endonucleases EcoRI / KpnI, in a forward-directed manner to obtain the pKANNIBAL-Nta11g04230-RNAi intermediate silencing vector. The RNAi silencing fragment of the Nta11g04230 gene was cloned into the pKANNIBAL-Nta11g04230-RNAi intermediate silencing vector, which was double-digested with restriction endonucleases ClaI / XbaI, in a reverse-directed manner to obtain the pKANNIBAL-Nta11g04230-RNAi hairpin structure silencing vector. The pKANNIBAL-Nta11g04230-RNAi silencing vector and the pART27 vector were digested with NotI, respectively. The linearized pART27 vector was dephosphorylated and then processed using T4... DNA ligase ligates the sequence containing the hairpin structure into the pART27 single-enzyme-digested linearized vector, thus obtaining the pART27-Nta11g04230-RNAi plant silencing vector.
[0054] Following the Agrobacterium electroporation competent cell transformation method, the pART27-Nta11g04230-RNAi plant silencing vector was transformed into GV3101 competent cells. The validated GV3101 strain was inoculated into liquid LB medium containing Kan and Rif, and cultured in shake flasks until orange-yellow. The bacterial suspension was collected (2500 rpm, 10 min), gently resuspended in LB liquid medium, and the resuspended medium concentration was adjusted to OD. 600 =0.4 was used for tobacco leaf disc transformation. Tobacco leaf disc preparation: Several 5mm long stem segments were cut from one-month-old tobacco tissue culture seedlings and placed on R3B solid plates. Two sheets of filter paper were placed on top of the stem segments, and 2 mL of PACM liquid medium was added to moisten the filter paper. The plates were then placed in a 23°C light incubator. After 24 hours of PACM treatment, the leaves were transferred to the infection solution and soaked for 5 min. The infected stem segments were then transferred to sterile filter paper to dry and placed on R3B solid plates for 48 hours. The stem segments were then transferred to MS solid medium (ZCVK) plates containing zeatin, cephalosporin, vancomycin, and kanamycin and incubated at 23°C light incubator. Every 20 days, the plates were transferred to fresh ZCVK solid medium plates to induce callus and bud differentiation until buds were induced. The plates were then transferred to MS medium containing kanamycin for growth. Transformants were identified after one month. DNA was extracted from the leaves of tobacco transformants grown on antibiotic-containing MS medium for one month. PCR amplification was performed using vector primers, and the vector insertion was detected by electrophoresis. RNA was extracted from leaves of stable tobacco transformants and wild-type tobacco Yunyan 87 grown on MS medium. Using NtNADH as an internal reference gene, the expression level of the Nta11g04230 gene in both transformants and wild-type Yunyan 87 was quantitatively detected. The results are shown below. Figure 3 As shown, two positive plants with the Nta11g04230 gene silence were obtained, labeled as RiNta11g04230-1 and RiNta11g04230-2.
[0055] The above-mentioned positively transformed plants were cultured in pots with wild-type tobacco (Yunyan 87) for five weeks. The plant phenotypes were then observed, and the results are as follows: Figure 4 As shown, the Nta11g04230 gene-silenced plants RiNta11g04230-1 and RiNta11g04230-2 showed no significant differences in growth phenotype compared to wild-type tobacco (Yunyan 87).
[0056] Example 5
[0057] This example tests the resistance of plants overexpressing the Nta11g04230 gene to Phytophthora indicum.
[0058] Five-week-old tobacco plants OE-Nta11g04230-2 and OE-Nta11g04230-3, which overexpress the Nta11g04230 gene, were used as the test group, while wild-type tobacco Yunyan 87 plants were used as the control group. The leaves of the test and control plants were inoculated with Phytophthora nicotineae for testing.
[0059] First, place fresh mycelial blocks of *Phytophthora tobaccoeris* on V8 solid medium and incubate at 22°C for 10-14 days. Examine the growth status and sporangia of *Phytophthora tobaccoeris* under a microscope. Select plates with well-grown *Phytophthora tobaccoeris* and add pre-cooled dH2O (4°C) to submerge the mycelium. Incubate at 4°C for 1-2 hours, then place on ice for inoculation of tobacco leaves. Cut healthy leaves from the middle section of tobacco plants that have grown in the substrate for five weeks. Wrap the petioles with moistened absorbent cotton and place the detached leaves face down in a plastic dish. Inoculate approximately 1000 zoospores in the center of the veins on the underside of the tobacco leaf. Incubate at 22°C for 3-5 days, observe and count the disease incidence on the leaves. The diameter of *Phytophthora tobaccoeris* lesions is shown in the figure below. Figure 6 As shown in b. Compared with wild-type tobacco (Yunyan 87), the tobacco phytosis lesions on the leaves of plants OE-Nta11g04230-2 and OE-Nta11g04230-3, which overexpress the Nta11g04230 gene, are larger in diameter, indicating greater susceptibility to tobacco phytosis infection.
[0060] Example 6
[0061] This example tests the resistance of Nta11g04230 gene-silenced plants to Phytophthora tobaccoii.
[0062] Five-week-old Nta11g04230 gene-silenced plants RiNta11g04230-4 and RiNta11g04230-6 were used as the test group, and wild-type tobacco Yunyan 87 plants (ATL) were used as the control group. The undersides of the leaves of the test group and the control group were inoculated with Phytophthora nicotineae for testing.
[0063] Five-week-old Nta11g04230 gene-silenced plants, RiNta11g04230-4 and RiNta11g04230-6, and wild-type tobacco (Yunyan 87) detached leaves were inoculated with zoospores of Phytophthora nicotinae. Disease incidence was observed and statistically analyzed. The inoculation method and staining and statistical methods for Phytophthora nicotinae lesions were the same as in Example 4. The statistical results of the diameter of Phytophthora nicotinae lesions are as follows: Figure 6 As shown in b. Compared with the wild-type Yunyan 87, the Nta11g04230 gene-silenced plants RiNta11g04230-4 and RiNta11g04230-6 showed smaller diameter of Phytophthora lesions on their leaves, indicating greater resistance to Phytophthora infection.
[0064] Example 7
[0065] This embodiment explores the mechanism by which the Nta11g04230 gene negatively regulates tobacco resistance to Phytophthora infestans.
[0066] SA content and the expression of the SA signaling pathway marker gene NtPR1 were measured in Nta11g04230 gene overexpression plants and Nta11g04230 gene silenced plants. Leaves were collected from Nta11g04230 gene silenced plants RiNta11g04230-1 and RiNta11g04230-2 and Nta11g04230 gene overexpression plants OE-Nta11g04230-1 and OE-Nta11g04230-3, as well as leaves from wild-type tobacco (control, Yunyan 87), and divided into two portions. One portion was used for SA content determination: 0.5g of fresh leaves were homogenized in liquid nitrogen, transferred to a 1.5mL centrifuge tube with 0.5mL of 90% methanol, centrifuged at 10000g for 15min, and the supernatant was collected and dried on a nitrogen blower. Dissolve in 0.25 mL of 5% trichloroacetic acid, shake, and then add 0.8 mL of a mixture of ethyl acetate and cyclohexane (V / V = 1:1). Extract twice, transfer the upper organic phase to a new centrifuge tube, dry under nitrogen, and dissolve in 0.6 mL of liquid mobile phase. Filter through a 0.45 μm filter and determine the SA content by high-performance liquid chromatography. One sample was used for NtPR1 gene expression determination: RNA was extracted, and the expression level of the SA signaling pathway marker gene NtPR1 was detected using real-time quantitative PCR. The results are as follows: Figure 8 As shown, the SA content and the expression level of the SA signaling pathway marker gene NtPR1 were higher in plants with silenced Nta11g04230 gene and lower in plants with overexpression of Nta11g04230 gene, indicating that Nta11g04230 gene negatively regulates the SA-dependent immune response in plants by inhibiting the SA signaling pathway.
[0067] As described above, the basic principles, molecular mechanisms, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.
Claims
1. The application of the Nta11g04230 gene as a negative regulator of tobacco immunity in improving tobacco disease resistance, characterized by, Silencing the Nta11g04230 gene can enhance tobacco resistance to black shank disease. The CDS sequence of the Nta11g04230 gene is shown in SEQ ID NO:
1.
2. The application according to claim 1, characterized in that, The Nta11g04230 gene can enhance tobacco's resistance to black shank by regulating the SA signaling pathway.
3. The application according to claim 2, characterized in that, The regulation of the SA signaling pathway involves upregulating the expression of the SA signaling pathway marker gene NtPR1.
4. The application according to claim 1, characterized in that, The tobacco black shank disease is caused by Phytophthora infestans.
5. The application of protein Nta11g04230 in improving tobacco resistance to black shank, characterized in that, Reducing the expression or weakening the activity of protein Nta11g04230 can improve resistance to tobacco black shank disease. The protein Nta11g04230 is encoded by the Nta11g04230 gene, and the amino acid sequence of the protein Nta11g04230 is shown in SEQ ID NO:
2.
6. A method for improving tobacco's resistance to black shank, characterized in that, A specific silencing fragment of the Nta11g04230 gene is expressed in tobacco, the CDS sequence of which is shown in SEQ ID NO:1; the nucleotide sequence of the specific silencing fragment of the Nta11g04230 gene is shown in SEQ ID NO:
3.
7. A method for breeding tobacco varieties resistant to black shank disease, characterized in that, This includes specific silencing fragments of the Nta11g04230 gene that are inactivated or expressed in tobacco, as shown in SEQ ID NO:
3.
8. The method according to claim 7, characterized in that, By constructing a gene silencing vector, the gene silencing vector was transformed into plants using Agrobacterium-mediated transformation to obtain Nta11g04230 gene-silenced plants.
9. The method according to claim 8, characterized in that, Black shank disease is caused by infection with Phytophthora indica.