Tobacco Nta05g14050 gene, protein and application of tobacco Nta05g14050 gene in tobacco black shank resistance

Abstract

The invention discloses a tobacco Nta05g14050 gene, a protein and application of the tobacco Nta05g14050 gene and the protein in tobacco black shank resistance. The amino acid sequence of the tobacco Nta05g14050 protein is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene for coding the tobacco Nta05g14050 protein is as shown in SEQ ID NO.1. The invention further discloses a preparation method of the tobacco Nta05g14050 protein. The Nta05g14050 gene has the advantages that the expression quantity of the Nta05g14050 gene is obviously increased after the Nta05g14050 gene is infected by tobacco black shank bacteria, the resistance of tobacco to the black shank can be obviously improved by overexpressing the Nta05g14050 gene in the tobacco, a new gene resource and a theoretical basis are provided for disease-resistant breeding of the tobacco black shank, and the Nta05g14050 gene has an important application value.

Description

Technical Field

[0001] This invention relates to a tobacco black shank resistance gene, specifically to a tobacco Nta05g14050 Genes, proteins and their application in tobacco black shank resistance. Background Technology

[0002] Tobacco black shank disease, also known as tobacco black root or black stem disease, is caused by Phytophthora indicum (… Phytophthora nicotianae This is an important disease caused by [unspecified pathogen], primarily infecting the roots and base of the stems of tobacco plants, forming black, sunken lesions. It generally occurs less frequently in the seedbed stage, mainly affecting tobacco plants in the field. Symptoms vary considerably depending on the growth stage of the tobacco plant and climatic conditions.

[0003] Tobacco black shank is prevalent worldwide, particularly severe in temperate, subtropical, and tropical regions. Rainfall and soil moisture are key factors in its spread. Tobacco black shank severely restricts the further improvement of tobacco yield and quality. Therefore, enhancing the resistance of tobacco varieties to black shank has become a crucial and urgent issue to be addressed in tobacco agricultural production.

[0004] Different tobacco varieties exhibit significant differences in resistance to black shank, and breeding and promoting disease-resistant varieties is the most economical and effective way to control this disease. Currently, research on the resistance mechanism of tobacco black shank mainly focuses on the physiological and biochemical levels, while systematic and in-depth research is still lacking in areas such as the cloning, functional verification, and expression regulation of resistance-related genes.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a tobacco Nta05g14050 This invention discovers genes, proteins, and their application in tobacco black shank resistance. Nta05g14050 The gene is an important gene in response to black shank infection, regulating... Nta05G14050 The expression of this substance can regulate the level of tobacco resistance to black shank.

[0007] To achieve the above objectives, the present invention provides tobacco Nta05g14050 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A second object of the present invention is to provide a gene encoding the tobacco Nta05g14050 protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] A third objective of this invention is to provide a recombinant vector containing the aforementioned gene.

[0010] A fourth objective of this invention is to provide recombinant bacteria containing the aforementioned recombinant vector.

[0011] The fifth objective of this invention is to provide the application of the tobacco Nta05g14050 protein, or the gene, or the recombinant vector, or the recombinant bacteria in tobacco black shank resistance.

[0012] Preferably, its application in molecular breeding of tobacco resistant to tobacco black shank disease.

[0013] The sixth objective of this invention is to provide a method for improving tobacco resistance to tobacco black shank disease by increasing the content of the Nta05g14050 protein in the tobacco, or by overexpressing the tobacco protein. Nta05g14050 Genes were developed to enhance tobacco's resistance to black shank.

[0014] The tobacco of the present invention Nta05g14050 The application of genes, proteins, and their role in tobacco black shank resistance has the following advantages: This invention utilizes omics technology to discover potential disease-resistant genes in highly resistant tobacco varieties, and discovers... Nta05G14050 The gene is an important gene in response to black shank infection, regulating... Nta05G14050 The expression of this gene can modulate the level of tobacco resistance to black shank. Therefore, Nta05G14050 Genes have broad application prospects in improving tobacco disease resistance, and can provide new gene resources and theoretical basis for molecular breeding of tobacco disease resistance. They can help accelerate the breeding of new tobacco varieties with high resistance to black shank, thereby effectively reducing yield loss and quality decline caused by black shank. This is of great significance to ensuring the sustainable development of the tobacco industry and has huge economic potential. Attached Figure Description

[0015] Figure 1 In Example 2 of this invention, the inoculation with Tobacco Blackleg Bacterium was carried out. Nta05G14050 A bar chart showing gene expression patterns in different resistant tobacco varieties (lines).

[0016] Figure 2 In Embodiment 3 of the present invention Nta05G14050 Schematic diagram of gene expression vector construction in plants.

[0017] Figure 3 For the transfer in Embodiment 3 of the present invention Nta05G14050 A bar chart showing the expression levels of genes in tobacco strains.

[0018] Figure 4 In Embodiment 4 of the present invention Nta05G14050Symptoms of disease in tobacco blackleg disease in tobacco overexpression strains inoculated with *Tobacco Blackleg* (A) and disease index bar chart (B). Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0021] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0022] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0023] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0024] Example 1 Nta05g14050 The discovery and cloning of genes 1. Infection response genes of Tobacco Blackleg Bacterium Nta05G14050 The discovery Tobacco black shank resistant variety G28 and susceptible variety XHJ (preserved by the Tobacco Molecular Breeding Laboratory of Henan Agricultural University) were inoculated with Tobacco Black Shank Fungi (pathogen race 1). Six days after inoculation, stem segments above the inoculation point of the tobacco plants were taken (with distilled water as a control mock) for transcriptome sequencing analysis.

[0025] Differential gene expression analysis revealed a gene with significant differential expression. Nta05g14050 Its transcriptional abundance in the resistant variety G28 (Log2) Fold change The expression level was 5.0 (significantly upregulated), while the transcriptional abundance in the susceptible variety XHJ was Log2. Fold change The expression level was -1.9 (significantly downregulated). Comparison with publicly available tobacco genome databases revealed no similar gene sequences or functional annotations, leading to the preliminary conclusion that this gene is a novel gene.

[0026] The above Nta05G14050 The nucleotide sequence of the gene (555 nucleotides, SEQ ID NO.1) is shown below: .

[0027] 2. Tobacco first-strand cDNA synthesis Total RNA from tobacco G28 stems was isolated and purified according to the TRIZOL kit (Thermo Fisher Scientific, USA) manufacturer's instructions. 2 μg of total RNA was pipetted into a 1.5 mL centrifuge tube and centrifuged according to the RevertAid instructions. TM The First Strand cDNA Synthesis Kit (Abm, Canada) product instructions state that reverse transcription is used to obtain the first-strand cDNA from tobacco stems.

[0028] 3. Nta05G14050 PCR amplification and sequencing verification of gene coding regions Using the first-strand cDNA obtained above as a template, tobacco was designed based on transcriptome sequencing information. Nta05G14050 Full-length cDNA primers (F / R) for the gene were used for... Nta05G14050 Genes are amplified by PCR to obtain amplification products.

[0029] Upstream primer (F, SEQ ID NO.3): 5′-ATGGCTTCATTCCATAGCTTG-3′; Downstream primer (R, SEQ ID NO.4): 5′-TTAGAAAGGAGGCATTGCCG-3′.

[0030] The PCR products were subjected to 1.2% agarose gel electrophoresis, and the amplification products were detected using a gel imaging system, yielding a 555 bp fragment. Subsequently, the PCR products were recovered and purified, and sequencing confirmed that the results were consistent with expectations.

[0031] The amino acid sequence (184 amino acids, SEQ ID NO.2) of the Nta05G14050 protein is as follows: MASFHSLKTLAIVALAISSFVQVTLGGIACENLNEDSCAFAISSNGKRCVLEKHLRRSGEEGYTCRTSEIEADKLKDWIETDECIEACGVDRNALGISSDALLESRFTNKLCSPACYKHCPNIVDLYFNLAAGEGVYLPKLCAEQGKSARREIAEIRSSGLVAPAPESEVKPSNFMIAPAMPPF.

[0032] Example 2 Nta05G14050 Gene expression analysis in different resistant tobacco varieties (lines) In this embodiment, quantitative real-time PCR (qPCR) was used to analyze the inoculation conditions of the pathogen (Phytophthora tobaccois race 1). Nta05G14050Gene expression in different resistant tobacco varieties (lines). The experimental samples were stem segments from four varieties (lines): G28, BH, XHJ, and Y2020. G28 and BH were disease-resistant varieties (lines), while XHJ and Y2020 were susceptible varieties (lines). From each sample, 2 µg of total RNA was reverse transcribed into first-strand cDNA as a template, following the method described in Example 1.

[0033] according to Nta05G14050 The full-length cDNA sequence of the gene was used to design specific primers for real-time PCR. Upstream primer (SEQ ID NO.5): 5'-GACGCTCTCCTGGAATCTCG-3'; Downstream primer (SEQ ID NO.6): 5'-CGCTATTTCTCGCCTTGCAC-3'.

[0034] The reaction was performed using a real-time quantitative PCR instrument, and the amplification product was 165 bp.

[0035] tobacco Tubulin The gene fragment is used as an internal reference gene (as a control group), and its amplification uses specific primers: Upstream primer (SEQ ID NO.7): 5'-CATCTTTGCGTACACTTTGC-3'; Downstream primer (SEQ ID NO.8): 5'-CATAAGCCCAAAACTAGCTG-3'.

[0036] According to Formula 2 -ΔΔC T calculate Nta05G14050 The relative expression levels of genes, of which: ΔC TNta05G14050 = C TNta05G14050 -C TTubulin ΔΔC TNta05G14050 =Experimental Group ΔC TNta05G14050 -Control group C TTubulin .

[0037] In the above formula, C TNta05G14050 C of the target gene t Value, C TTubulin C of the internal reference gene t value.

[0038] The results are as follows Figure 1 As shown, qPCR was used to detect... Nta05G14050 The expression levels of the gene in the stems of tobacco varieties (lines) with different disease resistance (G28, BH, XHJ, and Y2020) were studied. Results showed that under control (Mock) conditions... Nta05G14050 There was no significant difference in gene expression levels among different varieties. However, under inoculation conditions, the gene expression levels were higher in the resistant varieties (lines) G28 and BH (30.20 and 24.80, respectively), while the expression levels were lower in the susceptible varieties (lines) XHJ and Y2020 (0.25 and 0.30, respectively). Figure 1 This result indicates that... Nta05G14050 Gene expression levels are induced by Tobacco Blackleg and are closely related to the resistance levels of tobacco varieties.

[0039] Example 3 Nta05G14050 Gene-transformed tobacco and the creation of transgenic strains 1. Construction of tobacco transgenic expression vector Constructed in Example 2 Nta05G14050 Using the full-length cDNA fragment of the gene as a template, and with... Bam HI and Xba PCR amplification was performed using specific primers for the I adapter sequence, and the amplification product was then subjected to... Bam HI and Xba After double digestion and recovery, the enzyme was inserted forward into the cauliflower mosaic virus (CaMV) 35S promoter of the plant binary expression vector pWM101. Bam HI and Xba Between the I sites, the recombinant vector pWM101-35S- was obtained. Nta05G14050 ( Figure 2 ).

[0040] The primer sequences are as follows: Upstream primer (SEQ ID NO.9): 5'-AC GGATCC ATGGCTTCATTCCATAGC-3'; Downstream primer (SEQ ID NO.10): 5'-CA TCTAGA TTAGAAAGGAGGCATTGCC-3'.

[0041] 2. Agrobacterium-mediated genetic transformation of tobacco and identification of transgenic plants The recombinant plasmid was identified as correct by PCR, plasmid digestion, and sequencing, and then transformed into Agrobacterium using the freeze-thaw method. GV3101 After the recombinant colonies were confirmed to be correct by PCR, they were transformed into the susceptible tobacco strain Y2020 using the Agrobacterium-mediated leaf disc method. The specific process is as follows:

[0042] (1) Activation of Agrobacterium: single colonies containing the target gene were picked from the plate and inoculated into 5 mL of LB liquid medium. Rifampicin and kanamycin (Rif 50 μg / mL, Kan 50 μg / mL) were added and cultured at 28℃ and 200 rpm for 16 h until the OD600 reached 1.5. (2) Transplantation: The activated bacterial culture was transferred into LB liquid medium containing the corresponding antibiotic at a ratio of 1:100 and cultured at 28°C and 200 rpm for 4-5 h. When the OD600 reached 0.5-0.7, it could be used for transformation. (3) Infection: In a clean bench, take tender leaves of sterile tobacco seedlings, remove the midrib, and cut the leaves into 1 cm pieces. 2 Soak small pieces of the leaf in the bacterial solution for 5 minutes. Remove the leaf and place it on sterile filter paper to absorb the attached bacterial solution.

[0043] (4) Co-culture: The infected tobacco leaves were placed on a culture dish containing co-culture medium (MS basic solid medium + 2 mg / L 6-BA + 0.2 mg / L NAA), the culture dish was sealed with sealing film, and cultured in the dark at 28°C for 3 days; (5) Selective differentiation culture: The tobacco leaves after co-culture were transferred to a petri dish containing selective differentiation solid medium (co-medium + 400 mg / L Cef + 40 mg / L Hyg), the petri dish was sealed with sealing film, and cultured at a temperature of 25℃, a light intensity of 5000~10000 lux, and a light-dark condition of 16 h / 8 h. (6) Rooting culture: After about 3 weeks of differentiation culture, when the tobacco adventitious buds grow to 2cm, cut off the adventitious buds and transfer them to Erlenmeyer flasks containing rooting medium (MS basic solid medium + 0.3 mg / L NAA + 40 mg / L Hyg + 400 mg / L Cef) for rooting culture; (7) Transplanting: When the roots grow to 3-5 cm and the seedlings are 7-10 cm tall, transplant them into flower pots and cultivate them in a greenhouse.

[0044] 3. Extracting genomic DNA from transgenic tobacco seedlings PCR amplification was performed using the primers described in this embodiment to further detect and screen positive plants. Six positive plants were detected from nine regenerated tobacco plants.

[0045] 4. Quantitative real-time PCR analysis The control line Y2020 and 6 transgenic plants were extracted according to the method described in Example 2. Nta05G14050Total RNA from the T0 generation plants was analyzed by quantitative real-time PCR (qPCR) as described in Example 2 to detect gene expression in different transgenic lines. The tobacco internal control gene used in the quantitative real-time PCR was... Tubulin As described in Example 2.

[0046] The results are as follows Figure 3 As shown, the test results indicate that in 6 transformed plants Nta05G14050 In the T0 generation lines of the gene, compared with the control line Y2020, the expression levels of these six overexpression lines (OE-1, 2, 3, 4, 5, and 6) were significantly higher. Seeds from individual plants of the six identified high-expression lines were harvested, and each seed was sown separately. Hygromycin was used for further selection to observe segregation in the T1 generation. This process was repeated until the T3 generation to obtain genetically stable transgenic lines, resulting in a total of six stable transgenic lines. Nta05G14050 Transgenic lines with overexpressed genes.

[0047] Example 4 Overexpression Nta05G14050 Resistance of tobacco strains to black shank by genetic factors 1. Preparation method of spore suspension The fungus *Phytophthora indicum* (race 1 of *Phytophthora indicum*) was cultured on oat agar at 28 °C for 7 days. Two weeks before inoculation, mycelium was picked and inoculated into the culture medium. The culture was then incubated for two weeks at 28 °C with a shaker at 120 r / min, followed by a sudden cooling to prepare a zoospore suspension. The concentration of the zoospore suspension was adjusted to 1 × 10⁻⁶. 8 cfu / mL, for later use.

[0048] 2. Vaccination identification Three transgenic tobacco lines (OE-1, 2, and 5) and the control line Y2020 were selected and planted in pots in a greenhouse. Six boxes were prepared for each line, with one plant per box. Normal fertilization and watering were applied. When the seedlings reached 4-6 leaves, they were inoculated by root irrigation with a spore suspension. The stem base wound inoculation method was used: a wound was made at the base of the seedling stem, and the zoospore suspension was applied to the wound, which was then kept moist with absorbent cotton. Each plant was inoculated with 10 mL of the inoculum solution; a water control was included. A survey was conducted 12 days after inoculation, and the plants were graded according to the severity of disease, and a disease index was calculated.

[0049] 3. Experimental Results The results are as follows Figure 4 As shown, Figure 4A comparison of the disease incidence of three transgenic tobacco lines (OE-1, 2, and 5) and the control line (Y2020) after inoculation with *Phytophthora indica* was shown in Figure A. The figure indicates that the control line Y2020 was more severely susceptible to the disease, while the three transgenic lines showed lower disease severity, especially OE-1 and OE-2, which exhibited higher resistance to black shank. Disease index analysis showed that the disease index of the three transgenic lines was significantly lower than that of the control line (the disease indices of OE-1, 2, and 5 were 33.30, 28.90, and 64.40, respectively, compared to 91.11 for the control line). Figure 4 (B), which fully demonstrates overexpression Nta05G14050 Genes can significantly improve resistance to tobacco black shank disease, resulting in tobacco strains with high resistance to black shank disease.

[0050] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A tobacco Nta05gl4050 protein characterized in that, The amino acid sequence of the tobacco Nta05g14050 protein is shown as SEQ ID NO.

2.

2. A gene encoding the tobacco Nta05gl4050 protein according to claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

3. A recombinant vector containing the gene according to claim 2.

4. A recombinant bacterium containing the recombinant vector according to claim 3.

5. The tobacco Nta05g14050 protein according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant bacterium according to claim 4 in the resistance to tobacco black shank.

6. Use according to claim 5, characterized in that, Application in tobacco resistance to tobacco black shank molecular breeding.

7. A method of increasing resistance to Phytophthora parasitica in tobacco, characterized by, Increasing the content of the tobacco Nta05g14050 protein according to claim 1, or overexpressing the gene according to claim 2 to enhance the resistance of tobacco to black shank.

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