Application and method of NtDehydrain1 gene in regulation and control of drought tolerance and disease resistance of tobacco

Abstract

The invention belongs to the technical field of biology, and relates to genetic breeding of tobacco. In order to excavate functions of tobacco genes and cultivate drought-resistant and disease-resistant tobaccos, the invention provides application and a method of the NtDehydri1 gene in regulation and control of the drought resistance and the disease resistance of the tobaccos, and a nucleotide sequence of the NtDehydri1 gene is as shown in SEQ ID No.1. The invention further provides a method of the NtDehydri1 gene in regulation and control of the drought resistance and the disease resistance of the tobaccos. The drought tolerance and disease resistance of the tobacco are improved through the function of overexpressing the NtDehydrodrin1 gene. And a new thought is provided for cultivation of drought-resistant and disease-resistant tobaccos.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to genetic breeding of tobacco. Background Art

[0002] Nicotiana tabacum (scientific name: Nicotiana tabacum L. ) is an annual herbaceous plant of the genus Nicotiana in the Solanaceae family, native to South America, and widely cultivated in provinces and regions in the north and south of China. Tobacco mainly relies on division and sowing for reproduction. According to the "Compendium of Materia Medica", tobacco tastes spicy, is warm in nature, enters the lungs, wards off evil spirits, treats wind-cold dampness, stagnant qi and phlegm, mountain mist and fog, and is a product for dispersing. The water in the tobacco pipe can detoxify. Excellent tobacco varieties have the characteristics of high productivity and resistance to pests and diseases. The quality of tobacco varieties also affects the growth of tobacco in the later stage and the final results, so selecting good tobacco varieties is the key to tobacco cultivation and planting. For example, tobacco seeds with good performance are suitable for planting in dry land and paddy fields, while varieties with strong disease resistance are suitable for planting in places with sufficient water sources and fertile land, and varieties with strong disease resistance and strong adaptability are suitable for planting on multiple types of soil. Drought and disease are important factors affecting tobacco growth. The application of publication number CN117417946A discloses the application of gene NtBRL3 in improving tobacco drought resistance. The application of publication number CN107058340A discloses the application of gene NtSAP5 in improving tobacco drought tolerance. The application of publication number CN109112147A discloses a rice gene that is related to both bacterial leaf streak and drought stress. It can be seen that the role played by different genes in plant growth is unpredictable, and how to use genes to better cultivate plants with excellent traits is a technical problem that scientific researchers need to solve. Summary of the invention

[0003] In order to explore the functions of tobacco genes and cultivate drought-resistant and disease-resistant tobacco, the present invention proposes an application and method of NtDehydrin1 gene in regulating tobacco drought resistance and disease resistance, providing a new idea for the cultivation of drought-resistant and disease-resistant tobacco.

[0004] The technical solution of the present invention is achieved in this way: Application of NtDehydrin1 gene in regulating tobacco disease resistance.

[0005] The above-mentioned disease resistance is the property of resistance to diseases caused by Fusarium.

[0006] The above application is to improve the disease resistance of tobacco by overexpressing the function of the NtDehydrin1 gene.

[0007] Application of NtDehydrin1 gene overexpression vector in improving tobacco resistance to diseases caused by Fusarium spp.

[0008] Application of NtDehydrin1 gene in improving tobacco drought tolerance.

[0009] The above application is to improve the drought resistance of tobacco by overexpressing the function of the NtDehydrin1 gene.

[0010] Application of NtDehydrin1 gene overexpression vector in improving tobacco drought tolerance.

[0011] The nucleotide sequence of the above NtDehydrin1 gene is shown in SEQ ID No.1.

[0012] A method for cultivating disease-resistant and drought-tolerant tobacco, comprising the following steps: (1) Using tobacco cDNA as a template and NtDehydrin1 gene as a target gene, an overexpression vector was constructed; (2) The overexpression vector of step (1) is introduced into tobacco plants through Agrobacterium transformation to obtain disease-resistant and drought-tolerant tobacco.

[0013] The nucleotide sequence of the above NtDehydrin1 gene is shown in SEQ ID No.1.

[0014] In crop breeding, in order to improve disease resistance and cultivate crops with Fusarium resistance characteristics, researchers generally improve disease resistance by overexpressing certain disease resistance-related genes. For example, disease resistance-related genes closely related to plant immune response are selected, such as PR genes (PR1, PR2 and PR3, etc.) encoding pathogenesis-related proteins, R genes encoding disease resistance proteins, or Chitinase genes encoding chitinases, LysM genes encoding receptor kinases, etc., to help plants identify pathogens and initiate immune responses by decomposing pathogen cell walls, chitin, etc. In addition, transcription factors such as WRKY and NAC are also used to activate the expression of plant immune response-related genes, thereby improving plant disease resistance. The use of these genes can improve plant disease resistance, but there are also some important side effects. For example, due to the high expression level of exogenous resistance genes, crops will highly express resistance-related genes and activate immune-related responses without being invaded by pathogens, which will affect the normal growth and development of plants to a certain extent. In some cases, excessive activation of immunity may also lead to a serious decrease in the tolerance of plants to other pathogens or environmental stresses.

[0015] The present invention has the following beneficial effects: The gene described in this study is a dehydrin protein. The protein encoded by this gene has a relatively small molecular weight and is widely present in different tissues and organs of plants, but is only active in specific structures or cells such as guard cells, meristematic cells or pollen sacs. Under conditions of drought, low temperature, reactive oxygen species and other stresses, dehydrin proteins can protect cell membranes and maintain the efficiency of plant photosynthesis. After overexpression of the dehydrin protein encoding gene, the protein is only active in specific organs and tissues and has no adverse effects on other traits of the plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the relative expression analysis of different genes before and after inoculation.

[0018] Figure 2 Schematic diagram of the NtDehydrin1 gene structure and the conserved regions of the 12 members of the NtDehydrin gene family.

[0019] Figure 3 This is the domain prediction result of the protein encoded by the NtDehydrin1 gene.

[0020] Figure 4 for NtDehydrin1 Expression level characteristics of genes in different parts of tobacco at different stages.

[0021] Figure 5 for NtDehydrin1 overexpression vector.

[0022] Figure 6 PCR screening of transgenic lines.

[0023] Figure 7 These are the results of fluorescence screening of the progeny of the transgenic lines.

[0024] Figure 8 For overexpression NtDehydrin Inoculation identification results of the positive strains' resistance to Fusarium spores.

[0025] Fig. 9 This is the germination status of T2 generation seeds under PEG drought simulation conditions.

[0026] Fig.10Shown are the growth conditions of wild type and transgenic strains 7 days after inoculation with Fusarium.

[0027] Fig.11 This is the growth of wild type and transgenic strains of Fusarium after 14 days. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] Embodiment 1: NtDehydrin Gene acquisition process, sequence information and preliminary bioinformatics analysis results By inoculating pathogens and investigating the disease, our research group identified a small number of germplasm materials resistant to Fusarium root rot from a large number of germplasm resources. Through RNA-Seq sequencing analysis (transcriptome sequencing analysis) of disease-resistant and disease-susceptible tobacco after and before inoculation with pathogens, we screened out a gene that responds quickly to pathogen invasion. NtDehydrin (Nitab4.5_0000723g0160.1).

[0031] The specific steps of the transcriptome sequencing analysis are as follows: tobacco varieties that are resistant to Fusarium root rot and susceptible to Fusarium root rot are used as research objects, tobacco seedlings are cultivated using the floating seedling method, and when they grow to 4-5 true leaves, Fusarium root rot pathogenic bacteria ( Fusarium oxysporum ) is inoculated into the tobacco root system. The inoculation method steps are as follows: The spore solution root irrigation method is preferably used to identify tobacco resistance to Fusarium. Specific steps for inoculating pathogens: Preparation of spore solution: The pathogen of tobacco Fusarium root rot (Fusarium) was provided by the Plant Protection Office of the Tobacco Research Institute of Henan Academy of Agricultural Sciences. The pathogen was first cultured on oatmeal medium. After the mycelium grew to cover the entire plate, a round bacterial cake with a diameter of 5 mm was punched on the medium where the Fusarium mycelium was cultured, and inoculated into the liquid culture medium. After shaking culture for 3 days (180 rpm, 28°C), the spores were filtered through four layers of sterile gauze and centrifuged at 5000 rpm to collect the spores, which were resuspended in sterile water to prepare the spore solution and adjusted to a concentration of 1 × 10 7 -10 8 pcs / ml, used for inoculation.

[0032] Preparation of tobacco seedlings: Select strong seedlings with 5-6 true leaves, set up 6 biological replicates, and use spore solution root irrigation method to inoculate pathogens. Each tobacco seedling is irrigated with 25 ml of spore solution. After the inoculation, observe the status of tobacco seedlings and the disease situation every day. On the 7th day after inoculation, investigate the disease degree of tobacco seedlings according to the national standard GB / T 23222─2008, calculate the incidence rate, disease index, and take photos to record the disease situation.

[0033] Sampling and transcriptome after inoculation: 3-5 days after inoculation, the lower leaves of the susceptible strains showed obvious wilt, while the resistant strains showed no phenotype. At this time, fully expanded leaves of different strains were taken, and 3 biological replicates were taken for each treatment. The leaves were quick-frozen with liquid nitrogen, stored in a -80 degree refrigerator, and then placed in dry ice and mailed to Kidio for RNA extraction, library construction, transcriptome sequencing, data filtering, and bioinformatics analysis according to standard procedures.

[0034] The criteria of p value < 0.01 and log2 (FC) absolute value greater than 2 were used to screen the differentially expressed genes in the disease-resistant and susceptible materials after inoculation of pathogens. As a result, 848 differentially expressed genes were screened in the disease-resistant materials, of which 453 genes were up-regulated and 295 genes were down-regulated. The metabolic pathways, biological processes and functions of the differentially expressed genes were classified and annotated through GO (Gene Ontology) enrichment analysis and KEGG (Kyotoencyclopedia of genes and genomes) enrichment analysis. The results showed that the differentially expressed genes were mainly enriched in MAPK, plant hormones, phenylpropanoid metabolism and other pathways. Among them, in the disease-resistant materials, NtDehydrin For the coding gene, the fold change (FC) of the relative expression level before and after inoculation was taken, and the Log2(FC) was 6.253, the p value was <0.01, and the FDR was <1, suggesting that the expression change of this gene was very significant, which may be involved in the plant response to pathogen invasion and is closely related to disease resistance.

[0035] Ten genes including NtDehydrin1 were selected from the above-mentioned significantly differentially expressed genes, specific primers were designed, and the relative expression levels of these genes before and after tobacco inoculation were verified by fluorescence quantitative PCR analysis.

[0036] Fluorescence quantitative PCR method verification steps: The method of inoculating tobacco with pathogens was as described above. Before and after inoculation, samples were taken, frozen with liquid nitrogen, and quickly ground into powder in a sterile mortar. RNA was extracted using an RNA extraction kit and reversely transcribed into cDNA. The Actin gene was used as an internal reference to analyze the relative expression of differentially expressed genes (including NtDehydrin1 gene, a total of 10 genes). The results showed that the expression of this gene changed most significantly after inoculation, such as Figure 1 As shown. Figure 1 It can be seen that in addition to genes related to disease resistance such as PR1, ERF and PYL4, the Dehydrin gene may be involved in the response of tobacco to pathogen invasion and is closely related to the resistance of tobacco to Fusarium.

[0037] Based on the genome information of tobacco in the Solanaceae genome database (https: / / solgenomics.sgn.cornell.edu / organism / Nicotiana_tabacum / genome), after bioinformatics analysis, NtDehydrin1 The gene is located on the antisense strand, and the gene sequence contains one intron and two coding regions (CDS), with one untranslated region (UTR) at the 5' and 3' ends. The positions of its start codon ATG, 5'UTR, CDS, intron, 3'UTR and stop codon TGA are as follows: Figure 1 A. The Advanced Hmm search tool of TBtools-II software was used to search for NtDehydrin Gene analysis showed that the dehydrin family has 12 members, each of which contains a dehydrin conserved domain. Figure 1 As shown in B.

[0038] NtDehydrin The coding region sequence of the gene is shown in SEQ ID No. 1, which is 543 bp in total and encodes a protein containing 180 amino acids. The protein sequence is shown in SEQ ID No. 2. The sequence information of the 2000 bp upstream of the gene is shown in SEQ ID No. 3. NtDehydrin The protein domain prediction results are as follows Figure 3 As shown by Figure 3 It was found that the protein contained a structural domain unique to dehydrin proteins.

[0039] Embodiment 2: NtDehydrin Analysis of gene expression characteristics at different growth stages and before and after inoculation Based on the chip data of gene expression in different parts of Honghua Dajinyuan throughout the growth period, the expression of this gene in different parts of tobacco at several key periods was analyzed. The results are as follows Figure 4 Extracted from the chip data of gene expression levels in different parts of Honghua Dajinyuan throughout the growth period NtDehydrin The expression data of different periods and different parts were analyzed, and the results showed that the expression of this gene in leaves, stems, stem nodes and roots was significantly different during the clustering, vigorous growth, budding and topping stages. In particular, the highest expression was found in the upper leaves and fast-growing parts.

[0040] Embodiment 3: NtDehydrin Gene overexpression and functional verification in tobacco 1. Vector Construction Using tobacco cDNA as a template, Vazyme high-fidelity enzyme (P505) was used to amplify the target gene. After completion, the agarose electrophoresis gel method was used to observe the bands, and the Tiangen gel recovery kit was used to recover the target bands.

[0041] The amplification system is: The amplification procedure is: Pre-denaturation at 95°C for 1 minute; denaturation at 95°C for 30 seconds; annealing at 55-60°C for 30 seconds; extension at 72°C for 30 seconds; Denaturation, annealing, and extension were cycled 30 times; final extension: 72°C for 5-10 minutes.

[0042] Using P2300 as the background vector, Vazyme's one-step cloning and homologous recombination kit was used to digest and connect NtDehydrin The gene will be linked between SacI and BamHI to construct a gene overexpression vector and transform E. coli. Figure 5 shown.

[0043] Among them, the enzyme digestion system is: 37℃ for 30min, 80℃ for 5min, and 4℃ for enzyme digestion; after the digestion is completed, the digestion products are separated by agarose gel electrophoresis, the gel at the position of the target band is cut under a gel imager, the band is recovered using a gel recovery kit, the system is prepared according to the following ratio, and homologous recombination is carried out at 37℃.

[0044] 37°C for 30 min, 4°C, the vector was connected, and the primers used in the vector construction and subsequent screening process are shown in Table 1 Table 1. Primer information used in the present invention Through bacterial liquid PCR, using the primers used for screening in Table 1, the E. coli with the correct band position (about 550bp) was selected for sequencing, and the vector with completely correct sequence was selected. According to the instructions for use of Agrobacterium competent cells, the vector was transferred into Agrobacterium GV3101.

[0045] 2. Screening of gene overexpression strains The vector was introduced into tobacco by Agrobacterium-mediated genetic transformation, and regenerated plants were obtained by regeneration and resistance screening. Then, positive seedlings were screened by PCR. The electrophoresis of PCR products is shown in Figure 6 The results showed that strains 2, 3, 7, 9, 10, 12, 13, 14, 15, 18, 19, and 20 were positive strains in which both 35S and the reporter gene (NPT II gene) were normally expressed. They can be used for subsequent phenotypic identification experiments.

[0046] Since the vector carries GFP fluorescent protein as a reporter gene, in the process of screening and identifying the offspring of transgenic lines, in order to facilitate non-destructive and rapid screening of positive plants at the seedling stage, the whole seedling can be gently clamped, placed on a slide, and placed on the stage of an inverted biofluorescence microscope (such as Sunny Optical Technology Co., Ltd., model ICX41) to observe and screen the expression of fluorescent protein in the whole seedling. The results showed that the 2nd, 3rd, and 20th lines were able to stably express strong green fluorescent protein, such as Figure 7 shown.

[0047] The results of disease resistance phenotype identification are as follows Figure 8 As shown by Figure 8 It can be seen that after inoculation with the fungus, the leaves of ordinary tobacco wilt and turn yellow, or the growth stops and dies. However, the positive strains obtained after overexpressing the NtDehydrin gene have slightly wilted and yellowed leaves in the lower part after inoculation, but the overall growth is better than the control. The growth rate and growth are better than the control, and no plants wither and die due to wilting.

[0048] Example 4: Preliminary identification of drought tolerance of gene overexpression strains After overexpression in Nicotiana benthamiana and obtaining positive strains, the preliminary identification results of their drought tolerance are shown in Table 2.

[0049] Using T1 generation seeds as experimental materials and water treatment as control, the tolerance of the overexpression strains to drought stress was analyzed by 15% PEG treatment. The results are shown in Table 2: Table 2. Germination rate of seeds overexpressing NtDehydrin gene under 15% PEG stress As shown in Table 2, PEG stress reduced the germination rate of the control seeds by 32.56%; in the strains overexpressing the NtDehydrin gene, the inhibition rate of PEG stress on the germination rate was reduced to 8.82%, indicating that overexpressing the NtDehydrin gene can improve the tolerance of tobacco to PEG stress.

[0050] T2 seeds were used as experimental materials, and water was used as control. After 15% PEG treatment, the WT and NtDehydrin - Germination of OE strains, such as Fig. 9 As shown. Fig. 9 Overexpression NtDehydrin The gene can improve the tolerance of tobacco to PEG stress during germination.

[0051] Embodiment 5: NtDehydrin Effects of Gene Overexpression in Tobacco on Resistance to Fusarium spp. Use the pathogen of tobacco Fusarium root rot (Fusarium, Fusarium oxysporum ), provided by the Plant Protection Office of the Tobacco Research Institute of Henan Academy of Agricultural Sciences. The pathogen was first cultured on oatmeal medium. After the mycelium grew to cover the entire plate, a round fungus cake with a diameter of 5 mm was punched on the medium where the Fusarium mycelium was cultured, and inoculated into the liquid culture medium. After shaking culture for 3 days (180 rpm, 28°C), it was filtered through four layers of sterile gauze and centrifuged at 5000 rpm to collect spores, which were resuspended in sterile water to prepare spore solution and adjusted to a concentration of 1×10 7 -10 8 pcs / ml, used for inoculation.

[0052] Select strong seedlings with 5-6 true leaves, set up 6 biological replicates, and use the spore solution root irrigation method to inoculate the pathogen. Each tobacco seedling is irrigated with 25 ml of spore solution. After the inoculation, the status of the tobacco seedlings and the disease situation are observed every day. On the 7th day after inoculation, the disease degree of tobacco seedlings was investigated according to the national standard GB / T 23222-2008, and the incidence rate and disease index were calculated, and the disease situation was recorded by taking photos.

[0053] The diameter of the lesions is shown in Table 3: Photos of infected spots Fig.10 As shown in Table 3, Fig.10 It can be seen that after inoculation, the diameter of the lesions on the leaves of different transgenic strains was significantly smaller than that of the control leaves, indicating that the tobacco strains overexpressing the Dehydrin gene have better disease resistance to Fusarium than the control.

[0054] On the 14th day after inoculation, the results were as follows Fig.11 As shown. Fig.11It can be seen that 2 weeks after inoculation with the pathogen, the control leaves were seriously infected with the pathogen, all of them were chlorotic and necrotic, while the area of ​​lesions caused by the pathogen on the transgenic leaves (overexpressing dehydrin protein) was small and the disease was very mild.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of NtDehydrin1 gene in regulating tobacco disease resistance.

2. The use according to claim 1, characterized in that: The disease resistance is the performance of resisting diseases caused by Fusarium.

3. The use according to claim 1 or 2, characterized in that: Improving tobacco disease resistance by overexpressing the function of the NtDehydrin1 gene.

4. Application of NtDehydrin1 gene overexpression vector in improving tobacco resistance to diseases caused by Fusarium spp.

5. Application of NtDehydrin1 gene in improving tobacco drought tolerance.

6. The use according to claim 5, characterized in that: The purpose of improving tobacco drought tolerance is achieved by overexpressing the function of the NtDehydrin1 gene.

7. Application of NtDehydrin1 gene overexpression vector in improving tobacco drought tolerance.

8. The use according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No.

1.

9. A method for cultivating disease-resistant and drought-tolerant tobacco, characterized in that: The steps are: (1) Using tobacco cDNA as a template and NtDehydrin1 gene as a target gene, an overexpression vector was constructed; (2) The overexpression vector of step (1) is introduced into tobacco plants through Agrobacterium transformation to obtain disease-resistant and drought-tolerant tobacco.

10. The method for cultivating disease-resistant and drought-tolerant tobacco according to claim 9, characterized in that: The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No.1.

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