Application and method of ntdehydrin1 gene in regulating drought tolerance and disease resistance of tobacco

Abstract

The application belongs to the technical field of biology and relates to genetic breeding of tobacco. In order to excavate the function of tobacco genes and cultivate drought-tolerant and disease-resistant tobacco, the application provides application and a method of NtDehydrin1 gene in regulating drought tolerance and disease resistance of tobacco, wherein the nucleotide sequence of the NtDehydrin1 gene is shown as SEQ ID No. 1. The drought tolerance and disease resistance of tobacco are improved by overexpressing the function of the NtDehydrin1 gene. The application provides a new idea for cultivating drought-tolerant and disease-resistant tobacco.

Description

Technical Field

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

[0002] Tobacco (scientific name: Nicotiana tabacum L. Tobacco (Nicotiana macrophylla) is an annual herbaceous plant belonging to the genus Nicotiana in the Solanaceae family. It originated in South America and is widely cultivated throughout China. Propagation is primarily through division and sowing. According to the *Compendium of Materia Medica*, tobacco is pungent and warm in nature, entering the lungs, dispelling evil, treating wind-cold-dampness syndrome, stagnation of qi and phlegm, and miasma. It is considered a dispersing agent, and the water in the tobacco pipe can detoxify. Superior tobacco varieties are characterized by high yield and resistance to pests and diseases. The quality of the tobacco seeds also affects the later growth and final yield of the tobacco. Therefore, selecting good tobacco seeds is crucial for tobacco cultivation. For example, high-performance tobacco seeds are suitable for both dryland and paddy fields, while varieties with strong disease resistance are suitable for planting in areas with abundant water and fertile soil. Varieties with strong disease resistance and adaptability are suitable for cultivation in various soil types. Drought and disease are important factors affecting tobacco growth. Application CN117417946A discloses the application of the gene NtBRL3 in improving the drought resistance of tobacco. Application CN107058340A discloses the application of the gene NtSAP5 in improving drought resistance in tobacco. Application CN109112147A discloses a rice gene that is simultaneously associated with bacterial leaf streak and drought stress. It is evident that the roles of different genes in plant growth are unpredictable, and how to utilize genes to better cultivate plants with superior traits is a technical problem that researchers need to solve. Summary of the Invention

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

[0004] The technical solution of this invention is implemented as follows:

[0005] Application of the NtDehydrin1 gene in regulating tobacco disease resistance.

[0006] The above-mentioned disease resistance refers to the ability to resist diseases caused by Fusarium.

[0007] The above application enhances the disease resistance of tobacco by overexpressing the NtDehydrin1 gene.

[0008] Application of NtDehydrin1 gene overexpression vector in improving tobacco resistance to Fusarium diseases.

[0009] Application of NtDehydrin1 gene in improving drought tolerance of tobacco.

[0010] The application is to improve the drought tolerance of tobacco by overexpressing the function of NtDehydrin1 gene.

[0011] Application of NtDehydrin1 gene overexpression vector in improving drought tolerance of tobacco.

[0012] The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No. 1.

[0013] A method for cultivating disease-resistant and drought-tolerant tobacco, comprising the following steps:

[0014] (1) constructing an overexpression vector by using tobacco cDNA as a template and NtDehydrin1 gene as a target gene;

[0015] (2) introducing the overexpression vector of step (1) into tobacco plants by Agrobacterium transformation to obtain disease-resistant and drought-tolerant tobacco.

[0016] The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No. 1.

[0017] In crop breeding work, in order to improve disease resistance and cultivate crops with characteristics of resistance to Fusarium graminearum, researchers generally improve disease resistance by overexpressing some 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 chitinase, LysM genes encoding receptor kinases, etc., which help plants to recognize pathogens and start immune response by decomposing cell walls and chitin of pathogens. 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. Using 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 express resistance-related genes and activate immune-related responses in the absence of pathogen invasion, which will affect the normal growth and development of plants to some extent, and in some cases, excessive activation of immunity may also lead to a serious decrease in the tolerance of plants to other pathogens or environmental stress.

[0018] The present application has the following beneficial effects:

[0019] This study describes a gene encoding ahydroin protein. The protein encoded by this gene has a relatively small molecular weight and is widely distributed in different tissues and organs of plants, but it is active only in specific structures or cells such as guard cells, meristematic cells, or pollen sacs. Under stress conditions such as drought, low temperature, and reactive oxygen species, the ahydroin protein can protect cell membranes and maintain the efficiency of plant photosynthesis. Overexpression of the ahydroin protein encoding gene results in the protein being active only in specific organs and tissues, without adverse effects on other plant traits. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Analysis of the relative expression levels of different genes before and after inoculation.

[0022] Figure 2 This is a schematic diagram of the NtDehydrin1 gene structure and a schematic diagram of the conserved regions of the 12 members of the NtDehydrin gene family.

[0023] Figure 3 The domain prediction results for the protein encoded by the NtDehydrin1 gene.

[0024] Figure 4 for NtDehydrin1 The expression levels of genes in different parts of tobacco at different stages.

[0025] Figure 5 for NtDehydrin1 Overexpression vectors.

[0026] Figure 6 PCR screening for transgenic lines.

[0027] Figure 7 The results show the fluorescence screening of the offspring of the transgenic lines.

[0028] Figure 8 For overexpression NtDehydrin The results of inoculation identification of the resistance of positive strains to Fusarium.

[0029] Figure 9 Germination of T2 generation seeds under PEG drought simulation conditions.

[0030] Figure 10Growth of wild type and transgenic lines 7 days after inoculation with F. oxysporum.

[0031] Figure 11 Growth of wild type and transgenic lines 14 days after inoculation with F. oxysporum. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0033] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0034] Example 1 NtDehydrin Obtaining process of gene, sequence information and preliminary bioinformatics analysis results

[0035] Through inoculation of pathogenic bacteria and investigation of the incidence, the present research group identified a small amount of germplasm material resistant to F. oxysporum root rot from a large number of germplasm resources. Through RNA-Seq sequencing analysis (transcriptome sequencing analysis) after inoculation of pathogenic bacteria and before inoculation of pathogenic bacteria of resistant and susceptible tobacco, we screened a gene that can quickly respond to pathogen invasion NtDehydrin (Nitab4.5_0000723g0160.1).

[0036] The specific steps of the above transcriptome sequencing analysis are as follows: tobacco varieties resistant and susceptible to F. oxysporum root rot are used as research objects, tobacco seedlings are cultivated using the floating seedling method, and when the seedlings grow to 4-5 true leaves, the pathogenic bacteria F. oxysporum of F. oxysporum root rot are inoculated into the roots of the tobacco, and the inoculation method steps are as follows: Fusarium oxysporum

[0037] The spore liquid irrigation method is preferably used to identify the resistance of tobacco to F. oxysporum. The specific steps of inoculation of pathogenic bacteria are as follows:

[0038] Preparation of spore liquid: the pathogenic bacteria (F. oxysporum) of tobacco F. oxysporum root rot are provided by the Plant Protection Room of the Tobacco Institute of Henan Academy of Agricultural Sciences. The pathogenic bacteria are first cultured using oat medium, and when the mycelium grows to cover the entire plate, a 5 mm diameter circular cake of mycelium is punched on the culture medium using a puncher, inoculated into liquid medium, shaken and cultured for 3 days (180 rpm, 28°C), filtered through four layers of sterile gauze, centrifuged at 5000 rpm, the spores were collected, resuspended with sterile water to prepare spore liquid, and the concentration was adjusted to 1 × 10​7 -10 8 Individuals / milliliter, for bacteria.

[0039] Preparation of tobacco seedlings: Choose 5-6 leaf strong seedlings, set 6 biological repeats, when inoculating pathogenic bacteria by spore solution root irrigation, each tobacco seedling is irrigated with 25 ml of spore solution, after completing inoculation, observe the state of tobacco seedlings and incidence every day. On the 7th day after inoculation, investigate the incidence of tobacco seedlings according to the national standard GB / T 23222-2008, count the incidence rate, disease index, and take photos to record the incidence.

[0040] Sampling and transcriptome after inoculation: 3-5 days after inoculation, the lower leaves of the susceptible strain show obvious wilting, while the resistant strain has no phenotype. At this time, fully expanded leaves of different strains are taken, 3 biological repeats are taken for each treatment, the leaves are quickly frozen with liquid nitrogen, then stored in a-80 degree refrigerator, and then placed in dry ice, and then sent to Genedio Company for RNA extraction, library construction, transcriptome sequencing, data filtering and bioinformatics analysis according to standard procedures.

[0041] The genes differentially expressed in the resistant and susceptible materials after inoculation of pathogens are screened according to the standards of p value <0.01 and log2(FC) absolute value greater than 2. As a result, 848 differentially expressed genes are screened in the resistant material, of which 453 genes are up-regulated and 295 genes are down-regulated. GO (Gene Ontology) enrichment analysis and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis are used to classify and annotate the metabolic pathways, biological processes and functions of the differentially expressed genes. The results show that the differential genes are mainly enriched in MAPK, plant hormones, phenylpropanoid metabolism and other pathways. Among them, in the resistant material NtDehydrin Coding gene, the change multiple (Fold change, FC) of relative expression before and after inoculation is taken logarithm, Log2(FC) is 6.253, p value <0.01, FDR <1, which indicates that the expression change of the gene is very significant, and it may be involved in plant response to pathogen invasion and closely related to disease resistance.

[0042] From the above significantly differentially expressed genes, 10 genes containing NtDehydrin1 gene are selected, specific primers are designed, and the relative expression of these genes before and after inoculation of tobacco is analyzed and verified by fluorescence quantitative PCR technology.

[0043] Quantitative PCR method verification steps: the method of inoculating pathogenic bacteria into tobacco was as described above, before and after inoculation, sampling, quick freezing with liquid nitrogen, grinding into powder in a sterile mortar, extracting RNA with an RNA extraction kit, reverse transcription into cDNA, and analyzing the relative expression of the expression amount of differentially expressed genes (containing NtDehydrin1 gene, a total of 10 genes) with Actin gene as an internal reference. The results showed that the expression amount of the gene changed most significantly after inoculation, as shown in Figure 1 From Figure 1 it can be seen that in addition to PR1, ERF and PYL4 and other genes related to disease resistance, Dehydrin gene may be involved in the response of tobacco to pathogen invasion, and is closely related to the resistance of tobacco to fusarium.

[0044] Based on the genomic information of tobacco in the Solanaceae plant genome database (website https: / / solgenomics.sgn.cornell.edu / ) (website https: / / solgenomics.sgn.cornell.edu / organism / Nicotiana_tabacum / genome), bioinformatics analysis was performed, NtDehydrin1 The gene is located on the antisense strand, and the gene sequence contains one intron (intron) and two coding regions (CDS), and each has one untranslated region (UTR) at the 5' and 3' ends. The positions of the start codon ATG, 5'UTR, CDS, intron, 3'UTR and stop codon TGA are shown in Figure 1 A. The Advanced Hmm search tool of TBtools-II software was used to analyze the NtDehydrin gene in the whole genome of tobacco, and the results showed that there were 12 members in the Dehydrin family, and each member contained one Dehydrin conserved domain. The conserved regions of different family members are shown in Figure 1 B.

[0045] NtDehydrin The coding region sequence of the gene is shown in SEQ ID No. 1, which is 543 bp, encoding a protein containing 180 amino acids, and the protein sequence is shown in SEQ ID No. 2. The sequence information of the upstream 2000bp of the gene is shown in SEQ ID No. 3. The domain prediction results of the protein are shown in NtDehydrin From Figure 3 it can be seen that the protein contains a domain specific to dehydrin protein. Figure 3

[0046] Example 2: NtDehydrin Expression characteristics analysis of the gene at different growth stages and before and after inoculation ​

[0047] Based on microarray data of gene expression levels in different parts of the entire growth cycle of *Safflower 'Da Jin Yuan'*, the expression levels of this gene in different parts at several key stages of tobacco growth were analyzed. The results are as follows: Figure 4 As shown. Gene expression levels were extracted from microarray data of different parts of *Safflower 'Da Jin Yuan'* throughout its entire growth cycle. NtDehydrin Analysis of expression levels at different stages and in different parts of the plant revealed significant differences in gene expression levels across leaves, stems, stem nodes, and roots during the crowning, vigorous growth, budding, and topping stages. The highest expression levels were observed in the upper leaves and rapidly growing parts.

[0048] Example 3: NtDehydrin Overexpression and functional verification of genes in tobacco

[0049] 1. Carrier Construction

[0050] Using tobacco cDNA as a template, the target gene was amplified using Vazyme high-fidelity enzyme (P505). After amplification, the bands were observed using agarose gel electrophoresis, and the target bands were recovered using the Tiangen gel recovery kit.

[0051] The amplification system is as follows:

[0052]

[0053] The amplification procedure is as follows:

[0054] Pre-denaturation: 95℃ for 1 minute; Denaturation: 95℃ for 30 seconds; Annealing: 55-60℃ for 30 seconds; Extension: 72℃ for 30 seconds;

[0055] Denaturation, annealing, and extension, cycle 30 times; final extension: 72℃ for 5-10 minutes.

[0056] Using P2300 as the background vector, and employing Vazyme's one-step cloning and homologous recombination kit, enzyme digestion and ligation were used to clone... NtDehydrin The gene will be linked between SacI and BamHI to construct a gene overexpression vector, which will then be transformed into E. coli. The vector structure is as follows: Figure 5 As shown.

[0057] The enzyme digestion system is as follows:

[0058]

[0059] Enzyme digestion was performed at 37℃ for 30 min, 80℃ for 5 min, and 4℃. After digestion, the digestion products were separated by agarose gel electrophoresis. The gel at the location of the target band was cut under a gel imaging instrument. The band was recovered using a gel recovery kit. The system was prepared according to the following ratio and placed at 37℃ for homologous recombination.

[0060]

[0061] 37℃ 30min, 4℃, proceed to connect the vector, the vector construction and subsequent screening process, the use of primers as shown in Table 1

[0062] Table 1. Information of primers used in the application

[0063]

[0064] By bacterial liquid PCR, using the primers for screening in Table 1, the band position is correct (about 550bp) E. coli is sent to sequencing, and the vector with completely correct sequence is screened out. According to the use instruction of Agrobacterium competence, the vector is transformed into Agrobacterium GV3101.

[0065] 2Screening of gene overexpression strains

[0066] The vector is introduced into tobacco by Agrobacterium-mediated genetic transformation method, and the regenerated plants are obtained by regeneration and resistance screening. Then the positive seedlings are screened by PCR method. The electrophoresis map of PCR product is shown in Figure 6 . The results show that the 2nd, 3rd, 7th, 9th, 10th, 12th, 13th, 14th, 15th, 18th, 19th and 20th strains can normally express 35S and reporter gene (NPT II gene). They can be used for subsequent phenotype identification experiments.

[0067] Because the vector carries GFP fluorescent protein as a reporter gene, in the screening and identification process of the offspring of transgenic strains, in order to facilitate non-destructive and rapid screening of positive plants at seedling stage, the whole seedling can also be gently clamped and placed on a glass slide on the object table of an inverted biological fluorescence microscope (such as Shunyu Optical Technology Co., Ltd., model ICX41) to observe and screen the expression of fluorescent protein of the whole seedling. The results show that the 2nd, 3rd and 20th strains can stably express strong green fluorescent protein, as shown in Figure 7 .

[0068] The results of disease resistance phenotype identification are shown in Figure 8 . As shown in Figure 8 , after inoculation, the whole leaves of Nicotiana tabacum withered and turned yellow, or the growth stopped and died, while the positive strains obtained by overexpressing NtDehydrin gene had better growth than the control after inoculation, and no plant withered and died due to wilting.

[0069] Example 4: Preliminary identification of drought resistance of gene overexpression strains

[0070] After overexpressing the gene in Nicotiana benthamiana and obtaining positive lines, the preliminary identification results of their drought resistance are shown in Table 2.

[0071] Using T1 generation seeds as experimental material and water treatment as a control, the drought tolerance of overexpressing lines was analyzed by treatment with 15% PEG. The results are shown in Table 2:

[0072] Table 2. Germination rate of seeds after overexpression of NtDehydrin gene under 15% PEG stress treatment

[0073]

[0074] Table 2 shows that PEG stress reduced the germination rate of control seeds by 32.56%; in lines overexpressing the NtDehydrin gene, the inhibition rate of germination rate by PEG stress decreased to 8.82%. This indicates that overexpression of the NtDehydrin gene can improve the tolerance of tobacco to PEG stress.

[0075] Using T2 generation seeds as experimental material and water as a control, the WT (weight and gluten content) was observed after 14 days of treatment with 15% PEG. NtDehydrin Germination status of -OE strains, such as Figure 9 As shown. By Figure 9 It can be seen that overexpression NtDehydrin Genes can enhance tobacco's tolerance to PEG stress during germination.

[0076] Example 5: NtDehydrin The effect of tobacco gene overexpression on Fusarium resistance

[0077] The pathogen causing tobacco Fusarium root rot (Fusarium, Fusarium oxysporum The pathogen was provided by the Plant Protection Department of the Tobacco Research Institute, Henan Academy of Agricultural Sciences. The pathogen was first cultured on oat medium. After the mycelium had grown to cover the entire plate, circular mycelial discs with a diameter of 5 mm were punched into the culture medium using a perforator and inoculated into liquid medium. After shaking and incubation for 3 days (180 rpm, 28℃), the mixture was filtered through four layers of sterile gauze, centrifuged at 5000 rpm, and the spores were collected. The spores were resuspended in sterile water to prepare a spore suspension, and the concentration was adjusted to 1×10⁻⁶. 7 -10 8 Cells / ml, used for inoculation.

[0078] Strong seedlings with 5-6 true leaves were selected, and six biological replicates were set up. The pathogen was inoculated using the spore solution drenching method. Each seedling was drenched with 25 ml of spore solution. After inoculation, the seedling condition and disease incidence were observed daily. On the 7th day after inoculation, the disease severity of the seedlings was investigated according to the national standard GB / T 23222─2008, and the incidence rate, disease index, and disease status were recorded by photograph.

[0079] The diameters of the infected spots are shown in Table 3:

[0080]

[0081] The photos of the infected spots are shown in Figure 2, and the diameters of the infected spots are shown in Table 3: Figure 10 Figure 10 It can be seen from Table 3 that the diameters of the infected spots on the leaves of the different transgenic lines are significantly smaller than those of the control, indicating that the disease resistance of the tobacco lines overexpressing the Dehydrin gene is better than that of the control.

[0082] The results on the 14th day after inoculation are shown in Table 4: Figure 11 Figure 11 It can be seen from Table 4 that the leaves of the control are severely infected by the pathogen, and all of them are wilted and necrotic, while the areas of the infected spots on the leaves of the transgenic lines (overexpressing Dehydrin) are small, and the disease is very light.

[0083] The above merely describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​​

Claims

1. The application of the NtDehydrin1 gene in improving the resistance of tobacco to diseases caused by Fusarium, characterized by: The application enhances the disease resistance of tobacco by overexpressing the NtDehydrin1 gene; The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No.

1.

2. The application of the NtDehydrin1 gene overexpression vector in improving tobacco resistance to Fusarium diseases, characterized by: The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No.

1.

3. A method for cultivating tobacco resistant to diseases caused by Fusarium, characterized in that, The steps are as follows: (1) Using tobacco cDNA as a template and NtDehydrin1 gene as the target gene, an overexpression vector was constructed; (2) The overexpression vector in step (1) is transformed into tobacco plants by Agrobacterium to obtain tobacco plants resistant to Fusarium diseases. The nucleotide sequence of the NtDehydrin1 gene is shown in SEQ ID No. 1.

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