Method for promoting stress resistance of tobacco

Abstract

The invention discloses a method for promoting the stress resistance of tobacco, which is characterized in that an NtDREB2C gene nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence with the same function as the SEQ ID NO.1 is over-expressed in tobacco, so that the phenotype, growth and physiological and biochemical indexes of plants can be obviously improved, the seedling height, the fresh weight, the chlorophyll relative content, the root activity, the catalase activity and the peroxidase activity are remarkably improved compared with those of a wild type, and are particularly prominent under low-temperature and low-oxygen stress. The over-expression vector constructed by the invention is used for transforming a receptor plant to obtain a transgenic plant, so that the stress resistance of tobacco can be remarkably improved, and the over-expression vector has important significance on tobacco variety breeding, particularly lays an important technical foundation for floating seedling stable production of tobacco under low-temperature and low-oxygen stress, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and to a method for promoting the stress resistance of tobacco. Background Technology

[0002] tobacco( Nicotiana tabacum L. Tobacco (Nicotiana spp.), an annual herbaceous plant belonging to the genus Nicotiana in the Solanaceae family, is an important economic crop and a crucial model plant for research in plant physiology and molecular biology. Tobacco is widely cultivated in my country, and floating seedling raising is currently one of the main methods of tobacco seedling cultivation. However, during floating seedling raising, tobacco plants frequently suffer from environmental stresses such as low temperature and low oxygen, which severely affects the yield and quality of tobacco leaves.

[0003] Tobacco is a warm-season crop and is sensitive to temperature. Low temperature stress can lead to stunted emergence, poor seedling development, reduced leaf number, yellowing and wilting, and in severe cases, seedling death. In addition, during floating seedling cultivation, the roots are immersed in nutrient solution for a long time, and the high density of seedlings and high oxygen demand can lead to hypoxia stress, which can easily cause root rot, affect the absorption of water and nutrients, and thus reduce the quality of seedling cultivation.

[0004] DREB (dehydration responsive element binding protein) transcription factor, belonging to the AP2 / EREBP family of transcription factors, contains a conserved AP2 domain and can specifically bind to the DRE / CRT (dehydration responsive element / C-repeat) cis-regulatory element. It has been widely reported in plant responses to abiotic stresses. Previous studies have reported… DREB Overexpression of genes can improve plant phenotype, activate the expression of stress-related genes and metabolites, increase the content of osmotic regulators, enhance antioxidant enzyme activity and water use efficiency, thereby improving plant tolerance to environmental stress.

[0005] However, currently regarding tobacco DREB2C No existing technologies have reported on gene function studies, particularly those concerning their ability to significantly enhance tobacco's stress resistance and stabilize floating seedling production under low-temperature and low-oxygen stress conditions. Therefore, research using modern biotechnology to study tobacco... DREB2C The potential link between genes and their resistance to abiotic stresses is of great significance and practical value for enhancing the stress resistance of tobacco seedlings and ensuring yield and quality traits in tobacco production. Summary of the Invention

[0006] The present invention provides a first aspect NtDREB2C The use of genes or related biological materials in at least one of the following: A1) Regulating the stress resistance of tobacco or preparing products that regulate the stress resistance of tobacco; A2) Cultivate tobacco with improved stress resistance or prepare products that improve the stress resistance of tobacco; A3) Preparation of genetically modified tobacco; The NtDREB2C The coding sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the biomaterial includes one or more of the following: B1) NtDREB2C The gene-encoded protein and / or containing the above NtDREB2C Recombinant vectors of genes, recombinant microorganisms, or transgenic tobacco cell lines; B2) contains negative regulation NtDREB2C Nucleic acid molecules expressing genes, recombinant vectors, recombinant microorganisms, or transgenic tobacco cell lines.

[0008] Furthermore, the regulation of tobacco's stress resistance includes: regulating tobacco's resistance to low temperature and low oxygen.

[0009] Furthermore, the regulation of tobacco's resistance to low temperature and low oxygen includes: through positive regulation NtDREB2C Gene expression is used to enhance the stress resistance of tobacco; Or, through negative regulation NtDREB2C Gene expression reduces the stress resistance of tobacco.

[0010] Furthermore, the aforementioned NtDREB2C The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0011] Furthermore, the containing NtDREB2C Gene recombination vectors include basic vectors and NtDREB2C The basic carrier includes the pBWA(V)HS carrier.

[0012] Furthermore, the containing NtDREB2C Recombinant microorganisms include Agrobacterium GV3101.

[0013] In a second aspect, the present invention provides a positive regulation NtDREB2C Application of gene-expressing biomaterials in the cultivation of tobacco with enhanced stress resistance: NtDREB2C The coding sequence of the gene is shown in SEQ ID NO.1.

[0014] Further, the biomaterial includes: NtDREB2C The gene encodes a protein and / or contains positive regulatory functions. NtDREB2C Recombinant vectors for gene expression, recombinant microorganisms, or transgenic tobacco cell lines.

[0015] Furthermore, the improvement in stress resistance includes: enhancing the resistance of tobacco to low temperature and low oxygen.

[0016] In a third aspect, the present invention provides a method for improving the stress resistance of tobacco, comprising: Positive regulation NtDREB2C Gene-expressing biological materials are transferred into tobacco.

[0017] Furthermore, the aforementioned positive regulation NtDREB2C One method for transferring gene-expressing biological materials into tobacco is through Agrobacterium-mediated transformation.

[0018] In a fourth aspect, the present invention provides a method for cultivating tobacco with enhanced stress resistance, comprising: Positive regulation NtDREB2C Gene-expressing biological materials are transferred into tobacco and cultivated.

[0019] Furthermore, the aforementioned positive regulation NtDREB2C One method for transferring gene-expressing biological materials into tobacco is through Agrobacterium-mediated transformation.

[0020] Furthermore, the aforementioned NtDREB2C The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0021] Furthermore, the containing NtDREB2C Gene recombination vectors include basic vectors and NtDREB2C The basic carrier includes the pBWA(V)HS carrier.

[0022] Furthermore, the containing NtDREB2C Recombinant microorganisms include Agrobacterium GV3101.

[0023] Furthermore, the tobacco variety is 'K326'.

[0024] Furthermore, the treatment temperature for the low-temperature stress is 4~6℃, and the dissolved oxygen concentration in the low-oxygen stress environment is 1.0mg / L~2.0mg / L.

[0025] Furthermore, the improved growth phenotype of tobacco seedlings under the combined stress of low temperature and low oxygen includes: increased leaf size and increased root length.

[0026] Furthermore, the physiological and biochemical indicators of tobacco seedlings under the combined stress of low temperature and low oxygen are improved, including: increased aboveground seedling height, seedling fresh weight, relative chlorophyll content, root activity, CAT enzyme activity, and POD enzyme activity.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes tobacco cloning NtDREB2CGenes were then genetically transformed in the main tobacco varieties and overexpressed using genetic engineering techniques. NtDREB2C This gene can significantly promote the resistance of tobacco crops to combined low temperature and low oxygen stress, thus helping to improve their stress resistance. Experiments have shown that the method of this invention can improve the growth phenotype of tobacco seedlings under combined low temperature and low oxygen stress, and enhance physiological and biochemical indicators, including: increased aboveground seedling height, seedling fresh weight, relative chlorophyll content, root activity, CAT enzyme activity, and POD enzyme activity, indicating... NtDREB2C Genes have certain application potential in promoting tobacco plants' resistance to combined stresses of low temperature and low oxygen. Attached Figure Description

[0028] Figure 1 for NtDREB2C Map of gene overexpression vectors.

[0029] Figure 2 The expression levels of the positive overexpression material in wild-type and overexpression lines of tobacco 'K326' are shown; where WT represents wild-type. NtDREB2C OE-7 and NtDREB2C OE-9 indicates NtDREB2C Two strains of the overexpressing plant.

[0030] Figure 3 for NtDREB2C Phenotype of tobacco seedlings after low temperature and low oxygen treatment (overexpression).

[0031] Figure 4 Wild-type plants and NtDREB2C OE-7 and NtDREB2C The aboveground height of seedlings of the two OE-9 strains after low temperature and low oxygen treatment.

[0032] Figure 5 Wild-type plants and NtDREB2C OE-7 and NtDREB2C Fresh weight of seedlings of the two OE-9 strains after low temperature and low oxygen treatment.

[0033] Figure 6 Wild-type plants and NtDREB2C OE-7 and NtDREB2C The relative chlorophyll content of seedling leaves of two OE-9 strains after low temperature and low oxygen treatment.

[0034] Figure 7 Wild-type plants and NtDREB2C OE-7 and NtDREB2C Root vigor of seedlings of the two OE-9 strains after low temperature and low oxygen treatment.

[0035] Figure 8 Wild-type plants and NtDREB2C OE-7 and NtDREB2CCAT activity in the leaves of seedlings from two OE-9 strains after low-temperature and low-oxygen treatment.

[0036] Figure 9 Wild-type plants and NtDREB2C OE-7 and NtDREB2C POD activity in the leaves of seedlings of the two OE-9 strains after low temperature and low oxygen treatment. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0038] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0040] The present invention described NtDREB2C The coding sequence of the gene is shown in SEQ ID NO.1; NtDREB2C The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0041] SEQ ID NO.1:

[0042] SEQ ID NO.2: MPSDYSERNQKMAILDQAPKMTSLPLDCSRKRKSRSRRDGTKNVEETLAKWKEYNQKLDCVDDEGKTVRKVPAKGSKKGCMKGKGGPENSRCNYRGVRQRTWGKWVAEIREPNRGSRLWLGTFGTAIEAALAYDEAARAMYGPSARLNLPNYPSSKESSKDDSSWATTSASDSTAGSSLSEVCPA AEQKGISEIKFEDGEGESRIDGVTTAIHEVSTPLTSEKHEAKSKMGVVEAKEEPRSIESINQDMLKSGRDYLDNLNWDELFDVEELLGMLDSIPAGAPAFMQDFGSIAGQKEQYDAYNNNQLSNSSFQHQNADLKLLGGTQQMEQQAPIAVDYGFDFLKPGREEDLNFSLDDLALMDLDSELGV.

[0043] Example 1: Tobacco pBWA(V)HS-osgfp- NtDREB2C Construction of overexpression vectors 1. Design primers for target gene amplification The primer sequences are as follows: Forward primer: cattGGTCTCactacatgccttctgattattctgagagaaatcaaaagatgg (SEQ ID NO.3); Reverse primer: cattGGTCTCatacattagaccccaagttcagagtccaaatcc (SEQ ID NO.4); Target gene sequencing primers: ctgttgtcacgccatcaattct (SEQ ID NO.5).

[0044] 2. PCR system and procedure Table 1 PCR system

[0045] Table 2 PCR Procedure

[0046] Electrophoresis on a 1% agarose gel for 20 minutes under UV light. NtDREB2CThe 1110 bp electrophoretic fragment was excised and the DNA was dissolved and recovered according to the kit instructions from Wuhan Boyuan Biotechnology Co., Ltd. The vector was digested with restriction endonucleases, and the digested products and recovered fragments were purified using a PCR purification kit. After recombination, the ligation product was transformed into competent cells. Positive clones were selected for colony PCR identification, and positive bands were sequenced. After successful sequencing, Agrobacterium was transformed, and colonies were selected for PCR identification, followed by genetic transformation. The overexpression vector map is shown below. Figure 1 As shown.

[0047] Example 2 NtDREB2C Obtaining homozygous tobacco plants with gene overexpression 1. Preparation of Agrobacterium 1.1 Plasmid transformation: Take 1 μL of plasmid, add 50 μL of GV3101 Agrobacterium competent cells, mix thoroughly, electroporate, add 1000 μL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Shake at 30℃ and 180 rpm for 30 min. Take 50 μL of the activated Agrobacterium culture and inoculate it onto LB solid medium. Incubate in the dark at 30℃ for 2 days.

[0048] 1.2 Agrobacterium detection Table 3 PCR amplification system

[0049] Gel electrophoresis was performed using 1% agarose gel. The electrophoretic bands of the positive control and the sample were clear and of the correct size, while the negative control showed no bands. Genetic transformation was then performed.

[0050] 2. Genetic transformation of tobacco Sterilized tobacco seeds were pre-cultured on germination medium. After 2-3 days of pre-culture, tobacco leaves were inoculated with Agrobacterium suspension for 10-15 minutes. The inoculated tobacco leaves were then inoculated onto filter paper, dried, and inoculated onto a co-culture medium for 2 days in the dark. The leaves were then transferred to induction medium for about 10 days to induce callus growth. Callus tissue meeting the criteria was selected and inoculated onto the corresponding resistance selection medium. Vigorous positive callus tissue from the second screening was inoculated onto differentiation medium to await differentiation and rooting.

[0051] 3. Obtaining gene-edited homozygous plants Leaves were selected from T0 generation transgenic tobacco seedlings after approximately 25 days of floating seedling cultivation, when they reached the two-leaf-one-heart stage. DNA was extracted using the CTAB method, with primer sequences shown below, and RT-qPCR was performed. Results are as follows... Figure 2 As shown, the results revealed that NtDREB2C OE-7 and NtDREB2CThe expression levels of OE-9 were significantly higher than those of WT.

[0052] Forward primer: 5'-GGTGAATCAAGAATTGATGGCG-3' (SEQ ID NO.6); Reverse primer: 5'-ATCCCGTCCAGACTTGAGC-3' (SEQ ID NO.7).

[0053] Example 3 NtDREB2C Low-temperature and low-oxygen treatment and determination of physiological and biochemical indicators of overexpressed tobacco seedlings 1. Material preparation Wild-type and overexpression strains of tobacco 'K326' were collected from tobacco seeds and floating seedling nutrient solution.

[0054] 2. Seed treatment Select plump and uniform tobacco seeds, disinfect them with NaClO, rinse them three times with distilled water, and then blot the water off with filter paper to obtain the disinfected seeds.

[0055] 3. Experimental material cultivation Sterilized tobacco seeds were placed in glass petri dishes with three layers of filter paper and germinated in a 25℃ light incubator (16h light / 8h darkness). After 7 days, seedlings of uniform size and growth were selected and transferred to hydroponic boxes for floating seedling cultivation. When the seedlings reached the 2-leaf-1-heart stage, the tobacco seedlings were subjected to low-temperature and low-oxygen combined stress treatment.

[0056] 4. Test treatment 4.1 Preparation of floating seedling nutrient solution: Prepare 50ppm floating seedling nutrient solution, with substrate provided by Yunnan Yuxi Zhongyan Seed Co., Ltd.; 4.2 Low temperature and low oxygen treatment method: When tobacco seedlings are at the 2-leaf and 1-heart stage, add anhydrous sodium sulfite to the nutrient solution until the dissolved oxygen concentration is below 2.0 mg / L. At the same time, place the hydroponic box in a 5℃ light incubator for 3 days.

[0057] 5. Measurement of various indicators After 3 days of low-temperature and low-oxygen treatment, tobacco seedlings were sampled. Aboveground seedling height was measured with a ruler, fresh weight was measured using a 0.01% balance, relative chlorophyll content in leaves was measured using a portable SPAD analyzer, and root activity was determined using the TTC (Triphenyltetrazolium chloride) method. Fresh leaves were collected for the determination of catalase (CAT) and peroxidase (POD) activities, with three biological replicates.

[0058] The results are as follows Figure 3As shown, after 3 days of low temperature and low oxygen treatment, the tobacco seedlings of the overexpressing transgenic lines grew better than those of the wild type (WT), with larger leaves and longer roots than those of the WT.

[0059] The results are as follows Figures 4 - 9 As shown, the aboveground height, fresh weight, relative chlorophyll content in leaves, root activity, and CAT and POD activities of tobacco seedlings overexpressing transgenic lines were significantly higher than those of WT, indicating that... NtDREB2C Overexpression of the gene enhances the stress resistance of tobacco.

[0060] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. NtDREB2C Use of a gene or biological material related thereto in at least one of the following: A1) modulating stress tolerance of tobacco or preparing a product for modulating stress tolerance of tobacco; A2) breeding a tobacco with improved stress tolerance or preparing a product for improving stress tolerance of tobacco; A3) preparing a transgenic tobacco; The NtDREB2C The coding sequence of the gene is shown in SEQ ID NO.

1.

2. Use according to claim 1, wherein The biological material comprises one or more of the following: B1 ) the NtDREB2C gene encoding a protein and / or a recombinant vector, a recombinant microorganism or a transgenic tobacco cell line containing the NtDREB2C gene; B2) nucleic acid molecules, recombinant vectors, recombinant microorganisms or transgenic tobacco cell lines comprising a negative regulator NtDREB2C of gene expression.

3. Use according to claim 1 or 2, characterized in that, The modulating stress tolerance of tobacco comprises modulating cold and low oxygen tolerance of tobacco.

4. The use according to claim 3, wherein the compound is ###0002### The regulation of the low temperature and low oxygen resistance of tobacco comprises: improving the stress resistance of tobacco by positively regulating NtDREB2C the expression of the gene; or, by down-regulating NtDREB2C the expression of the gene to reduce the stress tolerance of the tobacco.

5. Upregulation NtDREB2C Application of biomaterials for gene expression in the cultivation of tobacco with improved stress tolerance: the NtDREB2C The coding sequence of the gene is shown in SEQ ID NO.

1.

6. The use according to claim 5, wherein the compound is ###0002### The biological material comprises: the NtDREB2C gene encoding a protein and / or a recombinant vector, a recombinant microorganism or a transgenic tobacco cell line containing a positively regulated NtDREB2C gene expression.

7. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The improved stress tolerance comprises improving cold and low oxygen tolerance of tobacco.

8. A method for increasing stress tolerance in tobacco, characterized by, Comprising: upregulating NtDREB2C biomaterials that modulate gene expression were introduced into tobacco.

9. A method for breeding tobacco with improved stress tolerance, characterized by, Comprising: upregulating NtDREB2C biomaterials that modulate gene expression were introduced into tobacco.

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