Application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance, and in the improvement of tobacco germplasm resources

Abstract

This invention discloses the application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance, and in improving tobacco germplasm resources. It belongs to the field of plant genetic engineering technology. The nucleotide sequence of the coding region of the tobacco NtGBSS2 gene is shown in SEQ ID NO.1. This invention overexpressed the NtGBSS2 gene in tobacco. Detection revealed that, compared to normal control tobacco plants, the content of resistant starch and amylose in the transgenic positive T1 generation was significantly increased. Furthermore, observation showed that wild-type control tobacco plants grew slowly and had yellowing leaves under drought stress, while the growth status of transgenic positive T2 generation tobacco plants was significantly improved compared to wild-type control tobacco plants, indicating that overexpression of the NtGBSS2 gene in tobacco enhances the tobacco's tolerance to drought stress.

Description

Technical Field

[0001] This invention relates to the application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance, and in the improvement of tobacco germplasm resources, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Tobacco is one of my country's important economic crops, and its production efficiency largely depends on the quality of the tobacco leaves. Starch is an important basic organic compound in tobacco leaves, generally containing about 25-40% in fresh tobacco leaves. It undergoes significant changes during the curing process; the degradation, transformation, consumption, and accumulation of carbohydrates determine the intrinsic quality and commercial grade of the tobacco leaves. If the starch content in tobacco leaves is too high, it will negatively affect the color, aroma, and flavor; if the starch content is too low, it will negatively impact the total sugar and reducing sugar content of the cured tobacco leaves. Therefore, the starch content in tobacco leaves is closely related to their quality.

[0003] Resistant starch, also known as resistant starch or indigestible starch, is affected by many factors, such as temperature and moisture. Generally, the content of resistant starch is positively correlated with the ratio of amylose to amylopectin; the higher the ratio, the higher the resistant starch content. Amylose molecules, especially those with lower molecular weights, can form a well-ordered arrangement and may be the main component forming the crystalline regions of resistant starch granules. Changes in starch accumulation and structure are often caused by the interaction and differences of starch synthases. Existing research has shown that genetic regulation is an important factor affecting the content of resistant starch. Starch synthesis involves ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE), starch isomerase (ISA), and granule-bound starch synthase (GBSS). Currently, there is limited research on the functions of various enzymes in the tobacco starch synthesis pathway.

[0004] On the other hand, global warming, desertification, and other climate problems have led to a decrease in soil water retention capacity and moisture content, resulting in frequent extreme heat waves and droughts in major tobacco-producing areas. Tobacco has a high water requirement throughout its entire growth cycle, with different requirements at different stages. When field water holding capacity is less than 50%, it affects tobacco growth and development, severely impacting leaf yield and quality. Unlike corn, rice, and wheat, which are primarily studied for their seeds and fruits, tobacco is mainly harvested from its leaves, making it an important economic and commercial crop. Therefore, research on the resistant starch content in tobacco leaves and the genetic regulation genes for drought resistance in tobacco is of great significance for improving tobacco quality and producing high-quality tobacco leaves. Summary of the Invention

[0005] The first objective of this invention is to provide the application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance, in order to address the problem of unknown function of granule-bound starch synthase in the existing tobacco starch synthesis pathway.

[0006] The second objective of this invention is to provide the application of the tobacco NtGBSS2 gene in the improvement of tobacco germplasm resources, so as to solve the problem of poor drought resistance of tobacco in the prior art.

[0007] To achieve the above objectives, the technical solution adopted in this invention for the application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance is as follows:

[0008] The application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance, wherein the nucleotide sequence of the coding region of the tobacco NtGBSS2 gene is shown in SEQ ID NO.1.

[0009] The beneficial effects of the above technical solution are as follows: The application of the tobacco NtGBSS2 gene in regulating starch synthesis and / or drought resistance is a pioneering invention. This invention clones the tobacco NtGBSS2 gene from tobacco seedlings, with a coding sequence (CDS) length of 1842 bp encoding 614 amino acids. Real-time quantitative PCR analysis shows that the NtGBSS2 gene is most highly expressed in tobacco leaves, especially young leaves. Further, a recombinant expression vector was constructed and transformed into tobacco to overexpress the NtGBSS2 gene. Detection revealed that, compared to normal control tobacco plants, the content of resistant starch and amylose in the transgenic positive T1 generation was significantly increased. Furthermore, observation showed that wild-type control tobacco plants grew slowly and had yellowing leaves under drought stress, while the growth status of transgenic positive T2 generation tobacco plants was significantly improved compared to wild-type control tobacco plants, indicating that overexpression of the NtGBSS2 gene in tobacco enhances the tobacco's tolerance to drought stress.

[0010] As a further improvement, the NtGBSS2 gene was overexpressed in tobacco using genetic engineering techniques to increase the content of resistant starch and amylose in tobacco, thereby enhancing the tobacco's tolerance to drought stress.

[0011] Specifically, the tobacco NtGBSS2 gene was transferred into plants using Agrobacterium-mediated transformation to obtain plants with overexpression of the tobacco NtGBSS2 gene. More specifically, the tobacco NtGBSS2 gene was constructed into a plant expression vector, transformed into Agrobacterium, and then transformed into tobacco to obtain transgenic tobacco plants with stable genetic inheritance.

[0012] As a further improvement, the overexpression of the NtGBSS2 gene is achieved by constructing a recombinant expression vector and transforming it into tobacco to overexpress the NtGBSS2 gene.

[0013] As a further improvement, the recombinant expression vector is constructed by ligating the cloned NtGBSS2 gene into the PCAMBIA1300-GFP vector and then sequencing it for identification.

[0014] To achieve the above objectives, the technical solution adopted in this invention for the application of the tobacco NtGBSS2 gene in tobacco germplasm resource improvement is as follows:

[0015] Application of the tobacco NtGBSS2 gene in the improvement of tobacco germplasm resources, wherein the coding region nucleotide sequence of the tobacco NtGBSS2 gene is as shown in SEQ ID NO.1.

[0016] The beneficial effects of the above technical solution are as follows: The application of the tobacco NtGBSS2 gene in the improvement of tobacco germplasm resources is a pioneering invention. Through observation, this invention has found that wild-type control tobacco plants grow slowly and their leaves turn yellow under drought stress, while transgenic positive T2 generation tobacco plants show a significant improvement in growth compared to wild-type control plants. This indicates that overexpression of the NtGBSS2 gene in tobacco enhances its tolerance to drought stress. Utilizing genetic engineering techniques to genetically regulate tobacco has improved tobacco germplasm resources, significantly increasing the efficiency of genetic improvement breeding compared to traditional methods. This invention has found that overexpression of the NtGBSS2 gene in tobacco plants can increase their drought resistance, which is of great significance for tobacco quality improvement and the production of high-quality tobacco leaves.

[0017] As a further improvement, the tobacco germplasm resources are improved to enhance the tolerance of tobacco to drought stress. Attached Figure Description

[0018] Figure 1 This refers to the expression level of the NtGBSS2 gene in different tissues in Example 2 of the present invention.

[0019] Figure 2 To identify the expression level of NtGBSS2 gene overexpressing tobacco plants in Example 5 of this invention (where wt is a normal wild control tobacco plant, and oelines1-3 are three individuals of NtGBSS2 gene overexpressing tobacco plants; * represents P<0.05, ** represents P<0.01);

[0020] Figure 3 The starch content of NtGBSS2 gene-overexpressing tobacco plants was detected in Example 6 of this invention (where wt represents normal wild-type tobacco plants, and eoelines1-3 represent three individuals of NtGBSS2 gene-overexpressing tobacco plants; ** represents P<0.01).

[0021] Figure 4 The content of amylose in tobacco plants overexpressing the NtGBSS2 gene was detected in Example 6 of this invention (where wt represents normal wild-type tobacco plants, and eoelines1-3 represent three individuals of tobacco plants overexpressing the NtGBSS2 gene; * represents P<0.05).

[0022] Figure 5 This invention provides an example of identifying the drought resistance of tobacco plants overexpressing the NtGBSS2 gene in Example 7 of this invention (where CK is the normal control group, PEG is the drought stress group; WT represents wild tobacco plants, and OE represents tobacco plants overexpressing the NtGBSS2 gene). Detailed Implementation

[0023] Granule-bound starch synthases are involved in the synthesis of amylose. There are two main types, both responsible for extending glucan chains. GBSS1 genes primarily control amylose synthesis in storage organs such as seeds, embryos, and endosperm, while GBSSII genes primarily control amylose synthesis in vegetative organs such as roots, stems, and leaves. To further investigate the function of granule-bound starch synthases in tobacco, this invention cloned a tobacco granule-bound starch synthase encoding gene, NtGBSS2. Real-time quantitative PCR analysis showed that the NtGBSS2 gene was expressed at the highest level in tobacco leaves, especially young leaves. A recombinant expression vector was further constructed and transformed into tobacco to overexpress the NtGBSS2 gene. Detection revealed that, compared to normal control tobacco plants, the transgenic positive T1 generation showed significantly increased resistant starch and amylose content. Furthermore, observations revealed that wild-type control tobacco plants grew slowly and had yellowing leaves under drought stress, while transgenic positive T2 generation tobacco plants showed significantly improved growth compared to wild-type control plants, indicating that overexpression of the NtGBSS2 gene in tobacco enhances its tolerance to drought stress.

[0024] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0025] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0026] Specific embodiments of the application of the tobacco NtGBSS2 gene of the present invention in regulating starch synthesis and / or drought resistance and tobacco germplasm resource improvement:

[0027] Example 1: Cloning of the tobacco NtGBSS2 gene

[0028] In this embodiment, the NtGBSS2 gene was cloned from tobacco seedling leaf tissue. The specific implementation steps are as follows:

[0029] Leaf tissue samples were taken from young Tobacco safflower seedlings and immediately flash-frozen in liquid nitrogen, then stored at -80°C. The leaf tissues were thoroughly ground in liquid nitrogen, and total RNA was extracted using a plant RNA extraction kit (Imagene, RE120-02), strictly following the kit instructions. During extraction, DNase I (Omega, #E1091-01) was used to digest and remove genomic DNA. After extraction, RNA quality was assessed using agarose gel electrophoresis, and RNA concentration was determined using Nanodrop. Samples with good quality and concentration were selected for cDNA first-strand synthesis and reverse transcription using PrimeScript reagent. TM II. 1st Strand cDNA Synthesis Kit (TAKARA, 6210A): Follow the kit instructions to obtain cDNA samples, determine sample concentration, and use for subsequent gene cloning.

[0030] The Ntab0773790 gene (i.e., the tobacco NtGBSS2 gene) was retrieved using the China Tobacco Genome Database V4.0, and its full-length CDS sequence was obtained. Based on the sequence in the database, specific amplification primers containing restriction enzyme sites were designed:

[0031] NtGBSS2-1300-F:5'-ATACACCAAATCGACTCTAGATGGCAAGCATCACAGCTTCAC-3' (shown in SEQID NO.3);

[0032] NtGBSS2-1300-R: 5'-GCCCTTGCTCACCATGGTACCTTAGGGAGCGGCCACATTTTCC-3' (shown in SEQ ID NO. 4).

[0033] Using tobacco leaf cDNA as a template, PCR amplification was performed using a high-fidelity PCR enzyme (KFX-101). The PCR reaction system consisted of: 2 μL DNA template, 1.0 μL each of forward and reverse primers, 1 μL KOD enzyme, 25 μL 2×KOD buffer, 5 μL dNTPs, and 15 μL ddH2O. The amplification program was as follows: 98℃ for 5 min; 98℃ for 30 s, 58℃ for 30 s, 68℃ for 2 min, 35 cycles; 68℃ for 10 min. All the amplified PCR products were spotted into the wells of a 1% agarose gel and electrophoresed at 130V for 30 min. The target band was cut under UV light and placed in a 1.5 mL centrifuge tube. The target fragment was recovered using the AxyPrep DNA Gel Recovery Kit (Axygen, AP-GX-250G), and the recovery results were detected by electrophoresis again.

[0034] In this embodiment, the NtGBSS2 gene was cloned from tobacco seedling leaf tissue. The gene is 3040 bp in length and includes 12 introns. Its coding region is 1845 bp in length and encodes a total of 614 amino acids. The coding sequence of the NtGBSS2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0035] Example 2: Analysis of the expression pattern of the tobacco NtGBSS2 gene

[0036] This embodiment analyzes the expression of the NtGBSS2 gene, and the specific implementation steps are as follows:

[0037] Tissue samples from tobacco plants at different growth stages, including leaves, stems, buds, roots, ovaries, and leaf veins, were collected. All samples were immediately flash-frozen in liquid nitrogen after collection and then stored at -80°C. Total RNA was extracted from each tissue using a plant RNA rapid extraction kit (Imagene, RE120-02) and analyzed using PrimeScript. TM II. The 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) was used to obtain cDNA samples from various tissues. After the concentration of the cDNA samples was determined by Nanodrop, they were used for qPCR to quantitatively detect the expression level of the NtGBSS2 gene.

[0038] The primer sequences for real-time quantitative PCR of the NtGBSS2 gene are as follows:

[0039] GBSS2-F:5'-CCCTCGCGTTTGCTGAGA-3' (shown in SEQ ID NO.5);

[0040] GBSS2-R:5'-CCCCTTCAACACCGGCTT-3' (shown as SEQ ID NO.6).

[0041] NtGAPDH was selected as the internal reference gene for real-time quantitative PCR, and the primer sequences are as follows:

[0042] GAPDH-F: 5'-TGGGTGTCAACGAGAAGGAA-3' (shown in SEQ ID NO. 7);

[0043] GAPDH-R: 5'-TCTGGGTGGCAGTAAGGGA-3' (shown in SEQ ID NO. 8).

[0044] The real-time quantitative PCR reaction system is: 5 μL 2×SYBR I Master, upstream and downstream...

[0045] Add 0.5 μL of each primer and 50 ng of cDNA to a final volume of 10 μL using ddH2O.

[0046] Reaction conditions: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 40 cycles.

[0047] After the reaction is complete, based on the cycle threshold (CT value), use 2- ΔΔCT The method calculates the relative expression level of genes. The detection results are as follows: Figure 1 As shown, the expression level of the tobacco NtGBSS2 gene is highest in leaves, especially young leaves, followed by the ovary, while the expression level is not high in other tissues.

[0048] Example 3 Construction of recombinant expression vector

[0049] To further demonstrate the function of the NtGBSS2 gene, this embodiment uses the NtGBSS2 gene fragment obtained in the previous embodiment to construct a recombinant expression vector for the NtGBSS2 gene. The specific implementation steps are as follows:

[0050] The PCAMBIA1301 plasmid was digested with XbaⅠ and KpnⅠ and incubated at 37℃ for 3 h. The linearized PCAMBIA1300-GFP plasmid was recovered by agarose gel electrophoresis. The following ligation mixture was prepared in a microcentrifuge tube: 1 μL PCAMBIA1300-GFP, 0.1–0.3 pmol DNA fragment, 1 μL MultiS, 2 μL 5×CE buffer (Vazyme, C113-01), and up to 10 μL ddH2O. Ligation was carried out at 37℃ for 30 min. The entire 10 μL ligation product was added to competent *E. coli* cells, heat-shocked, and positive clones were selected for sequencing. Plasmids were extracted from correctly sequenced positive clones. After PCR identification, the plasmid was transformed into competent *Agrobacterium* cells for subsequent tobacco transgenic experiments.

[0051] Example 4 Tobacco Conversion

[0052] In this embodiment, the recombinant expression vector of the NtGBSS2 gene constructed in Example 3 was transformed into tobacco using the leaf disc method to construct NtGBSS2 gene overexpression plants. The specific implementation steps are as follows:

[0053] Disinfect the leaves of the vigorous red-flowered Da Jin Yuan tobacco plant and cut them into 1cm pieces. 2 Small pieces were placed in MS differentiation medium and pre-cultured for 2 days at 28°C, light intensity of 2000 Lx, and light duration of 16 h / d. Then, they were inoculated into Agrobacterium bacterial suspension containing the target plasmid (the recombinant expression vector constructed in Example 3) for 10-15 min, with the suspension shaken several times during this period. Excess bacterial suspension was then blotted dry with sterile filter paper. The inoculated plants were then transferred to MS differentiation medium and co-cultured at 28°C in the dark for 3-5 days. The co-cultured plants were washed three times with sterile water, blotted dry with sterile paper, and transferred to MS differentiation medium containing hygromycin and carbenicillin for constant temperature culture. The medium was changed every 10 days. When the adventitious shoots reached 1-2 cm in length, the clustered adventitious shoots were cut into individual shoots and transferred to MS rooting medium containing hygromycin, carbenicillin, and activated carbon to promote rooting. Once the root system has developed well, remove the tissue culture seedlings, wash the culture medium off the roots with clean water, cut off a small number of lower leaves, transfer them to flowerpots filled with loose, sterile soil, and cultivate them according to conventional management methods.

[0054] Example 5: Identification and Expression Level Detection of Transgenic Plants

[0055] This embodiment identifies the tobacco plants transformed in Example 4 and detects the NtGBSS2 gene expression level in the successfully transformed tobacco plants. The specific implementation steps are as follows:

[0056] When the overexpressing transgenic seedlings grew to 8 true leaves, DNA was extracted from the leaves of the T0 generation plants and verified by PCR to identify positive transgenic plants. The T0 generation positive plants were planted and seeds were harvested. These were then planted again to obtain the T1 generation plants. Using the genomic DNA of the T1 generation plants as a template, PCR amplification was performed using the vector-specific upstream primer NtGBSS2-JC-F+ and the gene-specific downstream primer NtGBSS2-JC-R. Plants that amplified a specific single band were identified as overexpressing positive plants.

[0057] NtGBSS2-JC-F:5'-ACCAATACATACACTAGCATC-3' (as shown in SEQ ID NO.9);

[0058] NtGBSS2-JC-R:5'-CGCTAGTATCCCAAGCATCT-3' (shown in SEQ ID NO.10).

[0059] RNA was further extracted from the overexpression positive plants, and the expression level of the NtGBSS2 gene was detected by real-time PCR. The specific operation of real-time PCR is as described in Example 2.

[0060] Test results as follows Figure 2 As shown in the figure, the expression level of the NtGBSS2 gene is significantly increased in the overexpression positive T1 generation plants.

[0061] Example 6: Detection of starch content

[0062] This embodiment detects the starch content in the positive T1 generation overexpression plants from Example 5. The specific implementation procedure is as follows:

[0063] 1. Determination of resistant starch content

[0064] The resistant starch content was determined by enzymatic method using the Suzhou Greens Resistant Starch Content Enzymatic Assay Kit (catalog number: G0571W48). Sample pretreatment was as follows: 2g of sample was dried (50℃) to constant weight, ground, and passed through a 0.5mm sieve to obtain a uniform powder sample. 10mg of powder was placed in a 2mL EP tube, and 1mL of 80% ethanol was added. The tube was allowed to stand at room temperature for 30min (with shaking every 5min). It was then centrifuged at 5000rpm for 10min at room temperature, the supernatant was discarded, and the precipitate was allowed to evaporate at room temperature. Then, 0.4mL of reagent two from the above kit was added to the EP tube, vortexed, and placed horizontally in a shaking incubator at 37℃ and 200rpm for 16 hours. Add 0.4 mL of anhydrous ethanol to the precipitate, vortex, and centrifuge at 5000 rpm for 10 min at room temperature. Add 0.2 mL of reagent III to the precipitate, vortex, add another 0.6 mL of reagent III, vortex to mix, and centrifuge at 5000 rpm for 10 min at room temperature. Carefully collect all the supernatant into a 10 mL tube and repeat the operation once. Under ice bath conditions, slowly add 0.2 mL of reagent IV to the EP tube containing the precipitate while shaking by hand until completely dissolved. Place in an ice bath or freeze at 4°C for 10 min. Add 0.8 mL of reagent VI, invert to mix (do not vortex), immediately add 10 μL of reagent VII, mix well, and incubate at 50°C for 30 min (mixing intermittently 5-6 times). After incubation, cool to room temperature, centrifuge at 3000 rpm for 5 min at room temperature, and use the supernatant for resistant starch detection. Perform sample detection and calculation according to the instructions of the resistant starch detection kit G0571W48 (Suzhou Grees Biotechnology Co., Ltd.).

[0065] Test results as follows Figure 3 As shown in the figure, compared with the control plants, the resistant starch content in tobacco plants overexpressing the NtGBSS2 gene was significantly increased, with the resistant starch content of tobacco plants overexpressing the NtGBSS2 gene increasing by 1.28 to 2.63 times.

[0066] 2. Determination of amylose content

[0067] Amylose content was determined using an enzymatic method. This method utilizes the characteristic that concanavalin A binds only to amylopectin and not to amylose, separating them. An enzyme complex that hydrolyzes starch only then converts the starch into glucose, and the amylose content is determined by detecting the glucose content. The Suzhou Greens Amylose / Amylopectin / Total Starch Content (Enzymatic Method) Kit (catalog number: G0548W48) was used for detection. Sample pretreatment was as follows: 2g of sample was dried (50℃) to constant weight, ground, and sieved (e.g., through a 0.5mm sieve) to obtain a uniform powder sample. 10mg of the powder sample or 10mg of the quality control sample was added to a 2mL EP tube, along with 0.5mL of DMSO, and vortexed to disperse and suspend the sample in the liquid (avoid sedimentation at the bottom of the tube or lumpy suspension). Boil in a water bath until the sample is dispersed and dissolved (approximately 2 minutes, ensuring no gel lumps remain); vortex at high speed, then boil in a water bath for 15 minutes (vortexing intermittently every 2-3 minutes to ensure complete dispersion and dissolution; if gel lumps remain, increase the boiling time and vortexing frequency until completely dissolved); remove the sample and allow it to cool to room temperature for approximately 5 minutes, then add 1 mL of anhydrous ethanol and immediately vortex at high speed to prevent polymerization (it is recommended to process each sample individually), then add 0.5 mL of anhydrous ethanol, invert the EP tube, let stand for 5 minutes, centrifuge at 5000 rpm at room temperature for 5 minutes, discard the supernatant and retain the precipitate; add 1 mL of DMSO to the precipitate, vortex to mix, and boil in a water bath for 15 minutes, vortexing intermittently every 2-3 minutes to ensure complete dispersion and dissolution. Transfer 0.5 mL of the clear stock solution to a new 2 mL EP tube, then add 0.5 mL of reagent diluent; the diluted solution is the test solution. Then perform the sample analysis and calculations according to the instructions.

[0068] The results are as follows Figure 4 As shown in the figure, compared with the control plants, the amylose content in tobacco plants overexpressing the NtGBSS2 gene was significantly increased, and the resistant starch content of tobacco plants overexpressing the NtGBSS2 gene increased by 39.11% to 80.10%.

[0069] Example 7 Drought Resistance Identification

[0070] This embodiment tests the drought resistance of T2 generation plants that are positive for NtGBSS2 gene overexpression. The specific implementation procedure is as follows:

[0071] Seeds of T2 generation plants overexpressing the NtGBSS2 gene positive and seeds of common tobacco (Tobacco Dajinyuan) were sterilized with 70% alcohol and sown in 1 / 2 MS medium. The plants were cultured at 28℃, 2000 Lx light intensity, and 16 h / d photoperiod. After 35 days of culture, seedlings with uniform growth were transplanted to MS medium containing 10% PEG for simulated drought treatment, and simultaneously transplanted to MS medium without PEG. Results were observed after 7 days of treatment. Figure 5 As shown, compared with normal conditions, tobacco plants treated with 10% PEG grew slowly. Besides significant leaf yellowing, wild-type plants were smaller than the overexpressing lines, while tobacco plants overexpressing the NtGBSS2 gene grew better than wild-type plants. Therefore, the data indicate that overexpression of the NtGBSS2 gene enhances the tolerance of tobacco to drought stress.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. 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 or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Tobacco NtGBSS2 The application of genes in regulating drought resistance is characterized by: The tobacco NtGBSS2 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1; overexpression NtGBSS2 Genes that enhance tobacco's tolerance to drought stress.

2. The tobacco according to claim 1 NtGBSS2 The application of genes in regulating drought resistance is characterized by: The overexpression NtGBSS2 The gene was used to construct a recombinant expression vector and transform it into tobacco. NtGBSS2 The gene is overexpressed.

3. The tobacco according to claim 2 NtGBSS2 The application of genes in regulating drought resistance is characterized by: The method for constructing the recombinant expression vector is to obtain the cloned vector. NtGBSS2 The gene was ligated into the PCAMBIA1300-GFP vector and identified by sequencing.

4. Tobacco NtGBSS2 The application of genes in the improvement of tobacco germplasm resources is characterized by: The tobacco NtGBSS2 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1; the improvement of the tobacco germplasm resources is to enhance the tolerance of tobacco to drought stress.

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