Application of NtCER1-1 gene in improving drought resistance of tobacco

Abstract

The application discloses application of NtCER1-1 gene in improving drought resistance of tobacco, and through whole genome identification, a NtCER1 gene family member is screened out in tobacco, and NtCER1-1 gene is gathered for function research. Bioinformatics analysis reveals that a promoter region contains many adversity response elements, and qRT-PCR technology analysis is combined to determine that the NtCER1-1 gene is involved in drought stress response. NtCER1-1 overexpression plants are created, and it is found that the NtCER1-1 gene promotes tobacco leaf epidermis wax accumulation, reduces tobacco leaf water loss rate and chlorophyll leaching rate, and meanwhile, after drought treatment, the NtCER1-1 gene shows stronger antioxidant enzyme activity, higher proline content and lower malondialdehyde content. In summary, the NtCER1-1 gene improves drought resistance of tobacco by participating in tobacco leaf wax synthesis and enhancing antioxidant capacity.

Description

Technical Field

[0001] This invention relates to the application of the NtCER1-1 gene in improving the drought resistance of tobacco, and belongs to the field of genetic engineering technology. Background Technology

[0002] Tobacco is an important economic crop in my country, ranking first in the world in terms of both acreage and total output, making a significant contribution to national development. Drought stress severely restricts the growth and development of tobacco, leading to a significant reduction in both yield and quality. Discovering drought-resistant genes and applying them to tobacco drought-resistant molecular breeding through genetic engineering is the fundamental way to solve the problem of tobacco drought resistance. However, currently, there is a severe shortage of functionally validated drought-resistant genes discovered directly from tobacco, resulting in a lack of suitable target genes when using genetic engineering to improve tobacco drought resistance.

[0003] The surface of plants such as tobacco is covered by a nearly sealed protective cuticle, which plays a dominant role in preventing water loss, especially at night or under drought stress when stomata are closed. Water retention relies primarily on the water-holding capacity of the cuticle. Therefore, cuticle-mediated drought resistance has become an important strategy for plants to cope with drought stress. Studies have shown that the cuticle is mainly composed of cuticle and wax. Cuticle is its skeletal component, while the presence of wax enables the cuticle to prevent water loss from the plant surface, enhancing drought resistance. Therefore, in-depth analysis of the biosynthesis and regulation mechanism of tobacco wax is of significant theoretical and practical value for precisely regulating wax synthesis and thus improving tobacco drought resistance.

[0004] Alkanes are a class of high-carbon-chain secondary metabolites widely found in the waxy mixtures of plant aboveground organs, primarily synthesized by aldehyde decarbonylase encoded by the CER1 gene. Studies have shown that alkanes are important components in the water-retention function of epidermal waxes in plants such as Arabidopsis, rice, rapeseed, wheat, and tomato. For example, AtCER1 in Arabidopsis, OsCER1 in rice, BnCER1-2 in rapeseed, TaCER1-1A and TaCER1-6A in wheat, and SlCER1-1 in tomato promote wax accumulation by participating in the synthesis of alkanes in leaf waxes, thereby reducing the permeability of the cuticle and ultimately improving the drought resistance of transgenic plants. However, although alkanes are the most abundant component of tobacco epidermal wax, the CER1 gene family in tobacco has not yet been identified in the current technology, nor has the drought resistance function of any CER1 family gene been studied. In addition, it is unclear whether the CER1 family genes in tobacco respond to drought stress and whether the gene expression product alkanes enhance the drought resistance of tobacco. These problems and deficiencies limit the application of this important gene resource in molecular breeding for drought resistance in tobacco. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of the NtCER1-1 gene in improving drought resistance in tobacco. Through analysis of the tobacco CER1 gene family, 21 tobacco CER1 genes were identified, and their evolutionary relationships were clarified. Furthermore, through cloning, promoter cis-acting element analysis, expression pattern analysis, detection of wax components and content in overexpressing plants, and analysis of drought resistance physiological indicators in overexpressing plants, the function of the NtCER1-1 gene in tobacco drought resistance was elucidated. This provides a foundation for clarifying the evolution and molecular function of CER1 genes and offers new genetic and theoretical basis for improving drought resistance in tobacco and other crops. It overcomes the shortcomings of existing technologies.

[0006] The objective of this invention is achieved through the following technical solution: This invention discloses the application of the NtCER1-1 gene in improving the drought resistance of tobacco. The nucleotide sequence of the NtCER1-1 gene is shown in SEQ ID No.1.

[0007] SEQ ID No. 1: The amino acid sequence of the protein encoded by the NtCER1-1 gene is shown in SEQ ID No. 2.

[0008] SEQ ID No. 2: MASKPGILTEWPWTWLGNFKYVVLAPYVAHSLHSFFMSEDESKRDITYLIIFPFLLFRMLHNQIWISLSRYRTAKGDNRIVDKSIEFDQVDRERNWDDQIILNGLLFYYGYTKLEQSHHMPIWRTDGIIMTALLQTGPVEFLYYWLHRALHHHFLY SRYHSHHHSSIVTEPITSVIHPFAEHIAYFLLFAIPLLTTVLTGTASIVSFGGYITYIDFMNNMGHCNFEIIPKWMFSSFPPLKYLMYTPSYHSLHHTQFRTNYSLFMPMYDYIYDTLDKSSDTLYEKSLERQGKSPDVVHLTHLTTPESIYHLRLG FASFASEPYTSKWYFWLMWPVTLWSMMITWIYGHTFTVERNVFKSLNLQTWAIPKYRIQYFMQWQRETINNFIEEAIMEADRKGIKVLSLGLLNQEEQLNNNGELYIRRHPQLKVKVVDGSSLAVAVVLNSIPKGTTQVVLGGHLSKVANAIALAL CQGGVKVVTLREEEYKKLKSSLTPEVAINLVPSKTYASKIWLVGDGLSEDEQLKAPKGTLFIPFSQFPPRKARKDCLYFHTPAMITPKHFENVDSCENWLPRRVMSAWRVAGILHALKGWNEHECGNMIFDIEKVWKASLDHGFSPLTMASASESKA As mentioned above, overexpression of the NtCER1-1 gene is preferred to positively improve the drought resistance of tobacco.

[0009] As mentioned above, overexpression of the NtCER1-1 gene positively enhances the drought resistance of tobacco by increasing the epidermal wax content of tobacco leaves.

[0010] As mentioned above, overexpression of the NtCER1-1 gene is preferred to positively improve the drought resistance of tobacco by reducing the permeability of the cuticle of tobacco leaves.

[0011] The cuticle permeability mentioned above preferably includes leaf water loss rate and chlorophyll leaching rate.

[0012] As mentioned above, overexpression of the NtCER1-1 gene is preferred in that it positively improves the drought resistance of tobacco by enhancing the activity of antioxidant enzymes in tobacco plants under drought treatment.

[0013] The antioxidant enzymes mentioned above preferably include SOD, POD, and CAT.

[0014] As mentioned above, overexpression of the NtCER1-1 gene is preferred to positively improve the drought resistance of tobacco by reducing malondialdehyde content.

[0015] As mentioned above, overexpression of the NtCER1-1 gene preferably improves the drought resistance of tobacco by increasing proline content.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to complete the NtCER1 gene family analysis in tobacco and the drought resistance function study of its family member NtCER1-1. It clarifies that overexpression of the NtCER1-1 gene positively improves the drought resistance of tobacco by promoting the synthesis of tobacco wax and enhancing antioxidant capacity, thus expanding the application scope of CER1 family genes in tobacco drought resistance research.

[0017] 2. The provided NtCER1-1 gene and its overexpressing plants can serve as candidate genes and important germplasm resources for breeding new drought-resistant tobacco varieties, contributing to more efficient cultivation of drought-resistant tobacco varieties and supporting safe tobacco production. Furthermore, the overexpressing plants provided by this invention can also serve as ideal research materials for in-depth analysis of the drought-resistant molecular mechanism of the NtCER1-1 gene. This demonstrates the transformation of this invention from basic research to application, possessing clear application value.

[0018] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will now be described in further detail with reference to the accompanying drawings, wherein: Figure 1 The phylogenetic relationships of CER1 protein sequences in tobacco, tomato, Arabidopsis thaliana, rapeseed, cucumber, rice, wheat, and Kentucky bluegrass are shown in the figure; the eight groups are represented by different colors, with the NtCER1-1 gene highlighted in red.

[0020] Figure 2 Analysis of cis-regulatory elements in the NtCER1-1 gene promoter. In the figure, rectangles of different colors represent different types of cis-regulatory elements on the promoter.

[0021] Figure 3 The figure shows the expression pattern of the NtCER1-1 gene under drought stress. Different lowercase letters in the figure indicate significant differences at the P<0.05 level (t-test). All data are the mean ± standard deviation of three biological replicates.

[0022] Figure 4 This study analyzed the expression levels of the NtCER1-1 gene in different lines. WT represents cultivated tobacco K326; OE#1, OE#2, and OE#3 are all NtCER1-1 overexpressing lines; asterisks indicate significant differences from WT plants (t-test). All data are the mean ± standard deviation of three biological replicates. .

[0023] Figure 5 For the analysis of the waxy phenotype of tobacco plants overexpressing NtCER1-1, WT in the figure represents cultivated tobacco K326; OE#1, OE#2, and OE#3 are all overexpressing lines of NtCER1-1; (A) waxy phenotype of WT and NtCER1-1-OE plants under normal growth conditions; (B) waxy phenotype of WT and NtCER1-1-OE plants under drought stress; asterisks indicate significant differences from WT plants (t-test), and all data are the mean ± standard deviation of three biological replicates. .

[0024] Figure 6 To determine the water loss rate and chlorophyll leaching rate of detached leaves from tobacco plants overexpressing NtCER1-1, WT in the figure represents cultivated tobacco K326; OE#1, OE#2, and OE#3 are all overexpression lines of NtCER1-1; (A) Determination of water loss rate of detached leaves from overexpression plants under normal growth conditions; (B) Determination of chlorophyll leaching rate of detached leaves from overexpression plants under normal growth conditions; All data are the mean ± standard deviation of three biological replicates.

[0025] Figure 7 To compare the phenotypes of tobacco plants overexpressing NtCER1-1 and WT plants after seven days of drought treatment, WT in the figure represents cultivated tobacco K326; OE#1, OE#2 and OE#3 are all overexpressing lines of NtCER1-1.

[0026] Figure 8To detect the antioxidant enzyme activity, proline content, and malondialdehyde (MDA) content of tobacco plants overexpressing NtCER1-1 before and after drought stress; WT in the figure represents cultivated tobacco K326; OE#1, OE#2, and OE#3 are all overexpression lines of NtCER1-1; (A) Superoxide dismutase (SOD) activity; (B) Peroxidase (POD) activity; (C) Catalase (CAT) activity; (D) Proline (Pro) content; (E) Malondialdehyde (MDA) content. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be understood that the preferred embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] like Figures 1-8 As shown, this invention discloses the application of the NtCER1-1 gene in improving drought resistance in tobacco. In this invention, the nucleotide sequence of the NtCER1-1 gene is shown in SEQ ID No. 1. In this invention, the amino acid sequence of the protein encoded by the NtCER1-1 gene is shown in SEQ ID No. 2.

[0029] SEQ ID No. 1: SEQ ID No. 2: MASKPGILTEWPWTWLGNFKYVVLAPYVAHSLHSFFMSEDESKRDITYLIIFPFLLFRMLHNQIWISLSRYRTAKGDNRIVDKSIEFDQVDRERNWDDQIILNGLLFYYGYTKLEQSHHMPIWRTDGIIMTALLQTGPVEFLYYWLHRALHHHFLY SRYHSHHHSSIVTEPITSVIHPFAEHIAYFLLFAIPLLTTVLTGTASIVSFGGYITYIDFMNNMGHCNFEIIPKWMFSSFPPLKYLMYTPSYHSLHHTQFRTNYSLFMPMYDYIYDTLDKSSDTLYEKSLERQGKSPDVVHLTHLTTPESIYHLRLG FASFASEPYTSKWYFWLMWPVTLWSMMITWIYGHTFTVERNVFKSLNLQTWAIPKYRIQYFMQWQRETINNFIEEAIMEADRKGIKVLSLGLLNQEEQLNNNGELYIRRHPQLKVKVVDGSSLAVAVVLNSIPKGTTQVVLGGHLSKVANAIALAL CQGGVKVVTLREEEYKKLKSSLTPEVAINLVPSKTYASKIWLVGDGLSEDEQLKAPKGTLFIPFSQFPPRKARKDCLYFHTPAMITPKHFENVDSCENWLPRRVMSAWRVAGILHALKGWNEHECGNMIFDIEKVWKASLDHGFSPLTMASASESKA In this invention, overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco; overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco by increasing the epidermal wax content of tobacco leaves; overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco by reducing the permeability of the cuticle of tobacco leaves; the cuticle permeability preferably includes leaf water loss rate and chlorophyll leaching rate; overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco by enhancing the activity of antioxidant enzymes in tobacco plants under drought treatment; the antioxidant enzymes preferably include SOD, POD and CAT; overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco by reducing malondialdehyde content; overexpression of the NtCER1-1 gene preferably positively enhances the drought resistance of tobacco by increasing proline content.

[0030] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1. Identification and analysis of the tobacco NtCER1 gene family (1) Identification of the tobacco NtCER1 gene family The protein sequence of Arabidopsis thaliana CER1 was downloaded from the TAIR database as a seed sequence. BLASTP alignment of the seed sequence with default parameters was performed in the common tobacco protein database to obtain candidate sequences. The InterPro database (https: / / www.ebi.ac.uk / interpro / ) was used to screen candidate sequences. After removing protein sequences with redundant and incomplete conserved domains, protein sequences possessing both a fatty acid hydroxylase superfamily (FA_hydroxylase) domain and a Wax2_C-terminal domain were identified as members of the NtCER1 gene family.

[0032] (2) Construction of phylogenetic tree CER1 homologous protein sequences from tobacco, tomato, Arabidopsis thaliana, rapeseed, cucumber, rice, wheat, and Kentucky bluegrass were downloaded from the Phytozome database (https: / / phytozome-next.jgi.doe.gov / ). Multiple sequence alignment of tobacco CER1 and CER1 sequences from other species was performed using MEGA11 software. A phylogenetic tree was constructed using the neighbor-joining method and a 1000-repeat test. Finally, the constructed phylogenetic tree was enhanced using the iTOL online tool (https: / / itol.embl.de / ).

[0033] The results are as follows Figure 1As shown, the constructed phylogenetic tree divides the CER1 family into six main branches (Group I–VI). Group II includes CER1 proteins from dicotyledonous plants (Arabidopsis thaliana AtCER1 and AtCER1-like1, Brassica napus BnCER1, and cucumber CsCER1) as well as CER1 proteins from monocotyledonous plants (rice OsCER1 and OsWDA1, wheat TaCER1-1A, and Kentucky bluegrass PpCER1), suggesting that CER1 may play a conserved function in wax synthesis in both monocotyledonous and dicotyledonous plants. The remaining branches are mainly composed of members of the tobacco NtCER1 and tomato SlCER1 families, with tobacco NtCER1 distributed across multiple branches, exhibiting species-specific expansion. The target gene NtCER1-1 (marked in red in the figure) and the tomato wax synthesis gene SlCER1-1 cluster in the same phylogenetic branch (Group III), indicating that NtCER1-1 may also have a similar function to SlCER1-1 and may be involved in tobacco wax synthesis.

[0034] (3) Analysis of promoter cis-acting elements The nucleotide sequence of the NtCER1-1 gene upstream of the start codon was extracted from the tobacco genome database. Cis-regulatory elements in the promoter region of the NtCER1-1 gene were mined using the PlantCARE online database (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The element analysis results were visualized using TBtools software.

[0035] The results are as follows Figure 2 As shown, the NtCER1-1 promoter region contains many cis-acting elements related to environmental stress responses, including response hormones (abscisic acid, gibberellin, salicylic acid, jasmonic acid), anaerobic induction, and photoresponse elements. This suggests that NtCER1-1 may be related to abiotic stress responses such as drought, hypoxia, and photoperiod, and may also be finely regulated by multiple hormonal signals.

[0036] (4) Analysis of expression patterns under drought stress Tobacco K326 seeds were germinated in petri dishes lined with filter paper. Seedlings with uniform growth were then placed in hydroponic sponges for further growth, using Hoagland solution. When the seedlings reached five true leaves, the Hoagland solution was replaced with 20% PEG6000 solution, and cultivation continued. The second leaf below the growth point was collected at 0h, 1h, 3h, 6h, 12h, 24h, and 48h after treatment. Total RNA was extracted from these materials using Trizol reagent from Yisheng (Shanghai) according to their method, and then reverse transcribed into cDNA using Yisheng (Shanghai)'s genomic DNA reverse transcription kit. Primers for quantitative real-time PCR were designed using Primer software, with tobacco NtL25 (GeneIDinNCBI:107796789) as the internal reference gene, and three biological replicates and technical replicates were set up. Subsequently, qRT-PCR experiments were performed using Yisheng (Shanghai)'s quantitative real-time PCR reagent (SYBR GreenMasterMix). Gene expression levels were calculated, and data were statistically analyzed and plotted using EXCEL and GraphpadPrism 10.1.2 software, respectively. Significance differences were analyzed using t-tests, with lowercase letters indicating statistical significance at the P < 0.05 level. The primers used in the experiment are listed in Table 1.

[0037] The results are as follows Figure 3 As shown, the expression level of NtCER1-1 gradually increased over time before 6 hours of treatment and gradually decreased after 6 hours, but it was still higher than the expression level at 0 hours, indicating that NtCER1-1 was induced by drought stress and thus participated in the drought resistance response process of tobacco.

[0038] Table 1 Primer sequences required for qRT-PCR ; Example 2. Creation of tobacco NtCER1-1 overexpressing plants (1) Cloning of the NtCER1-1 gene Based on the NtCER1-1 gene sequence from the tobacco genome database, primers were designed to amplify its coding sequence (CDS), and a vector sequence was added to the 5' and 3' ends of the primers. The primer sequences are shown in Table 2, where lowercase letters represent the vector sequence. Using leaf cDNA obtained in Example 1 as a template, the reaction system was prepared according to Table 3, and the CDS of the target gene was amplified according to the PCR amplification procedure in Table 4. After the reaction, the PCR products were placed in a 1% agarose gel containing nucleic acid dye for electrophoresis. After electrophoresis, the products were observed and photographed using a UV imager. Single target bands of the expected size were cut out and purified using a gel extraction kit (Sangon Biotech, Shanghai) for later use.

[0039] Table 2 Primers for PCR amplification of the target gene CDS ; Table 3. PCR reaction procedure for the target gene ; Table 4. PCR amplification procedure for the target gene. ; (2) Linearization of overexpression vectors The overexpression vector pCAMBIA1300 plasmid was extracted according to the instructions of the plasmid extraction kit (Sangon Biotech, Shanghai). 3 μg of plasmid was used to prepare the enzyme digestion reaction system according to Table 5, and then incubated at 37°C for 15 min. After the reaction, the digestion products were placed on a 1% agarose gel containing nucleic acid dye for electrophoresis. After electrophoresis, the products were observed and photographed using a UV imager. Single target bands of the expected size were cut out and purified using a gel extraction kit (Sangon Biotech, Shanghai) for later use.

[0040] Table 5 Enzyme digestion reaction system ; (3) Ligation of the target gene and the vector The target gene was ligated to the linearized vector using a one-step rapid cloning kit (Yisheng, Shanghai). After preparing the system according to Table 6, the mixture was gently mixed by pipetting and briefly centrifuged to concentrate the reaction solution at the bottom of the tube. The system was then placed in a PCR instrument at 50°C for 20 min. After the reaction, it was immediately cooled on ice for 5 min to avoid the high temperature affecting the subsequent transformation efficiency of competent cells. The resulting reaction solution can be used directly for transformation experiments.

[0041] Table 6 Recombination Reaction System ; (4) The recombinant products are converted and coated. Thaw DH5α competent cells on ice; add 10 μL of pre-cooled recombinant product to 100 μL of competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, and immediately place on ice for 2 min; add 900 μL of LB liquid medium, incubate at 37℃ for 10 min, then shake at 200 rpm and 37℃ for 45 min; centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant, resuspend the cells in the residual medium, and spread evenly on LB solid medium containing 50 mg / L kanamycin (Kan). After the bacterial suspension is absorbed, invert the culture dish and incubate overnight at 37℃.

[0042] (5) Cloning identification Ten single clones were picked from overnight culture plates and inoculated into 1 mL LB liquid medium containing 1 μL of 50 mg / L Kan. The cultures were incubated at 37°C and 200 rpm for 12–16 h with shaking. Colony PCR amplification was performed according to the system in Table 3 and the procedure in Table 4, for a total of 34 cycles. After 1% agarose gel electrophoresis, positive clones with the target band were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the reference sequence using DNAMAN software. Bacterial cultures with correct alignment were mixed with 50% glycerol at a 1:1 ratio and stored at -80°C for later use. The primer sequences used are shown in Table 2.

[0043] (6) Recombinant plasmid transformation of Agrobacterium 20 μL of the correctly sequenced single-clone bacterial culture was transferred to 10 mL LB broth containing 10 μL of Kan (50 mg / L) and cultured at 37°C and 200 rpm for 12 h with shaking. After culture, the recombinant plasmid pCAMBIA1300-NtCER1-1 was obtained according to the instructions of the plasmid mini-extraction kit (Sangon Biotech, Shanghai). The pCAMBIA1300-NtCER1-1 recombinant plasmid was transformed into Agrobacterium GV3101 competent cells using the Agrobacterium freeze-thaw transformation method, as detailed below: Thaw GV3101 competent cells stored at -80℃ completely on ice, add 2 μL of plasmid, gently tap the tube wall to mix, and incubate on ice for 30 min; flash freeze in liquid nitrogen for 1 min, heat shock in a 37℃ water bath for 5 min, then quickly incubate on ice for 2 min, avoiding shaking the centrifuge tube throughout the process; add 900 μL of antibiotic-free YEP liquid medium. Incubate at 28°C and 200 rpm with shaking for 1–3 h, centrifuge at 8000 rpm for 1 min, discard the supernatant, and resuspend the cells in 100 μL of LYEP medium; spread 50 μL of the resuscitated bacterial solution onto YEP solid medium containing 50 mg / L Kan and 25 mg / L rifampicin (Rif), and incubate at 28°C inverted mode for 2–3 days; pick single clones and incubate in 1 m of LYEP liquid medium containing 1 μL Kan (50 mg / L) and 1 μL Rif (25 mg / L), and incubate at 28°C and 200 rpm with shaking for 24 h. Positive single clones were identified by PCR according to Tables 11 and 12. The amplified products were subjected to 1% agarose gel electrophoresis and then observed and photographed on a UV imager to obtain positive clone bacterial solutions with the target band, namely Agrobacterium GV3101 strain containing the pCAMBIA1300-NtCER1-1 recombinant plasmid.

[0044] (7) Identification of genetically transformed tobacco and transgenic plants The constructed pCAMBIA1300-NtCER1-1 overexpression vector was transformed into cultivated tobacco K326 using the leaf disc method. The specific steps are as follows: Select plump tobacco seeds, disinfect them with 75% ethanol for 1 min, rinse them 3 times with sterile water, treat them with 3% H2O2 for 12 min, rinse them 5 times with sterile water, blot them dry with sterile filter paper, and then inoculate them onto 1 / 2 MS solid medium. Once the seedlings reach approximately 2 cm in height, transfer them individually to tissue culture flasks and culture them at 24°C under 16h light / 8h dark conditions. Streak 10 μL of Agrobacterium GV3101 containing the pCAMBIA1300-NtCER1-1 recombinant plasmid onto YEP solid medium containing Kan (50 mg / L) and Rif (25 mg / L), and incubate at 28°C inverted for 2-3 days to activate the strain. Select robust, sterile seedlings with 5 true leaves, cut leaves into approximately 1 cm² pieces avoiding the midrib, and incubate them in the dark on pre-cultured medium for 2 days. Resuspend the activated Agrobacterium in 1 / 2 MS liquid containing 40 mg / L acetylsyleugenol, shake at 150 rpm for 1-2 hours, and dilute to [a specific concentration]. The inoculum was prepared by allowing the cultured leaf pieces to stand at room temperature for 30 minutes. The pre-cultured leaf pieces were placed in the bacterial solution and inoculated at 28°C and 150 rpm for 18 minutes. The leaf pieces were then removed, the surface bacterial solution was blotted dry with sterile filter paper, and the leaf pieces were transferred to a co-culture medium and cultured in the dark for 3 days. The co-cultured leaf pieces were first cultured on an induction medium for 7 days, and then transferred to a selection medium for two rounds of selection (12 days each). Subsequently, the healthy explants were transferred to a differentiation medium until 3 cm adventitious buds were grown. The adventitious buds were cut and inoculated into a rooting medium to induce root formation. After the plants were vigorous, the cap was opened and a small amount of sterile water was added. The plants were then covered with a plastic bag for 3 days to adapt. After washing the culture medium from the roots, the plants were transplanted into a sterile substrate. The opening of the plastic bag was gradually torn open to allow the transformed seedlings to gradually adapt to the external environment.

[0045] DNA was extracted from transgenic plants using the CTAB method. PCR was performed according to Tables 3 and 4, and the primers used are shown in Table 2. The PCR amplification products were subjected to 1% agarose gel electrophoresis and then observed and photographed using a UV imager. Transgenic plants exhibiting the target band were selected as positive plants. Following the total RNA extraction, reverse transcription, and qRT-PCR methods described in Example 1, the expression level of the NtCER1-1 gene in the positive plants was analyzed. Three lines with significantly increased NtCER1-1 gene expression levels were selected for subsequent phenotypic analysis.

[0046] The results are as follows Figure 4 As shown, the expression levels of the NtCER1-1 gene in the three transgenic lines OE#1, OE#2, and OE#3 were significantly higher than those in WT (untransformed tobacco K326), being 373 times, 306 times, and 345 times higher, respectively, indicating that the NtCER1-1 gene had been successfully transferred into tobacco K326 and overexpressed.

[0047] Example 3. Waxy phenotype analysis of tobacco plants overexpressing NtCER1-1 The overexpression lines OE#1, OE#2 and OE#3 obtained in Example 2 were transferred to a greenhouse for cultivation (25℃ / 20℃, 16h light / 8h darkness). When buds appeared, they were covered with net bags to allow them to self-pollinate and produce seeds for later use.

[0048] (1) Sample preparation Seeds of wild-type K326 (WT) and NtCER1-1 overexpression lines (OE#1, OE#2, and OE#3) were sown in seedling substrate. When the seedlings reached the cross-shaped stage, 18 seedlings from each line were transplanted into one pot (10×12cm). When the plants reached 60 days of growth, two leaves from the middle section were taken for epidermal wax content determination.

[0049] (2) Extraction of epidermal wax Fresh tobacco leaves were extracted in a fume hood with 20–30 mL of chloroform at room temperature for 1 min. 20 μL of 1 mg / mL tetracosane (C24) was added to the extract as an internal standard. The extract was filtered and naturally concentrated to approximately 0.5 mL, then transferred to a GC vial. After solvent evaporation, 40 μL of pyridine and 40 μL of LSTFA were added, mixed thoroughly, and incubated at 70 °C for 1 h. The mixture was then dried under nitrogen, reconstituted with 500 μL of chloroform, and used for gas chromatography-mass spectrometry (GC-MS) analysis.

[0050] (3) Detection of epidermal wax composition and content The injection volume was set to 2 μL, the injection port temperature to 280℃, the split ratio to 2:1, and the detector temperature to 310℃. Helium was used as the carrier gas at a flow rate of 2 mL / min. The temperature program was as follows: hold at 50℃ for 2 min, increase to 200℃ at 20℃ / min and hold for 2 min, then increase to 310℃ at 1.6℃ / min and hold for 20 min. Full scan mode was used, with a scan range of 35–700 m / z. The wax components were qualitatively identified using mass spectrometry databases, literature, and standards. The absolute content of each component was calculated using the internal standard method, and three biological replicates were performed for each group.

[0051] The results are as follows Figure 5 As shown, compared with WT, under normal growth conditions, the wax content in the leaves of NtCER1-1 overexpressing lines (OE#1, OE#2, and OE#3) increased by 1.33, 1.18, and 1.21 times, respectively; under drought treatment, it increased by 1.55, 1.47, and 1.48 times, respectively. These changes were mainly due to a significant increase in alkane content. This phenotype confirms that NtCER1-1 overexpression can promote the synthesis of alkane waxes in tobacco leaves, thereby significantly increasing the accumulation of waxes in tobacco leaves under both normal and drought stress conditions.

[0052] (4) Determination of water loss rate and chlorophyll leaching rate of detached leaves Seeds of WT and NtCER1-1 gene overexpression lines (OE#1, OE#2, and OE#3) were sown in MS medium with or without 50 mg / L hygromycin for seedling cultivation. When the seedlings reached 2 cm in height, they were transplanted into pots for further growth. At 60 days of age, the water loss rate and chlorophyll leaching rate of detached leaves were measured. Leaf water loss rate and chlorophyll leaching rate are two key indicators for evaluating leaf cuticle permeability.

[0053] ① Determination of water loss rate of detached leaves Leaves from WT, OE#1, OE#2, and OE#3 plants grown for 60 days were selected, with four biological replicates per treatment. Three leaves were taken from each replicate, cut into 4-5 cm segments, and placed in tissue culture bottles, wrapped in aluminum foil, and incubated in the dark for 1 hour. Under dark conditions, the fresh weight of the leaves was measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours. The water loss rate at each time point was calculated using the fresh weight at 0 hours as the baseline. A graph was plotted with time on the x-axis and leaching rate on the y-axis.

[0054] ② Chlorophyll leaching rate determination Leaves from WT, OE#1, OE#2, and OE#3 plants grown for 60 days were selected, with each treatment replicated four times. Three leaves from each replicate were cut into 4-5 cm segments, completely submerged in 100 mL of 80% ethanol, and allowed to stand in the dark with gentle agitation. 200 μL of the extract was collected at 1–10 h and 24 h, and the absorbance at 647 nm and 664 nm was measured using a microplate reader. Total chlorophyll content was determined. The chlorophyll leaching rate was calculated as the ratio of the leaching amount at each time point to the total content over 24 hours. A graph was plotted with time on the x-axis and leaching rate on the y-axis.

[0055] The results showed that, compared with WT, the NtCER1-1 gene overexpression lines (OE#1, OE#2, and OE#3) exhibited lower detached leaf water loss and chlorophyll leaching rates. Figure 6 ), combined Figure 5 The wax analysis results show that NtCER1-1 can promote wax accumulation by participating in the synthesis of waxy alkane in tobacco leaves, thereby reducing the permeability of the leaf cuticle.

[0056] Example 4. Phenotypic analysis of drought resistance in tobacco plants overexpressing NtCER1-1 Seeds of WT and NtCER1-1 gene overexpression lines (OE#1, OE#2, and OE#3) were sown on MS medium with or without 50 mg / L hygromycin for seedling cultivation. When the seedlings reached 2 cm in height, they were transplanted into pots for further growth. At 60 days of age, the tobacco leaves were divided into a control group and a drought treatment group. The control group received normal watering, while the treatment groups received no watering and were grown under conditions of 25℃ / 20℃, 16h light / 8h darkness, and 40% relative humidity. Each group was subjected to at least three biological replicates. At 7 days of drought stress, the drought phenotype was evident. Samples were taken from tobacco leaves at 0 and 7 days of treatment, and drought resistance physiological indicators were measured (the second and third leaves from the top were taken, flash-frozen in liquid nitrogen, and stored at -80℃ for later use).

[0057] ① Observation of drought resistance phenotype Photographs were taken of the 60-day-old tobacco seedlings at 0-day and 7-day treatment. For example... Figure 7 As shown, after 7 days of continuous drought, WT exhibited obvious wilting and yellowing phenotypes, while the NtCER1-1 gene overexpressing lines (OE#1, OE#2, and OE#3) remained upright with bright green leaves, and their overall growth was significantly better than WT. Combined with... Figure 5 and Figure 6 The results show that NtCER1-1 can promote wax accumulation by participating in the synthesis of waxy alkane in tobacco leaves, thereby reducing the permeability of the leaf cuticle and ultimately improving the drought resistance of tobacco.

[0058] ② Determination of drought resistance physiological indicators Using the second and third leaves from the top of the 60-day-old tobacco seedlings at treatment 0 and 7 days as research materials, the SOD activity, POD activity, CAT activity, Pro content, and MDA content of the leaves were measured before and after treatment according to the instructions of the physiological index assay kit from Beijing Solarbio Technology Co., Ltd. Each physiological index measurement was performed in triplicate. Results are as follows: Figure 8 As shown, under normal growth conditions, there were no significant differences in various drought resistance physiological indicators among WT and OE#1, OE#2, and OE#3 plants. After drought treatment, the SOD activity, POD activity, CAT activity, Pro content, and MDA content of WT, OE#1, OE#2, and OE#3 plants all showed a gradual increasing trend. However, the increase in SOD activity, POD activity, CAT activity, and Pro content of OE#1, OE#2, and OE#3 plants was significantly higher than that of WT, while the increase in MDA content was significantly lower than that of WT. These results indicate that overexpression of NtCER1-1 can alleviate oxidative damage caused by drought stress and improve the drought resistance of tobacco by enhancing the activity of antioxidant enzyme systems.

[0059] In summary, overexpression of NtCER1-1 can significantly promote wax accumulation and enhance the antioxidant capacity of tobacco, thereby positively improving the drought resistance of tobacco.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The application of the NtCER1-1 gene in improving drought resistance in tobacco, characterized by, The nucleotide sequence of the NtCER1-1 gene is shown in SEQ ID No.

1.

2. The application of the NtCER1-1 gene according to claim 1 in improving drought resistance in tobacco, characterized in that, The amino acid sequence of the protein encoded by the NtCER1-1 gene is shown in SEQ ID No.

2.

3. The application of the NtCER1-1 gene according to claim 1 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively enhances drought resistance in tobacco.

4. The application of the NtCER1-1 gene according to claim 3 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively enhances the drought resistance of tobacco by increasing the epidermal wax content of tobacco leaves.

5. The application of the NtCER1-1 gene according to claim 4 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively enhances tobacco drought resistance by reducing the permeability of the tobacco leaf cuticle.

6. The application of the NtCER1-1 gene according to claim 5 in improving drought resistance in tobacco, characterized in that, The cuticle permeability includes leaf water loss rate and chlorophyll leaching rate.

7. The application of the NtCER1-1 gene according to claim 3 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively enhances the drought resistance of tobacco plants by increasing the activity of antioxidant enzymes under drought treatment.

8. The application of the NtCER1-1 gene according to claim 7 in improving drought resistance in tobacco, characterized in that, The antioxidant enzymes include SOD, POD, and CAT.

9. The application of the NtCER1-1 gene according to claim 3 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively improves drought resistance in tobacco by reducing malondialdehyde content.

10. The application of the NtCER1-1 gene according to claim 3 in improving drought resistance in tobacco, characterized in that, Overexpression of the NtCER1-1 gene positively enhances drought resistance in tobacco by increasing proline content.

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