Tobacco gene nt04g14970 and application thereof in affecting tobacco cold tolerance and enhancing carbon sink
By overexpressing the Nt04g14970 gene in tobacco, a low-temperature tolerant tobacco line was constructed, which solved the problems of insufficient tolerance to low temperatures and insufficient carbon sequestration capacity of tobacco, and achieved efficient growth and carbon absorption of tobacco under low-temperature conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUNNAN ACAD OF TOBACCO AGRI SCI
- Filing Date
- 2025-01-24
- Publication Date
- 2026-06-12
AI Technical Summary
Existing tobacco breeding techniques are insufficient to effectively improve tobacco's tolerance to low temperatures and enhance its carbon sequestration capacity, thus affecting tobacco growth, yield, and quality.
By overexpressing the tobacco gene Nt04g14970 in tobacco, a low-temperature resistant tobacco strain was constructed, which improved its biomass, photosynthetic rate, transpiration rate, stomatal conductance, antioxidant capacity, and soluble protein and proline content, thereby enhancing its carbon sequestration function.
It significantly improved the low-temperature tolerance and carbon sequestration capacity of tobacco, enhanced the biomass and photosynthetic efficiency of tobacco, increased antioxidant capacity and soluble protein content, and promoted the growth and carbon absorption of tobacco under low-temperature conditions.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, specifically to a tobacco gene Nt04g14970 and its application in preparing low-temperature resistant and carbon-enhancing tobacco lines. Background Technology
[0002] In the global carbon cycle, terrestrial ecosystems serve as a bridge to establish a global carbon balance. As the main body of terrestrial ecosystems, farmland ecosystems have a significant carbon sink effect. Through vegetation photosynthesis, they fix and absorb CO2 from the atmosphere, and their carbon sink function plays a crucial role in stabilizing and even reducing the concentration of greenhouse gases in the atmosphere.
[0003] Tobacco, as a special leaf-based economic crop, has a large leaf area, and the photosynthesis of its leaves is of great significance for carbon sequestration and emission reduction in tobacco field ecosystems. However, tobacco seedlings and its vigorous growth period are frequently affected by low-temperature disasters, which seriously impact leaf growth, yield, and quality. Current frost protection measures in tobacco fields mainly involve mulching, covering with materials such as straw and branches, and physical measures such as smoke fumigation. Developing tobacco varieties with strong low-temperature resistance is of great importance for carbon sequestration and emission reduction in the tobacco industry and tobacco fields.
[0004] The MADS-box gene family represents an important transcription factor family widely distributed in fungi, animals, and plants. The MADS-box gene family has been extensively characterized in many plant species, including Arabidopsis thaliana, rice, maize, and cotton. Currently, MADS-box genes are known to play crucial roles in various processes, including flower and seed development, controlling flowering time, and regulating fruit ripening. Simultaneously, MADS-box genes are also involved in plant responses to various abiotic and biotic stresses. For example, the rice genes OsMADS55 and OsMADS57 are induced by low temperature, and in pepper, the JX402926 gene is induced by low temperature; enhancing its expression improves the pepper's tolerance to low temperatures. However, there are no reports on whether enhanced expression of tobacco-related MADS-box family genes contributes to low temperature tolerance. Summary of the Invention
[0005] The purpose of this invention is to provide a tobacco gene Nt04g14970 and its application in influencing tobacco cold resistance and enhancing carbon sequestration. It can be used to prepare tobacco lines with superior traits and has promising applications in the tobacco planting field.
[0006] To achieve the above objectives, the present invention provides a tobacco gene Nt04g14970 that affects the cold resistance of tobacco, the nucleotide sequence of which is shown in SEQ ID NO.5.
[0007] The present invention also provides a primer for amplifying and identifying the above-mentioned tobacco gene Nt04g14970, the nucleotide sequence of which is shown in SEQ ID NO.1 and 2.
[0008] The tobacco gene Nt04g14970 provided by this invention can be used in the field of tobacco cultivation and includes:
[0009] It is used to prepare low-temperature resistant tobacco strains; increase tobacco biomass; enhance tobacco's antioxidant capacity; improve tobacco's photosynthetic rate, transpiration rate, and stomatal conductance; and increase the content of soluble protein and proline in tobacco.
[0010] The tobacco gene Nt04g14970 provided by this invention can be applied to enhance carbon sinks. By increasing the absorption and conversion of carbon dioxide, it can improve the biomass of tobacco and help enhance carbon sinks.
[0011] This invention also provides a method for preparing a low-temperature resistant tobacco strain, comprising the following steps:
[0012] S1. Construct an overexpression vector for the tobacco gene Nt04g14970, whose nucleotide sequence is shown in SEQ ID NO.5;
[0013] S2. The obtained overexpression vector was transformed into Agrobacterium to obtain an overexpressing Agrobacterium that could overexpress the tobacco gene Nt04g14970.
[0014] S3. The obtained Agrobacterium overexpression was transfected into tobacco. After screening and identification, tobacco plants overexpressing the tobacco gene Nt04g14970 were obtained, which are the low-temperature resistant tobacco strains.
[0015] Preferably, the overexpression vector in the above preparation method is selected from the pCAMBIA1305 plasmid.
[0016] Preferably, the Agrobacterium used in the above preparation method is selected from Agrobacterium tumefaciens, and more preferably from EHA105.
[0017] The present invention has the following advantages:
[0018] This invention discloses for the first time a tobacco gene Nt04g14970 related to low temperature tolerance in tobacco, providing a research basis for the gene function, expression pathway, and subsequent functions of this gene.
[0019] This invention also provides various applications for the tobacco gene Nt04g14970, such as preparing low-temperature resistant tobacco strains; increasing tobacco biomass; improving tobacco's antioxidant capacity; increasing tobacco's photosynthetic rate, transpiration rate, and stomatal conductance; and increasing the content of soluble protein and proline in tobacco, etc., which have significant application value in the field of tobacco cultivation.
[0020] The present invention also provides a method for preparing tobacco strains with excellent low-temperature tolerance. After screening, tobacco strains with significantly improved low-temperature tolerance can be obtained, which helps to build a tobacco resource library with excellent traits. Attached Figure Description
[0021] Figure 1 The results of phylogenetic analysis of the tobacco gene Nt04g14970.
[0022] Figure 2 The expression of Nt04g14970 in different parts of tobacco and the effect of low temperature stress on gene expression in each part.
[0023] Figure 3 The overexpression effects of wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2) under low temperature stress were investigated.
[0024] Figure 4 Effects of low temperature stress on the biomass of wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2).
[0025] Figure 5 The effects of low temperature stress on the photosynthesis of tobacco leaves of wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2).
[0026] Figure 6 The effects of low temperature stress on tobacco antioxidant enzymes in wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2).
[0027] Figure 7 The effects of low temperature stress on soluble tobacco proteins and proline in wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2). Detailed Implementation
[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0029] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0030] Experiment 1: Gene Acquisition
[0031] Local BLAST and HMM analyses were performed based on the tobacco genome database, and a rootless phylogenetic tree was constructed by combining the obtained MADS protein sequences with those of rice and tomato MADS-box family genes. Among the findings, the tobacco MADS-box family gene Nt04g14970 was identified as being evolutionarily closely related to cold-resistance genes such as OsMADS55 and OsMADS57 in rice. The nucleotide sequence of this gene is shown in SEQ ID NO.5, and its phylogenetic analysis results are as follows: Figure 1 As shown. To date, the gene function and expression pathway of this tobacco gene Nt04g14970 have not been reported.
[0032] Example 2: Construction of the overexpression vector for the tobacco gene Nt04g14970
[0033] The specific steps for constructing the tobacco gene Nt04g14970 overexpression vector are as follows:
[0034] 1) Extract total RNA from tobacco K326;
[0035] 2) The total RNA extracted in step 1) was reverse transcribed to synthesize total cDNA;
[0036] 3) Using the total cDNA obtained in step 2) as a template, PCR primers (Nt04g14970-F, Nt04g14970-R, specific sequences are as follows) were designed based on the cDNA sequence of tobacco Nt04g14970. The PCR product contains the complete Nt04g14970 reading frame (from start codon ATG to TAG). The amplified product is the cDNA of the Nt04g14970 gene. The specific nucleotide sequence is shown in SEQ ID NO.5.
[0037] Nt04g14970-F (SEQ ID NO.1):
[0038] 5'-ATGGGAAGGGGAAAGATAGTG-3'
[0039] Nt04g14970-R (SEQ ID NO.2):
[0040] 5'-CTACCTCGAATTGGATACTCCTGC-3'
[0041] Nt04g14970 gene (SEQ ID NO.5):
[0042] 。
[0043] 4) Based on the cDNA sequence of the wild-type Yunyan K326 Nt04g14970 gene, amplification primers were designed. The amplification product contained the complete Nt04g14970 reading frame (from the start codon ATG to the stop codon TAG). Restriction endonuclease sites SwaI and SacI, along with homologous arms, were introduced into the upstream and downstream primers, respectively. Homologous recombination PCR amplification primers (Nt04g14970 OE-F, Nt04g14970 OE-R, specific sequences are shown below) were designed. Using the Nt04g14970 gene cDNA obtained in 3) as a template, the target PCR product was separated by agarose gel electrophoresis and recovered. Simultaneously, the plant vector pCAMBIA1305 plasmid was digested with SwaI and SacI. Then, the recovered digested PCR product fragment and the vector were subjected to homologous recombination. The recombinant product was transformed into E. coli DH5α competent cells and plated on LB solid medium containing antibiotics. Positive colonies were selected for sequencing. The correctly sequenced bacterial cultures were then stored at -70°C with an equal volume of 30% glycerol for later use. The overexpression cloning plasmid was named Nt04g14970-OE.
[0044] Nt04g14970 OE-F(SEQ ID NO.3):
[0045] 5'-ACTTCTATTGCAGCAATTTAAATatgggaaggggaaagatagtgat-3'
[0046] Nt04g14970 OE-R(SEQ ID NO.4):
[0047] 5'-GCCCTTGCTCACCATTTAATTAActacctcgaattggatactcctgc-3'.
[0048] Example 3: Construction of tobacco lines overexpressing the Nt04g14970 gene
[0049] The overexpression cloning plasmid Nt04g14970-OE obtained in Experiment 2 was transformed into competent cells of Agrobacterium tumefaciens EHA105, and positive colonies were empirically obtained. Details are as follows:
[0050] Leaf pieces of Yunyan K326 tobacco were cut into small pieces and soaked in EHA105 Agrobacterium solution adjusted to an appropriate OD value. Transfection was performed according to standard Agrobacterium transfection methods. The leaves were then transferred to filter paper to absorb excess bacterial solution and placed on a co-culture medium for dark incubation to promote Agrobacterium absorption. The co-cultured leaves were then transferred to a differentiation medium containing a selector and cultured under light to screen for successfully transformed tobacco plants. The selected transformed tobacco plants were then transferred to a rooting medium to promote rooting. PCR identification: PCR was used to identify the rooted tobacco seedlings at the molecular level to confirm successful integration of the exogenous gene into the tobacco genome, resulting in tobacco plants with enhanced Nt04g14970-OE expression. Further cultivation and screening yielded the Nt04g14970-OE transgenic homozygous line, which is the Nt04g14970 gene overexpressing tobacco line.
[0051] Example 4: Verification of the characteristics of the overexpression lines
[0052] 1. Low-temperature tolerance verification
[0053] Wild-type Yunyan K326 (WT) tobacco seeds were disinfected and sown on 1 / 2 MS solid medium. After 30 days of culture, they were transferred to pots containing quartz sand and cultured at low temperature (0-4℃) for 7 days. Quantitative amplification primers targeting the Nt04g14970 gene sequence were designed for quantitative detection of Nt04g14970 gene expression. The expression of Nt04g14970 in tobacco roots, stems, and leaves was detected. The quantitative detection results are shown below. Figure 2 As shown, where, Figure 2 In this context, A represents the gene expression level before low-temperature treatment. Figure 2 In the figure, B represents the gene expression in different tissues at different time points after low-temperature treatment, with lowercase letters a, b, c, and d indicating the degree of significance of the differences. The results show that Nt04g14970 is induced by low temperature in tobacco, especially upregulated in leaves, and plays an important regulatory role in tobacco's adaptation to low temperatures. Overexpression of this gene in tobacco can help improve the low-temperature tolerance of tobacco and can also be used to create low-temperature tolerant tobacco lines, thus having application value in the creation of tobacco lines with superior traits.
[0054] To further verify the expression of the Nt04g14970 gene under different temperature conditions, the expression level of Nt04g14970 was determined by qRT-PCR. Wild-type tobacco (WT) and two overexpression transgenic materials (OE1 and OE2) were used to detect the expression of the Nt04g14970 gene under normal temperature (NT) and low temperature (0-4℃) (LT) conditions. The quantitative results are as follows: Figure 3As shown in the figure, lowercase letters a, b, c, and d indicate the degree of significance of the differences. The results showed that the relative expression level of the Nt04g14970 gene in Nt04g14970-OE transgenic plants was significantly higher than that in WT plants under normal temperature (NT) and low temperature (LT) conditions, and the expression level of the Nt04g14970 gene in wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2) was significantly upregulated under low temperature conditions.
[0055] 2. Impact on biomass
[0056] Further measurements were taken of wild-type tobacco (WT) and two overexpression transgenic materials (OE1 and OE2) and their fresh weight biomass at different temperatures (normal temperature NT and low temperature LT). The results are shown in [Table / Reference]. Figure 4 As shown, where, Figure 4 In this context, A represents the growth phenotype of the strain. Figure 4 In the figure, B represents the statistical quantitative result of wet biomass, and the lowercase letters a and b indicate the degree of significance of the difference. The results showed that under low temperature (LT) conditions, the biomass of Nt04g14970 overexpression materials (OE1 and OE2) was increased by 27.2% and 21.9% respectively compared with WT plants, and the difference was statistically significant.
[0057] 3. Effects on photosynthetic rate, transpiration rate and stomatal conductance of leaves
[0058] Using conventional methods, the photosynthetic rate, transpiration rate, and stomatal conductance of leaves from wild-type tobacco (WT) and two overexpression transgenic materials (OE1 and OE2) were further determined.
[0059] The test results are shown below. Figure 5 The above, wherein, Figure 5In the figures, A represents the photosynthetic rate under different temperature treatments, B represents the transpiration rate under different temperature treatments, and C represents the stomatal conductance under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significance of the differences. The results showed that under low temperature (LT) conditions, the photosynthetic rate of Nt04g14970 overexpression materials (OE1 and OE2) was significantly increased by 16.7% and 16.2% compared to WT plants; under LT conditions, the transpiration rate of Nt04g14970 overexpression materials (OE1 and OE2) was significantly increased by 56.6% and 48.2% compared to WT plants; and under LT conditions, the stomatal conductance of Nt04g14970 overexpression materials (OE1 and OE2) was significantly increased by 40.7% and 43% compared to WT plants.
[0060] 4. Effects on CAT and MDA content
[0061] Using conventional methods, the catalase (CAT) and malondialdehyde (MDA) contents of wild-type tobacco (WT) and two overexpression transgenic materials (OE1 and OE2) were further determined.
[0062] The test results are shown below. Figure 6 The above, wherein, Figure 6 In the figures, A represents the MDA content of plants under different temperature treatments, and B represents the CAT content of plants under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significance of the differences. The results showed that under low-temperature conditions, the CAT activity of the Nt04g14970 overexpression material was significantly higher than that of WT plants, while the MDA content was significantly lower, indicating that overexpression of Nt04g14970 in tobacco significantly improved the antioxidant capacity of the plant under low-temperature conditions.
[0063] 5. Effects on soluble protein and proline content
[0064] Using conventional methods, the soluble protein and proline content of wild-type tobacco (WT) and two overexpression transgenic materials (OE1 and OE2) were further determined.
[0065] The test results are shown below. Figure 6 The above, wherein, Figure 6In the figures, A represents the soluble protein content of plants under different temperature treatments, and B represents the proline content of plants under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significance of the differences. The results showed that under low temperature conditions, the soluble protein content of the Nt04g14970 overexpression material was significantly increased by 17.5% and 19.1% compared to the WT plants; simultaneously, the proline content of the Nt04g14970 overexpression material was significantly increased by 36.1% and 40.4% compared to the WT plants.
[0066] In summary, this invention provides a tobacco Nt04g14970 gene. Overexpression of this gene in tobacco can significantly improve the low-temperature tolerance of tobacco and also help increase its biomass. Measurements showed that tobacco lines overexpressing the Nt04g14970 gene, compared to wild-type tobacco, exhibited significantly increased biomass, photosynthetic rate, transpiration rate, stomatal conductance, antioxidant capacity, and soluble protein and proline content under low-temperature conditions, thus contributing to improved carbon sequestration and demonstrating significant application value in tobacco cultivation.
[0067] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. A tobacco gene that enhances the cold resistance of tobacco. Nt04g14970 Its characteristics are, The nucleotide sequence of this gene is shown in SEQ ID NO.
5.
2. The tobacco gene as described in claim 1 Nt04g14970 Its application in the tobacco cultivation field is characterized by, The application is as follows: Preparation of low-temperature resistant tobacco strains; Increase tobacco biomass; Enhance the antioxidant capacity of tobacco; To improve the photosynthetic rate, transpiration rate and stomatal conductance of tobacco; Increase the soluble protein and proline content of tobacco.
3. The tobacco gene as described in claim 1 Nt04g14970 Applications in enhancing carbon sequestration.
4. A method for preparing a low-temperature resistant tobacco strain, characterized in that, It includes the following steps: S1. Construct a tobacco gene with an expressible nucleotide sequence as shown in SEQ ID NO.
5. Nt04g14970 Overexpression vectors; S2. The obtained overexpression vector was transformed into Agrobacterium to obtain the tobacco gene that can be overexpressed. Nt04g14970 Overexpression of Agrobacterium; S3. The obtained overexpressing Agrobacterium was transfected into tobacco, and after screening and identification, the overexpressing tobacco gene was obtained. Nt04g14970 The tobacco plants that are resistant to low temperatures are the low-temperature resistant tobacco strains.
5. The preparation method according to claim 4, characterized in that, The overexpression vector was selected from the pCAMBIA1305 plasmid.
6. The preparation method according to claim 4, characterized in that, The Agrobacterium mentioned is selected from Agrobacterium tumefaciens.
7. The preparation method according to claim 6, characterized in that, The Agrobacterium tumefaciens was selected from EHA105.