Tobacco gene Nt04g14970 and application of tobacco gene Nt04g14970 in influence on cold resistance of tobacco and enhancement of carbon sink

Abstract

The invention discloses a tobacco gene Nt04g14970 and application of the tobacco gene Nt04g14970 in influence on tobacco cold resistance and enhancement of carbon sink, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the gene Nt04g14970 is as shown in SEQ ID NO. 5. Researches show that compared with wild tobacco, the Nt04g14970 gene overexpression tobacco strain obtained by transfecting the gene in the tobacco can significantly improve the low-temperature tolerance of the tobacco, is also helpful to improve the biomass of the tobacco, and has the advantages of low-temperature biomass, photosynthetic rate, transpiration rate, stomatal conductance and oxidation resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco, low-temperature resistance of the tobacco and low-temperature resistance of the tobacco. The content of soluble protein and the content of proline are remarkably increased, carbon sink can be improved, and the method has a remarkable application value in the field of tobacco planting.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a tobacco gene Nt04g14970 and an application thereof in preparing low-temperature-resistant and carbon-sink-enhanced tobacco strains. Background Art

[0002] In the global carbon cycle, terrestrial ecosystems are the bridge to build a global carbon balance. As the main body of terrestrial ecosystems, farmland ecosystems have an important carbon sink effect. They can fix and absorb CO2 in the atmosphere through vegetation photosynthesis. 2 The carbon sink function plays an important role in stabilizing and even reducing the concentration of greenhouse gases in the atmosphere.

[0003] As a special leaf-based economic crop, tobacco has a large leaf area, and the photosynthesis of its leaves is bound to be of great significance to the carbon sink and emission reduction of tobacco field ecosystems. However, tobacco often encounters low temperature disasters during the seedling period to the growing season, which seriously affects the leaf growth, yield and quality of flue-cured tobacco. The current anti-freeze measures for tobacco fields mainly include mulching, covering with materials such as straw and branches and leaves, and implementing physical measures such as fumigation and fogging. Cultivating tobacco varieties with strong resistance to low temperatures is of great significance to the carbon sink and emission reduction of the tobacco industry and tobacco fields.

[0004] The MADS-box gene family represents an important transcription factor that is widely present in fungi, animals and plants. The identification of the MADS-box gene family has been extensively characterized in many plant species, including Arabidopsis, rice, maize, cotton, etc. At present, it is known that MADS-box genes play key roles in various processes, including flower and seed development, controlling flowering time and regulating fruit ripening. At the same time, MADS-box genes are also involved in the response of plants to various abiotic and biotic stress factors. For example, the rice genes OsMADS55 and OsMADS57 are regulated by low temperature induction, and in pepper, the JX402926 gene is induced by low temperature, and enhancing its expression can improve the tolerance of pepper to low temperature. However, there are no reports on whether the enhanced expression of tobacco-related MADS-box family genes can tolerate low temperature. Summary of the invention

[0005] The purpose of the present invention is to provide a tobacco gene Nt04g14970 and its application in affecting tobacco cold resistance and enhancing carbon sequestration, which can be used to prepare tobacco strains with excellent traits and has application prospects in the field of tobacco planting.

[0006] In order to achieve the above object, the present invention provides a tobacco gene Nt04g14970 that affects the cold resistance of tobacco. The nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0007] The present invention also provides a primer for amplifying and identifying the tobacco gene Nt04g14970. The nucleotide sequence of the primer is shown in SEQ ID NOs. 1 and 2.

[0008] The tobacco gene Nt04g14970 provided by the present invention can be used in the field of tobacco planting, including:

[0009] Used to prepare low-temperature resistant tobacco strains; increase tobacco biomass; improve tobacco's antioxidant capacity; increase tobacco's photosynthetic rate, transpiration rate and stomatal conductance; and increase tobacco's soluble protein and proline content.

[0010] The tobacco gene Nt04g14970 provided by the present invention can be used to enhance carbon sink, and can help enhance carbon sink by increasing the absorption and conversion of carbon dioxide and improving the biomass of tobacco.

[0011] The present invention also provides a method for preparing a low-temperature resistant tobacco strain, comprising the following steps:

[0012] S1. Constructing an overexpression vector capable of expressing the tobacco gene Nt04g14970 having a nucleotide sequence as shown in SEQ ID NO.5;

[0013] S2, transferring the obtained overexpression vector into Agrobacterium to obtain overexpression Agrobacterium capable of overexpressing tobacco gene Nt04g14970;

[0014] S3. Transfect tobacco with the obtained overexpression Agrobacterium, and after screening and identification, obtain tobacco plants overexpressing the tobacco gene Nt04g14970, which are low-temperature tolerant tobacco strains.

[0015] Preferably, the overexpression vector in the above preparation method is selected from pCAMBIA1305 plasmid.

[0016] Preferably, the Agrobacterium in the above preparation method is selected from Agrobacterium tumefaciens, more preferably from EHA105.

[0017] The present invention has the following advantages:

[0018] The present invention discloses for the first time a tobacco gene Nt04g14970 related to low temperature tolerance in tobacco, and provides a research basis for the gene function, expression pathway, and subsequent functions of the gene.

[0019] The present invention also provides multiple uses of the tobacco gene Nt04g14970, such as preparing low-temperature resistant tobacco strains; increasing tobacco biomass; increasing tobacco antioxidant capacity; increasing tobacco photosynthetic rate, transpiration rate and stomatal conductance; increasing tobacco soluble protein and proline content, etc., and has significant application value in the field of tobacco planting.

[0020] The present invention also provides a method for preparing a tobacco strain with excellent low-temperature resistance traits. After screening, a tobacco strain with significantly improved low-temperature tolerance can be obtained, which is helpful for constructing a tobacco resource library with excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Phylogenetic analysis results of 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 various parts.

[0023] Figure 3 Overexpression effects of wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2) under low temperature stress.

[0024] Figure 4 Effect of low temperature stress on the biomass of wild-type tobacco (WT) and Nt04g14970 overexpressing materials (OE1 and OE2).

[0025] Figure 5 The effects of low temperature stress on photosynthesis of tobacco leaves of wild-type tobacco (WT) and Nt04g14970 overexpressing materials (OE1 and OE2).

[0026] Figure 6 Effects of low temperature stress on tobacco antioxidant enzymes in wild-type tobacco (WT) and Nt04g14970 overexpressing materials (OE1 and OE2).

[0027] Figure 7 The effects of low temperature stress on tobacco soluble proteins and proline in wild-type tobacco (WT) and Nt04g14970 overexpression materials (OE1 and OE2). DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] Experimental Example 1 Gene Acquisition

[0031] Based on the tobacco genome database, local BLAST and HMM analysis were performed, and an unrooted phylogenetic tree was established by combining the obtained MADS protein sequences with rice and tomato MADS-box family gene protein sequences. Among them, the study found that the tobacco MADS-box family gene Nt04g14970 had a close evolutionary relationship with rice genes OsMADS55, OsMADS57 and other low-temperature tolerance genes. The nucleotide sequence of this gene is shown in SEQ ID NO.5, and its phylogenetic analysis results are shown in Figure 1 So far, the gene function and expression pathway of the tobacco gene Nt04g14970 have not been reported.

[0032] Experimental Example 2 Construction of tobacco gene Nt04g14970 overexpression vector

[0033] The tobacco gene Nt04g14970 overexpression vector was constructed, and the specific steps were as follows:

[0034] 1) Extracting total RNA from tobacco Yunyan K326;

[0035] 2) Reverse transcription of the total RNA extracted in step 1) to synthesize total cDNA;

[0036] 3) Using the total cDNA obtained in step 2) as a template, PCR primers (Nt04g14970-F, Nt04g14970-R, the specific sequence is as follows) were designed according to the cDNA sequence of tobacco Nt04g14970, and the PCR product contained the complete Nt04g14970 reading frame (from the start codon ATG to TAG), and the amplified product was the cDNA of the Nt04g14970 gene, and the specific nucleotide sequence was 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) According to the cDNA sequence of the wild-type Yunyan K326 Nt04g14970 gene, amplification primers were designed, and the amplification product contained the complete Nt04g14970 reading frame (from the start codon ATG to the stop codon TAG), and restriction endonuclease sites SwaI and SacI and homology arms were introduced on the upstream and downstream primers, respectively, and PCR amplification primers for homologous recombination were designed (Nt04g14970 OE-F, Nt04g14970 OE-R, the specific sequence is as follows), and the cDNA of the Nt04g14970 gene obtained in 3) was used as a template. The target PCR product was separated by agarose electrophoresis and then recovered by gel cutting. At the same time, the plant vector pCAMBIA1305 plasmid was double-digested with SwaI and SacI, and then the recovered enzyme-digested PCR product fragments and the vector were homologously recombined. The recombinant product was transferred into Escherichia coli DH5α competent cells and spread on LB solid medium containing antibiotics. Positive colonies were selected for sequencing, and the bacterial solution with correct sequencing was stored at -70°C with an equal volume of 30% glycerol for later use. The overexpression clone 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] Experimental Example 3 Construction of Nt04g14970 gene overexpression tobacco strain

[0049] The overexpression clone plasmid Nt04g14970-OE obtained in Experimental Example 2 was transformed into competent cells of Agrobacterium tumefaciens EHA105, and positive colonies were obtained by empirical determination. The details are as follows:

[0050] Cut the leaves of Yunyan K326 into small pieces, soak them in EHA105 Agrobacterium solution adjusted to an appropriate OD value, and perform transfection according to the conventional Agrobacterium transfection method. Then transfer the leaves to filter paper to absorb the excess bacterial solution, and place them on the co-culture medium to promote the absorption of Agrobacterium by the leaves under dark culture conditions. The leaves after co-culture are transferred to a differentiation medium containing a selection agent and cultured under light to screen out the successfully transformed tobacco plants. The selected transformed tobacco plants are transferred to the rooting medium to promote their rooting. PCR identification: The rooted tobacco seedlings are identified at the molecular level by the PCR method to confirm whether the exogenous gene is successfully integrated into the tobacco genome, and the enhanced expression Nt04g14970-OE tobacco plants are obtained. Subsequently, the Nt04g14970-OE transgenic homozygous line is obtained by further cultivation and screening, which is the Nt04g14970 gene overexpression tobacco strain.

[0051] Experimental Example 4: Verification of the characteristics of the overexpression strain

[0052] 1. Verification of low temperature tolerance

[0053] Wild-type Yunyan K326 (WT) tobacco seeds were sterilized and sown on 1 / 2MS solid culture medium. After 30 days of culture, they were moved to a pot filled with quartz sand and cultured at low temperature (0-4°C) for 7 days. Quantitative amplification primers were routinely designed for the Nt04g14970 gene sequence for quantitative detection of the expression of the Nt04g14970 gene. The expression of Nt04g14970 in tobacco roots, stems and leaves was detected. The quantitative detection results are shown in Figure 2 As shown, Figure 2 A in the figure is the gene expression before low temperature treatment. Figure 2 B in the figure is the gene expression in each tissue at different time periods after low temperature treatment, and the lowercase letters a, b, c, and d indicate the degree of significant difference. The results show that Nt04g14970 is induced by low temperature in tobacco, especially up-regulated in leaves, and plays an important regulatory role in tobacco adaptation to low temperature. Overexpression of this gene in tobacco helps to improve the low temperature tolerance of tobacco, and can also be used to create low temperature resistant tobacco strains, which has application value in the creation of tobacco strains with excellent traits.

[0054] To further verify the expression of 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 taken to detect the expression of Nt04g14970 gene under normal temperature (NT) and low temperature 0-4℃ (LT). The quantitative results are shown in Figure 3As shown, lowercase letters a, b, c, and d indicate significant differences. The results showed that the relative expression 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 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] The fresh weight biomass of wild-type tobacco (WT) and two overexpressed transgenic materials (OE1 and OE2) at different temperatures (normal temperature NT and low temperature LT) was further measured. The results are shown in Figure 4 As shown, Figure 4 A in the figure is the growth phenotype of the strain. Figure 4 B in the figure is the statistical quantitative result of wet weight biomass, and the lowercase letters a and b indicate the significant difference. The results showed that under low temperature LT conditions, the biomass of Nt04g14970 overexpression materials (OE1 and OE2) increased by 27.2% and 21.9% compared with WT plants, and the difference reached a significant level.

[0057] 3. Effects on photosynthetic rate, transpiration rate and stomatal conductance of leaves

[0058] The conventional methods were used to further measure the photosynthetic rate, transpiration rate and stomatal conductance of leaves of wild-type tobacco (WT) and two overexpressing transgenic materials (OE1 and OE2).

[0059] The results of the test are shown in Figure 5 Said, wherein, Figure 5A is the photosynthetic rate under different temperature treatments, B is the transpiration rate under different temperature treatments, and C is the stomatal conductance under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significant difference. The results showed that under low temperature conditions (LT), the photosynthetic rate of Nt04g14970 overexpression materials (OE1 and OE2) increased by 16.7% and 16.2% compared with WT plants, and the difference reached a significant level; under low temperature conditions, the transpiration rate of Nt04g14970 overexpression materials (OE1 and OE2) increased by 56.6% and 48.2% compared with WT plants, and the difference reached a significant level; under low temperature conditions, the stomatal conductance of Nt04g14970 overexpression materials (OE1 and OE2) increased by 40.7% and 43% compared with WT plants, and the difference reached a significant level.

[0060] 4. Effect on CAT and MDA content

[0061] The catalase (CAT) and malondialdehyde (MDA) contents of wild-type tobacco (WT) and two overexpressing transgenic materials (OE1 and OE2) were further determined using conventional methods.

[0062] The results of the test are shown in Figure 6 Said, wherein, Figure 6 A in the figure is the MDA content of plants under different temperature treatments, and B is the CAT content of plants under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significant difference. The results showed that under low temperature conditions, the CAT activity of Nt04g14970 overexpression materials was significantly higher than that of WT plants, and the MDA content was significantly lower than that of WT plants, indicating that the overexpression of Nt04g14970 in tobacco significantly improved the antioxidant capacity of plants under low temperature conditions.

[0063] 5. Effects on soluble protein and proline content

[0064] Conventional methods were used to further determine the soluble protein and proline contents of wild-type tobacco (WT) and two overexpressing transgenic materials (OE1 and OE2).

[0065] The results of the test are shown in Figure 6 Said, wherein, Figure 6A is the soluble protein content of plants under different temperature treatments, and B is the proline content of plants under different temperature treatments. The lowercase letters a, b, and c indicate the degree of significant difference. The results showed that under low temperature conditions, the soluble protein content of Nt04g14970 overexpression materials increased by 17.5% and 19.1% compared with WT plants, and the difference reached a significant level; at the same time, the proline content of Nt04g14970 overexpression materials increased by 36.1% and 40.4% compared with WT plants, and the difference reached a significant level.

[0066] In summary, the present invention provides a tobacco Nt04g14970 gene, which can significantly improve the low temperature tolerance of tobacco when overexpressed in tobacco, and also helps to increase the biomass of tobacco. It was found through measurement that the tobacco strains overexpressing the Nt04g14970 gene, relative to wild-type tobacco, significantly increased biomass, photosynthetic rate, transpiration rate and stomatal conductance, antioxidant capacity, soluble protein and proline content under low temperature conditions, helped to improve carbon sink, and had significant application value in the field of tobacco planting.

[0067] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A tobacco gene Nt04g14970 affecting tobacco cold tolerance, characterized in that: The nucleotide sequence of the gene is shown as SEQ ID NO.

5.

2. A primer for amplifying and identifying the tobacco gene Nt04g14970 as claimed in claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NOs. 1 and 2.

3. Use of the tobacco gene Nt04g14970 as claimed in claim 1 in the field of tobacco planting.

4. The use according to claim 3, characterized in that: The app contains: preparing low temperature resistant tobacco strains; Increase tobacco biomass; Improve the antioxidant capacity of tobacco; Improve the photosynthetic rate, transpiration rate and stomatal conductance of tobacco; Increase the soluble protein and proline content of tobacco.

5. Application of a tobacco gene Nt04g14970 in enhancing carbon sequestration.

6. A method for preparing a low-temperature resistant tobacco strain, characterized in that: The following steps are included: S1. Constructing an overexpression vector capable of expressing the tobacco gene Nt04g14970 having a nucleotide sequence as shown in SEQ ID NO.5; S2, transferring the obtained overexpression vector into Agrobacterium to obtain overexpression Agrobacterium capable of overexpressing tobacco gene Nt04g14970; S3. Transfect tobacco with the obtained overexpression Agrobacterium, and after screening and identification, obtain tobacco plants overexpressing the tobacco gene Nt04g14970, which are low-temperature tolerant tobacco strains.

7. The preparation method according to claim 5, characterized in that: The overexpression vector is selected from pCAMBIA1305 plasmid.

8. The preparation method according to claim 5, characterized in that: The Agrobacterium is selected from Agrobacterium tumefaciens.

9. The preparation method according to claim 7, characterized in that: The Agrobacterium tumefaciens is selected from EHA105.

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