Tobacco salt-tolerant gene Nt-zfhd-6 and application thereof

Abstract

The invention discloses a tobacco salt-tolerant gene Nt-zfhd-6 and an application of the tobacco salt-tolerant gene Nt-zfhd-6. The nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 comprises a sequence as shown in SEQ ID No.1 (Sequence Identifier Number 1). The amino acid sequence coded by the tobacco salt-tolerant gene Nt-zfhd-6 is shown as SEQ ID No.2, and under the condition of salt stress, the expression quantity of the Nt-zfhd-6 gene in tobacco seedling leaves is increased by nearly 40 times. The Nt-zfhd-6 gene is transformed into tobacco through amplification, enzyme digestion, expression vector construction and agrobacterium infection, the root length of the obtained Nt-zfhd-6 gene transformed tobacco is obviously increased under the salt stress treatment condition compared with that of a wild type, and it is proved that the Nt-zfhd-6 gene is a salt-tolerant gene. The functional identification of the tobacco Nt-zfhd-6 gene enriches the gene resources of tobacco salt-tolerant breeding, and has important theoretical significance and application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a tobacco salt-tolerant gene Nt-zfhd-6 and an application thereof. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual herbaceous plant belonging to the Solanaceae family and the genus Nicotiana L. It is one of the important cash crops in my country and occupies an important position in the national economy. Abiotic stress caused by the external environment has an important impact on tobacco quality and yield, especially salt stress, which leads to disordered physiological and biochemical reactions in tobacco, seriously affecting the growth and development of tobacco, and causing a large reduction in agricultural production. According to Zimbabwe's largest tobacco dealer, TSL Tobacco Co., Ltd., it is expected that Zimbabwe's tobacco production will decrease by 10% to 15% in 2022 due to adverse drought weather. At the same time, due to the thin leaves and high moisture content of tobacco leaves, the color is difficult to fix during modulation, and the color and luster are dark, the oil content is low, the elasticity is poor, and the taste is light after modulation. The impact on the aroma quality and aroma of tobacco leaves is particularly serious, resulting in a sharp decline in the quality of tobacco leaves. Therefore, drought seriously restricts the further development of my country's tobacco industry.

[0003] Although my country has made significant breakthroughs in flue-cured tobacco dryland cultivation technology through long-term technical research, thus reducing the dependence on water in flue-cured tobacco cultivation to a certain extent, the construction of water conservancy facilities is relatively slow and requires large investments. After investigations in various tobacco-producing areas, it was found that the construction of water conservancy facilities in various places was significantly lagging behind, resulting in a lack of sufficient guarantee for tobacco production. During the growth of tobacco leaves, drought will cause the upper leaves to fail to fully open, and the annual tobacco leaf losses caused by water irrigation are still very huge. How to cope with adverse weather conditions caused by drought and improve tobacco yield and quality has become an urgent problem to be solved. Therefore, the cultivation of new salt-tolerant tobacco varieties is crucial to the development of the tobacco industry. Compared with traditional breeding, the use of molecular biology technology to cultivate new salt-alkali-tolerant tobacco varieties has the advantages of short cycle, high efficiency and strong targeting. The mining and utilization of salt-tolerant genes is of great practical significance for the cultivation of salt-tolerant varieties to ensure the quality and yield of tobacco.

[0004] At present, there are few genes in tobacco that have been clearly identified to have salt tolerance, and there is an urgent need to discover and provide a gene related to tobacco drought resistance. Summary of the invention

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a tobacco salt-tolerance gene Nt-zfhd-6 and an application thereof. The transgenic strain obtained by transforming the Nt-zfhd-6 gene into tobacco has a root length significantly better than that of the wild-type strain under salt stress conditions, proving that the gene can effectively improve the salt tolerance of plants.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tobacco salt-tolerance gene Nt-zfhd-6, the nucleotide sequence of which comprises the sequence shown in SEQ ID No.1.

[0008] The present invention screened a tobacco gene Nt-zfhd-6 whose expression level was significantly increased under salt stress through transcriptome sequencing analysis, verified by fluorescent quantitative PCR that the expression level of the gene in tobacco seedling leaves under salt stress was increased by nearly 40 times, and then the gene was cloned, an overexpression vector was constructed, and an overexpression recombinant strain was transformed into tobacco to obtain a transgenic tobacco plant Nt-zfhd-6-OE, and confirmed that the root length of Nt-zfhd-6-OE under salt stress was significantly increased compared with the wild type. This proves that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also helps to analyze the diverse functions of tobacco ZFHD transcription factors, which has important theoretical significance and application value.

[0009] SEQ ID No.1:

[0010] .

[0011] It can be understood that genes with similar functions obtained by performing certain base mutations (such as base substitution, deletion, etc.) on the tobacco salt-tolerance gene Nt-zfhd-6 discovered in the present invention should all be within the protection scope of the present invention.

[0012] Preferably, the amino acid sequence of the protein encoded by the tobacco salt tolerance gene Nt-zfhd-6 is shown as SEQ ID No.2.

[0013] SEQ ID No.2:

[0014] MALAGEDKEMRMQGSLGYHSLDQGNQHHHQPNNNNLQPQQDNEKSSSGTGAAPPVVVPYSSGGSTNNKFKITSRARYRECLKNHAASIGGNVTDGCGEFMPSGEEGTLEALKCAACNCHRNFHRKEQPNVDNNNAGIMVVHPLQ LPQPLPSPLPSMNHHHQHGRSVWSTMPPQPVKMAFGGSGGGGSGATDSSSEELNFNTYQQATSVPPQPQPPFMLAKKRFRTKFTQDQKEKMLEFAEKLGWRIPREDDTEVQRFCSQVGVKRQVFKVWMHNNKNPSAKKNPQEEP.

[0015] In a second aspect, the present invention provides the use of the tobacco salt-tolerance gene Nt-zfhd-6 and / or an agent for overexpressing the tobacco salt-tolerance gene Nt-zfhd-6 described in the first aspect in improving plant salt tolerance.

[0016] In a third aspect, the present invention provides a method for improving plant salt tolerance, the method for improving plant salt tolerance comprising the following steps:

[0017] (1) using primers to amplify the tobacco salt tolerance gene Nt-zfhd-6 described in the first aspect;

[0018] (2) cloning the tobacco salt tolerance gene Nt-zfhd-6, and connecting it to the expression vector after enzyme digestion and purification to obtain an overexpression recombinant vector;

[0019] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0020] (4) Transforming the overexpression recombinant strain into plants, screening and obtaining the Nt-zfhd-6 transgenic strain.

[0021] Preferably, the nucleic acid sequence of the primer in step (1) includes the sequences shown in SEQ ID No.3 and SEQ ID No.4.

[0022] SEQ ID No. 3: GGATCCATGGCATTAGCTGGTGAGGA.

[0023] SEQ ID No. 4: GAGCTCTTAAGGTTCTTCTTGTGGGTTCT.

[0024] Preferably, the amplification conditions in step (1) are: pre-denaturation at 96-98°C (e.g., 96°C, 97°C, 98°C) for 20-30s (e.g., 20s, 25s, 30s); denaturation at 96-98°C (e.g., 96°C, 97°C, 98°C) for 10-20s (e.g., 10s, 15s, 20s), annealing at 56-58°C (e.g., 56°C, 57°C, 58°C) for 10-20s (e.g., For example, 10s, 15s, 20s), 70-72°C (for example, 70°C, 71°C, 72°C) for 40-50s (for example, 40s, 45s, 50s), for 30-40 cycles (for example, 30 cycles, 35 cycles, 40 cycles); 70-72°C (for example, 96°C, 97°C, 98°C) followed by extension for 10-15min (for example, 10min, 13min, 15min).

[0025] Preferably, the expression vector in step (2) comprises a pCambia2300EC vector, wherein the pCambia2300EC vector contains a 35S promoter and a NOS terminator.

[0026] Preferably, the plant in step (4) comprises tobacco.

[0027] In a fourth aspect, the present invention provides the use of the tobacco salt-tolerance gene Nt-zfhd-6 described in the first aspect in breeding salt-tolerant plant varieties.

[0028] Preferably, the plant comprises tobacco.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention screened a tobacco gene Nt-zfhd-6 whose expression level was significantly increased under salt stress through transcriptome sequencing analysis, verified by fluorescent quantitative PCR that the expression level of the gene in tobacco seedling leaves under salt stress was increased by nearly 40 times, and then the gene was cloned, an overexpression vector was constructed, and an overexpression recombinant strain was transformed into tobacco to obtain a transgenic tobacco plant Nt-zfhd-6-OE, and confirmed that the root length of Nt-zfhd-6-OE under salt stress was significantly increased compared with the wild type. This proves that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also helps to analyze the diverse functions of tobacco ZFHD transcription factors, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is the result diagram of Nt-zfhd-6 gene expression under salt stress;

[0032] Figure 2 The figure is the salt tolerance result of the overexpression strain;

[0033] Figure 3 The figure shows the results of the malondialdehyde content test of the overexpression strain and wild-type tobacco;

[0034] Figure 4 The figure shows the results of superoxide dismutase activity detection in overexpression lines and wild-type tobacco. DETAILED DESCRIPTION

[0035] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] The present invention screens a tobacco gene Nt-zfhd-6 whose expression level is significantly increased under salt stress through transcriptome sequencing analysis, and its nucleotide sequence is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.2. Fluorescence quantitative PCR verification found that under salt stress conditions, the expression level of the Nt-zfhd-6 gene in tobacco seedling leaves increased by nearly 40 times, and it was preliminarily determined that the gene played an important role in tobacco's response to salt stress. Further, using tobacco leaf cDNA as the test material, PCR amplification was used to obtain the Nt-zfhd-6 gene fragment; after enzyme cutting and connection to construct an expression vector, it was transferred to tobacco by Agrobacterium infection, and a transgenic tobacco plant of Nt-zfhd-6 gene was obtained, and it was confirmed that the gene can significantly improve the resistance of transgenic tobacco plants to salt stress, thereby proving that the Nt-zfhd-6 gene is a salt-tolerant gene. Therefore, the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the genetic resources for tobacco salt tolerance breeding, but also helps to analyze the diverse functions of tobacco ZFHD transcription factors, which has important theoretical significance and application value.

[0038] Example 1

[0039] This example provides a method for cloning the tobacco salt tolerance gene Nt-zfhd-6.

[0040] (1) Using the coding region sequence of tobacco Nt-zfhd-6 gene as a reference, specific primers Nt-zfhd-6-F and Nt-zfhd-6-R were designed. The primer sequences are as follows:

[0041] Nt-zfhd-6-F (SEQ ID No. 3):

[0042] GGATCCATGGCATTAGCTGGTGAGGA;

[0043] Nt-zfhd-6-R (SEQ ID No. 4):

[0044] GAGCTCTTAAGGTTCTTCTTGTGGGTTCT.

[0045] (2) Extracting mRNA from the seedling leaves of tobacco cultivar "K326" and reversely transcribing it into cDNA, which is used as a template for PCR amplification. PCR amplification is performed using the primers in step (1) to obtain an amplified fragment of the Nt-zfhd-6 gene.

[0046] PCR amplification system: ddH2O 31μL, Mg 2+ 10 μL of 5×HF buffer; 2 μL of 2.5 mM dNTPs; 2 μL each of Nt-zfhd-6-F and Nt-zfhd-6-R at 5 μM concentration; 0.6 μL of DMSO; 0.5 μL of Phusion enzyme; and 2 μL of cDNA template.

[0047] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, and 72°C for 50 s, for 30 cycles; and post-extension at 72°C for 10 min.

[0048] (3) Construction of an overexpression vector of Nt-zfhd-6: The PCR amplified fragment obtained in step (2) was connected to the cloning vector pEASY-Blunt simple. After correct sequencing, the plasmid was extracted, double-digested with BamH I and Sac I, purified, and connected to the pCambia2300EC vector (which contains a 35S promoter and a NOS terminator) that had been digested with BamH I and Sac I. After correct sequencing, the Agrobacterium GV3101 was transformed by the freeze-thaw method, and the tobacco was genetically transformed by the dip-infection method to obtain the transgenic plant Nt-zfhd-6.

[0049] Example 2

[0050] This embodiment provides an application of a tobacco salt tolerance gene Nt-zfhd-6.

[0051] (1) Analysis of expression patterns in tobacco seedling leaves under salt stress

[0052] "K326" in the seedling stage was treated with 200mM NaCl solution, and the dynamic changes of the expression level of Nt-zfhd-6 gene at 0h, 6h, 24h and 48h were analyzed by fluorescence quantitative PCR technology.

[0053] Among them, the primer sequences of fluorescent quantitative PCR are:

[0054] Nt-zfhd-6-qF (SEQ ID No. 5):ATCAACACGGTCGCTCAGTT;

[0055] Nt-zfhd-6-qR (SEQ ID No. 6): GTCCGTAGCTCCACTTCCAC.

[0056] The results are as follows Figure 1 As shown, the expression level of Nt-zfhd-6 increased significantly after salt stress treatment, reaching a peak at 48 hours, an increase of nearly 40 times, which preliminarily verified that the Nt-zfhd-6 gene responded to salt stress, and further indicated that the Nt-zfhd-6 gene was involved in the salt tolerance response of tobacco.

[0057] (2) Identification of salt tolerance of transgenic tobacco

[0058] The T3 generation seeds of the transgenic plant Nt-zfhd-6-OE and wild-type (K326) tobacco seeds were evenly sown into the soil mixture and maintained under normal conditions. After 6 weeks of growth, they were treated with 200mM NaCl solution for 1 week, and wild-type tobacco seeds (WT) were used as controls. At the same time, the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of tobacco seedlings were detected by nitroblue tetrazolium (NBT) photoreduction method and TBARS-TCA method, respectively.

[0059] Results After salt treatment, the salt resistance of transgenic tobacco OE2 and OE3 plants was significantly increased compared with wild-type tobacco ( Figure 2 ), and the MDA content after salt treatment was significantly lower than that of WT ( Figure 3 ), SOD activity was significantly higher than WT ( Figure 4 ). This shows that the salt tolerance of transgenic tobacco is better than that of wild type, that is, the Nt-zfhd-6 gene can improve the salt tolerance of transgenic tobacco.

[0060] In summary, the present invention screened a tobacco gene Nt-zfhd-6 whose expression level was significantly increased under salt stress through transcriptome sequencing analysis, verified by fluorescent quantitative PCR that the expression level of the gene in tobacco seedling leaves under salt stress was increased by nearly 40 times, and then the gene was cloned, an overexpression vector was constructed, and the overexpression recombinant strain was transformed into tobacco to obtain the transgenic tobacco plant Nt-zfhd-6-OE, and it was confirmed that the root length of Nt-zfhd-6-OE under salt stress was significantly increased compared with the wild type. This proves that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also helps to analyze the diverse functions of tobacco ZFHD transcription factors, which has important theoretical significance and application value.

[0061] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A tobacco salt tolerance gene Nt-zfhd-6, characterized in that: The nucleotide sequence of the tobacco salt tolerance gene Nt-zfhd-6 includes the sequence shown in SEQ ID No.

1.

2. The tobacco salt tolerance gene Nt-zfhd-6 according to claim 1, characterized in that The amino acid sequence of the protein encoded by the tobacco salt tolerance gene Nt-zfhd-6 is shown in SEQ ID No.

2.

3. Use of the tobacco salt-tolerance gene Nt-zfhd-6 according to claim 1 or 2 and / or an agent for overexpressing the tobacco salt-tolerance gene Nt-zfhd-6 in improving plant salt tolerance.

4. A method for improving plant salt tolerance, characterized in that: The method for improving plant salt tolerance comprises the following steps: (1) using primers to amplify the tobacco salt tolerance gene Nt-zfhd-6 described in claim 1; (2) cloning the tobacco salt tolerance gene Nt-zfhd-6, and connecting it to the expression vector after enzyme digestion and purification to obtain an overexpression recombinant vector; (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) Transforming the overexpression recombinant strain into plants, screening and obtaining the Nt-zfhd-6 transgenic strain.

5. The method for improving plant salt tolerance according to claim 4, characterized in that: The nucleic acid sequences of the primers in step (1) include sequences shown as SEQ ID No.3 and SEQ ID No.

4.

6. The method for improving plant salt tolerance according to claim 4 or 5, characterized in that: The amplification conditions in step (1) are: pre-denaturation at 96-98°C for 20-30s; denaturation at 96-98°C for 10-20s, annealing at 56-58°C for 10-20s, and 70-72°C for 40-50s, for 30-40 cycles; and post-extension at 70-72°C for 10-15min.

7. The method for improving plant salt tolerance according to any one of claims 4 to 6, characterized in that: The expression vector in step (2) includes a pCambia2300EC vector, and the pCambia2300EC vector contains a 35S promoter and a NOS terminator.

8. The method for improving plant salt tolerance according to any one of claims 4 to 7, characterized in that: The plant in step (4) includes tobacco.

9. Use of the tobacco salt-tolerance gene Nt-zfhd-6 according to claim 1 or 2 in breeding salt-tolerant plant varieties.

10. The use according to claim 9, characterized in that: The plants include tobacco.

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