A tobacco drought-resistant gene Nt-zfhd-12 and its application

Abstract

This invention discloses a tobacco drought-resistant gene Nt-zfhd-12 and its applications. The nucleotide sequence of the tobacco drought-resistant gene Nt-zfhd-12 includes the sequence shown in SEQ ID No. 1. The amino acid sequence encoded by the tobacco drought-resistant gene Nt-zfhd-12 of this invention is shown in SEQ ID No. 2. Under drought stress conditions, the expression level of the Nt-zfhd-12 gene in tobacco seedling leaves increases nearly 60-fold. After amplification, enzyme digestion, construction of an expression vector, and Agrobacterium infection, the gene was transferred into tobacco. The resulting Nt-zfhd-12 transgenic tobacco showed a significant increase in root length under drought stress conditions compared to the wild type, proving that the Nt-zfhd-12 gene is a drought-resistant gene. The functional identification of the tobacco Nt-zfhd-12 gene enriches the gene resources for tobacco drought-resistant breeding and has important theoretical and applied value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a tobacco drought-resistant gene Nt-zfhd-12 and its applications. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) belongs to the Solanaceae family and is an annual herbaceous plant of the genus Nicotiana L. It is one of my country's important economic crops and plays a vital role in the national economy. my country has the world's largest tobacco planting area, reaching 13.318 million mu (approximately 984,667 hectares) in 2021, with annual output accounting for about 30% of global production, ranking first in the world. Abiotic stress caused by external environmental factors has a significant impact on tobacco quality and yield, especially drought stress, which disrupts the physiological and biochemical reactions within the tobacco plant, severely affecting its growth and development and causing significant yield reductions in agricultural production. Therefore, drought severely restricts the further development of my country's tobacco industry.

[0003] How to cope with unfavorable drought conditions and improve tobacco yield and quality has become an urgent problem to be solved. Therefore, cultivating new drought-resistant tobacco varieties is crucial for the development of the tobacco industry. Compared with traditional breeding, using molecular biology techniques to cultivate new drought-resistant and alkali-tolerant tobacco varieties has the advantages of shorter cycle, higher efficiency, and stronger targeting. The discovery and utilization of drought-resistant genes is of great practical significance for cultivating drought-resistant varieties to ensure the quality and yield of tobacco.

[0004] For example, CN107058340A discloses a tobacco drought-resistance gene NtSAP5, its cloning method, and its applications. Overexpression of this gene can improve the adaptability of tobacco to drought stress, and NtSAP5 has broad application prospects in the field of tobacco drought resistance. In drought-treated tobacco seedling leaves, the ubiquitin E3 ligase-encoding gene NtSAP5 was induced to express. The protein NtSAP5 is a key enzyme in the degradation pathway of plant ubiquitinated proteins, and regulating the expression of NtSAP5 can modulate plant resistance to drought.

[0005] Currently, there are few genes in tobacco that have been clearly identified as having drought resistance functions, and there is an urgent need to discover and provide a gene related to the drought resistance of tobacco. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a tobacco drought-resistant gene Nt-zfhd-12 and its application. Transgenic lines obtained by transforming tobacco with the Nt-zfhd-12 gene exhibit significantly longer root lengths than wild-type lines under drought stress conditions, demonstrating that this gene can effectively improve the drought resistance of plants.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a tobacco drought-resistant gene Nt-zfhd-12, the nucleotide sequence of which includes the sequence shown in SEQ ID No. 1.

[0009] This invention screened a tobacco gene, Nt-zfhd-12, whose expression level significantly increased under drought stress through transcriptome sequencing analysis. Quantitative real-time PCR verified that the gene expression level in tobacco seedling leaves increased nearly 60-fold under drought stress. The gene was then cloned, an overexpression vector was constructed, and a recombinant strain was overexpressed. This overexpression was then transformed into tobacco to obtain transgenic tobacco plants Nt-zfhd-12-OE. It was confirmed that Nt-zfhd-12-OE plants exhibited significantly increased root length under drought stress compared to the wild type. This demonstrates that the Nt-zfhd-12 gene is a drought-resistant gene. The functional identification of the tobacco Nt-zfhd-12 gene enriches the gene resources for tobacco drought-resistant breeding and is beneficial for elucidating the diverse functions of tobacco ZFHD-type transcription factors, possessing significant theoretical and practical value.

[0010] SEQ ID No. 1:

[0011] ATGGAACTTCCAATTCAAGAGGAGGAAATGCCAATGCCAATTAACAGCACATATGTTGGAGGAGGAGAAGGACATGGTCATGGCCACATAATACATCATGATCCTACACCAGTACCCAACAATACTAATCACATTATACCTTCTTCAGTAGCAAATTCCATCAATGGCCCTCCCATTGAAGCAACACCAGTATCAGCAGACCATCATGTACCTTACAAGAAGATTGTCAGGTACAAAGAATGCCTCAAGAACCATGCAGCTGCAATGGGTGGCAATGCAACTGATGGATGTGGTGAGTTTATGCCAAGTGGTGAAGAAGGTACAATTGAAGCACTCATTTGTTCAGCTTGCAATTGTCATAGAAATTTCCACAGAAAAGAGATGGAAGGTGAGCAGCAACTTCTTCAACTACCACCTTCCTCTTGTGATTACTATAATCTCAACAGGGGAGGAAAAAAAGTTTATTTAGGACACTCACACAACCATCACAAAGGCCTTTTGGGACCTGAACCATTTGGAACTATAATCCCTTCTAGAGCAGCAGCACCACATCATCATCAGATGATAATGTCATATAATAATATGGGGTCACTTCCTTCTGAGTCAGAAGAGCATGAAGATATTAATGGTGGTGGTGGGGTTATGGCTATGGCTATGGCTAGGCCATTACATCATCATAATCAGATAGTGAAGAAAAGGTTTAGAACAAAATTTACTCAAGAACAGAAGGAGAAAATGTTCAACTTTGCAGAGAAAGTTGGCTGGAAAATACAAAAACAAGAGGAAACTGTGGTGCAACAATTCTGTCAAGAACTTGGAATTAAAAGAAGAGTACTTAAAGTTTGGATGCACAACAATAAGCACAGTCTTGCCAAGAAAAACTCCAACAGTACCATTCCTCAAAATCAAGTTTAA。

[0012] It is understood that any gene with similar function obtained by making certain base mutations (such as base substitution, deletion, etc.) on the tobacco salt tolerance gene Nt-zfhd-12 discovered in this invention should be within the scope of protection of this invention.

[0013] Preferably, the amino acid sequence of the protein encoded by the tobacco drought-resistant gene Nt-zfhd-12 is shown in SEQ ID No. 2.

[0014] SEQ ID No. 2:

[0015] MELPIQEEEEMPMPINSTYVGGGEGHGHGHIIHHDPTPVPNNTNHIIPSSVANSINGPPIEATPVSADHHVPYKKIVRYKECLKNHAAAMGGNATDGCGEFMPSGEEGTIEALICSACNCHRNFHRKEMEGEQQLLQLPPSSCDYYNLNRGGK KVYLGHSHNHHKGLLGPEPFGTIIPSRAAAPHHHQMIMSYNNMGSLPSESEEHEDINGGGGVMAMAMARPLHHHNQIVKKRFRTKFTQEQKEKMFNFAEKVGWKIQKQEETVVQQFCQELGIKRRVLKVWMHNNKHSLAKKNSNSTIPQNQV.

[0016] In a second aspect, the present invention provides the application of the tobacco drought resistance gene Nt-zfhd-12 described in the first aspect and / or the reagent for overexpressing the tobacco drought resistance gene Nt-zfhd-12 in improving plant drought resistance.

[0017] Thirdly, the present invention provides a method for improving the drought resistance of plants, the method comprising the following steps:

[0018] (1) Amplify the tobacco drought resistance gene Nt-zfhd-12 described in the first aspect using primers;

[0019] (2) The tobacco drought resistance gene Nt-zfhd-12 was cloned, digested with enzymes, purified, and then ligated into an expression vector to obtain an overexpression recombinant vector;

[0020] (3) Transform the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0021] (4) Transform the overexpressing recombinant strain into plants, screen and obtain the Nt-zfhd-12 transgenic line.

[0022] 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.

[0023] SEQ ID No. 3: GGATCCATGGAACTTCCAATTCAAGAGGAG.

[0024] SEQ ID No. 4: GAGCTCTTAAACTTGATTTTGAGGAATGGT.

[0025] Preferably, the amplification conditions in step (1) are: pre-denaturation at 96-98℃ (e.g., 96℃, 97℃, 98℃) for 20-30 s (e.g., 20 s, 25 s, 30 s); denaturation at 96-98℃ (e.g., 96℃, 97℃, 98℃) for 10-20 s (e.g., 10 s, 15 s, 20 s); annealing at 56-58℃ (e.g., 56℃, 57℃, 58℃) ​​for 10-20 s (e.g., 10 s, 15 s, 20 s); and annealing at 70-72℃ (e.g., 70℃, 71℃, 72℃) for 40-50 s (e.g., 40 s, 45 s, 50 s). 30-40 cycles (e.g., 30, 35, 40 cycles); followed by 10-15 min extension at 70-72℃ (e.g., 96℃, 97℃, 98℃) (e.g., 10 min, 13 min, 15 min).

[0026] Preferably, the expression vector in step (2) includes the pCambia2300EC vector, which contains a 35S promoter and a NOS terminator.

[0027] Preferably, the plant mentioned in step (4) includes tobacco.

[0028] Fourthly, the present invention provides the application of the tobacco drought-resistant gene Nt-zfhd-12 described in the first aspect in the breeding of drought-resistant plant varieties.

[0029] Preferably, the plant includes tobacco.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention screened a tobacco gene, Nt-zfhd-12, whose expression level significantly increased under drought stress through transcriptome sequencing analysis. Quantitative real-time PCR verified that the gene expression level in tobacco seedling leaves increased nearly 60-fold under drought stress. The gene was then cloned, an overexpression vector was constructed, and a recombinant strain was overexpressed. This overexpression was then transformed into tobacco to obtain transgenic tobacco plants Nt-zfhd-12-OE. It was confirmed that Nt-zfhd-12-OE plants exhibited significantly increased root length under drought stress compared to the wild type. This demonstrates that the Nt-zfhd-12 gene is a drought-resistant gene. The functional identification of the tobacco Nt-zfhd-12 gene enriches the gene resources for tobacco drought-resistant breeding and is beneficial for elucidating the diverse functions of tobacco ZFHD-type transcription factors, possessing significant theoretical and practical value. Attached Figure Description

[0032] Figure 1 The graph shows the expression levels of the Nt-zfhd-12 gene under drought stress.

[0033] Figure 2 The graph shows the drought tolerance of the overexpression lines after 10 days of drought stress and 3 days of recovery.

[0034] Figure 3 Figure showing the survival rates of overexpression strains and wild-type tobacco.

[0035] Figure 4 Figure showing the water content of abscission leaves of the overexpression line and wild-type tobacco. Detailed Implementation

[0036] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0037] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0038] This invention screened a tobacco gene, Nt-zfhd-12, whose expression level significantly increased under drought stress through transcriptome sequencing analysis. Its nucleotide sequence is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No. 2. RT-qPCR verification revealed that under drought stress, the expression level of the Nt-zfhd-12 gene in tobacco seedling leaves increased nearly 60-fold, preliminarily confirming that this gene plays an important role in tobacco's response to drought stress. Furthermore, using tobacco leaf cDNA as experimental material, the Nt-zfhd-12 gene fragment was amplified by PCR; after enzyme digestion and ligation to construct an expression vector, it was transferred into tobacco plants using Agrobacterium infection, obtaining Nt-zfhd-12 gene-transgenic tobacco plants. This demonstrated that the gene significantly improved the resistance of transgenic tobacco plants to drought stress, thus proving that the Nt-zfhd-12 gene is a drought-resistant gene. Therefore, the functional identification of the tobacco Nt-zfhd-12 gene not only enriches the gene resources for tobacco drought-resistant breeding, but also helps to elucidate the diverse functions of tobacco ZFHD transcription factors, which has important theoretical significance and application value.

[0039] Example 1

[0040] This embodiment provides a method for cloning the tobacco drought-resistant gene Nt-zfhd-12.

[0041] (1) Using the coding region sequence of the tobacco Nt-zfhd-12 gene as a reference, specific primers Nt-zfhd-12-F and Nt-zfhd-12-R were designed. The primer sequences are as follows:

[0042] Nt-zfhd-12-F (SEQ ID No.3):

[0043] GGATCCATGGAACTTCCAATTCAAGAGGAG;

[0044] Nt-zfhd-12-R (SEQ ID No.4):

[0045] GAGCTCTTAAACTTGATTTTGAGGAATGGT.

[0046] (2) Extract mRNA from seedling leaves of tobacco cultivar “K326” and reverse transcribe it into cDNA, which is then used as a template for PCR amplification. PCR amplification is performed using the primers from step (1) to obtain the amplified fragment of the Nt-zfhd-12 gene.

[0047] The PCR amplification system was: 31 μL ddH2O, containing Mg. 2+10 μL of 5×HF buffer; 2 μL of 2.5 mM dNTPs; 2 μL each of Nt-zfhd-12-F and Nt-zfhd-12-R (5 μM each); 0.6 μL of DMSO; 0.5 μL of Phusion enzyme; and 2 μL of cDNA template.

[0048] The PCR reaction conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ for 50 s, for 30 cycles; extension at 72℃ for 10 min.

[0049] (3) Construction of overexpression vector of Nt-zfhd-12: The PCR amplification fragment obtained in step (2) was ligated into the cloning vector pEASY-Blunt simple. After the sequence was correct, the plasmid was extracted, digested with BamHI and SacI, purified, and ligated into the pCambia2300EC vector (which contains a 35S promoter and a NOS terminator) digested with BamHI and SacI. After the sequence was correct, Agrobacterium GV3101 was transformed by freeze-thaw method, and tobacco was genetically transformed by the infiltration method to obtain transgenic plant Nt-zfhd-12.

[0050] Example 2

[0051] This embodiment provides an application of the tobacco drought-resistant gene Nt-zfhd-12.

[0052] (1) Analysis of expression patterns in tobacco seedling leaves under drought stress

[0053] Seedlings of "K326" were treated with 200 mM PEG solution, and the dynamic changes in the expression level of the Nt-zfhd-12 gene at 0 h, 6 h, 24 h, and 48 h were analyzed using real-time PCR.

[0054] The primer sequences for real-time PCR are as follows:

[0055] Nt-zfhd-12-qF (SEQ ID No. 5): ATTCCATCAATGGCCCTCCC;

[0056] Nt-zfhd-12-qR (SEQ ID No. 6): CATCAGTTGCATTGCCACCC.

[0057] The results are as follows Figure 1As shown, the expression level of Nt-zfhd-12 increased significantly after drought stress treatment, reaching a peak at 48 h, which was nearly 60-fold higher. This preliminarily verifies that the Nt-zfhd-12 gene responds to drought stress, and further indicates that the Nt-zfhd-12 gene is involved in the drought resistance response of tobacco.

[0058] (2) Identification of drought resistance in transgenic tobacco

[0059] T3 generation seeds of the transgenic plant Nt-zfhd-12-OE and wild-type (K326) tobacco seeds were evenly sown in a soil mixture and maintained under normal conditions. Six weeks later, tobacco seedlings were subjected to drought stress for 10 days. Seedlings were then re-watered for 3 days, and seedling survival rates were calculated. Seedling leaves were collected, leaf water content was calculated, and leaf weight was recorded at five time points (0 min, 45 min, 90 min, 135 min, and 180 min). Water loss rate was calculated based on changes in leaf weight. The overexpression line Nt-zfhd-12-OE showed higher drought tolerance after 10 days of drought stress and 3 days of re-watering. Figure 2 After drought treatment and rehydration, the survival rate of Nt-zfhd-12-OE seedlings was significantly higher than that of WT seedlings. Figure 3 Over time, the leaf water content of Nt-zfhd-12-OE seedlings was significantly higher than that of WT ( Figure 4 ).

[0060] In summary, this invention screened a tobacco gene, Nt-zfhd-12, whose expression level significantly increased under drought stress through transcriptome sequencing analysis. Quantitative real-time PCR verified that the gene expression level in tobacco seedling leaves increased nearly 60-fold under drought stress. After cloning, constructing an overexpression vector, and overexpressing the recombinant strain, the gene was transformed into tobacco to obtain transgenic tobacco plants Nt-zfhd-12-OE. It was also confirmed that the root length of Nt-zfhd-12-OE plants under drought stress was significantly increased compared to the wild type. This demonstrates that the Nt-zfhd-12 gene is a drought-resistant gene. The functional identification of the tobacco Nt-zfhd-12 gene not only enriches the gene resources for tobacco drought-resistant breeding but also helps to elucidate the diverse functions of tobacco ZFHD-type transcription factors, possessing significant theoretical and practical value.

[0061] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the 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. Use of a tobacco drought-resistant gene Nt-zfhd-12 as shown in SEQ ID No. 1 in improving drought resistance of a plant, wherein the plant is tobacco.

2. Use of a reagent for overexpressing a tobacco drought-resistant gene Nt-zfhd-12 as shown in SEQ ID No. 1 in improving drought resistance of a plant, wherein the plant is tobacco.

3. A method for improving drought tolerance in plants, characterized by, The method for improving drought resistance of a plant comprises the following steps: (1) amplifying a tobacco drought-resistant gene Nt-zfhd-12 as shown in SEQ ID No. 1 using a primer; (2) cloning the tobacco drought-resistant gene Nt-zfhd-12 as shown in SEQ ID No. 1, and then connecting the tobacco drought-resistant gene Nt-zfhd-12 to an 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 a plant, and screening and obtaining an Nt-zfhd-12 transgenic line, wherein the plant is tobacco.

4. The method of increasing drought tolerance in a plant of claim 3, wherein, The nucleic acid sequence of the primer in step (1) is shown in SEQ ID No. 3 and SEQ ID No.

4.

5. The method of increasing drought tolerance in plants according to claim 3, wherein, The amplification conditions in step (1) are as follows: 96-98 °C pre-denaturation for 20-30 s; 96-98 °C denaturation for 10-20 s, 56-58 °C annealing for 10-20 s, 70-72 °C extension for 40-50 s, 30-40 cycles; and 70-72 °C post-extension for 10-15 min.

6. The method of increasing drought tolerance in plants of claim 3, wherein, The expression vector in step (2) comprises a pCambia2300EC vector, and the pCambia2300EC vector contains a 35S promoter and a NOS terminator.

7. Use of a tobacco drought-resistant gene Nt-zfhd-12 as shown in SEQ ID No. 1 in breeding a drought-resistant plant variety, wherein the plant is tobacco.

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