Tobacco potassium ion channel gene NtSKOR1B and its application in improving salt stress resistance of tobacco

Abstract

The present invention discloses the application of the tobacco potassium ion channel gene NtSKOR1B in improving the salt stress resistance of tobacco. The nucleotide sequence of the tobacco NtSKOR1B gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The present invention finds that knocking out this gene can increase the potassium ion content and biological accumulation of tobacco under salt stress, improve the K<supgt;+< / supgt; / Na<supgt;+< / supgt; ratio of tobacco cells, enhance the salt tolerance of plants; NtSKOR1B negatively regulates the salt stress resistance of tobacco.

Description

Technical Field

[0001] The present invention relates to the field of gene technology, and more specifically, to the application of the tobacco potassium ion channel gene NtSKOR1B in the salt stress resistance of tobacco. Background Art

[0002] Soil salinization causes huge losses to global agricultural production. Approximately 20% of the arable land and nearly 33% of the irrigated land in the world are being affected by salt stress. Developing crop varieties adapted to saline soil environments is one of the key strategies to address soil salinization. Understanding the salt stress response mechanism of plants and mining genes that affect the formation of plant salt stress resistance contribute to the cultivation of salt-tolerant varieties and are of great significance for the comprehensive utilization of saline-alkali land and the sustainable development of agriculture.

[0003] During evolution, plants have correspondingly developed a series of mechanisms to cope with high salt stress. Among them, maintaining a relatively stable K + / Na + ratio in cells is one of the important salt tolerance strategies of plants. Shaker family potassium channels are important proteins in plants responsible for K + absorption, transport, and maintenance of intracellular K + homeostasis. As an important member of the Shaker potassium channel family, the outward rectifying channel SKOR mediates the outward export of intracellular K + . Studies have found that heterologous expression of the melon Shaker outward rectifying potassium channel CmSKOR in Arabidopsis thaliana increased the K + / Na + ratio in plants and enhanced the salt tolerance of plants; similar to the results in melons, the rice SKOR homologous gene OSK5.2 can increase the K + / Na + ratio in xylem sap and enhance salt tolerance. The above studies indicate that SKOR potassium channels can improve the salt stress resistance of plants by increasing the intracellular K + / Na + homeostasis.

[0004] Identifying and mining genes that negatively regulate the salt tolerance of crops and using gene editing technology to improve salt-tolerant crop varieties are becoming effective strategies for the comprehensive utilization of saline-alkali land. Common tobacco (Nicotiana tabacum) is a field economic crop with relatively strong salt tolerance and is also a potential crop for the development and utilization of saline-alkali land. There is currently no report on the participation of tobacco SKOR genes in salt stress resistance. The present invention clones the NtSKOR1B gene from common tobacco, constructs tobacco NtSKOR1B knockout materials through gene editing technology, analyzes the salt tolerance of NtSKOR1B knockout materials, clarifies that this gene negatively regulates the salt tolerance of tobacco, and realizes the effective utilization of saline-alkali land by mining and screening genes related to negative regulation of salt tolerance in tobacco and carrying out breeding applications. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention proposes a new application of the tobacco potassium ion channel gene NtSKOR1B. The present invention has identified that the tobacco potassium ion channel gene NtSKOR1B has a new function of negatively regulating the salt stress resistance of tobacco, and has expanded the new use of this gene in improving the salt stress resistance of tobacco.

[0006] The technical problems to be solved by the present invention are achieved through the following technical solutions:

[0007] The present invention provides a tobacco potassium ion channel gene NtSKOR1B, characterized in that the nucleotide sequence of the gene comprises any one of the following (1) to (4):

[0008] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0009] (2) A nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO.1;

[0010] (3) A nucleotide sequence in which one or more bases are added, substituted, deleted or modified in the nucleotide sequence shown in SEQ ID NO.1 and which expresses the same functional protein; or,

[0011] (4) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions.

[0012] Preferably, the amino acid sequence of the protein encoded by the NtSKOR1B gene comprises any one of the following (5) to (7):

[0013] (5) The amino acid sequence shown in SEQ ID NO.2;

[0014] (6) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.2; or,

[0015] (7) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the amino acid sequence shown in SEQ ID NO.2 and which expresses the same functional protein.

[0016] The present invention also provides a biological material comprising the NtSKOR1B gene as claimed in claim 1, and the biological material is a recombinant expression vector, an expression cassette or a recombinant bacterium.

[0017] The present invention also provides the application of the above biological material in cultivating transgenic plants with different degrees of salt tolerance.

[0018] Preferably, the tobacco potassium ion channel gene NtSKOR1B provided by the present invention can negatively regulate the tolerance of tobacco to Na + and knocking out the NtSKOR1B gene can improve the tolerance of plants to high salt.

[0019] Preferably, the transgenic plant is tobacco.

[0020] The present invention also provides a method for cultivating transgenic plants with different degrees of salt tolerance. By introducing the recombinant expression vector of the above NtSKOR1B gene into a recipient plant to obtain a transgenic plant, or by using the CRISPR / Cas9-mediated gene editing technology, a CRISPR / Cas9 editing vector for knocking out the above NtSKOR1B gene is constructed, and a tobacco plant with edited NtSKOR1B gene is obtained after genetic transformation.

[0021] Preferably, the promoter contained in the recombinant expression vector is the 35S promoter, and the recombinant expression vector includes a binary Agrobacterium vector and a vector suitable for plant microprojectile bombardment; the recombinant expression vector contains the 3' untranslated region of the foreign gene.

[0022] Preferably, the recombinant expression vector is specifically pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, pORE-Cas9, pYLCRISPR / cas9-MT, pCas9gRNA7, pCas9gRNA7-GFP or other derivative plant expression vectors.

[0023] When using the gene to construct a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added before the transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A, etc. They can be used alone or in combination with other plant promoters.

[0024] In addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic.

[0025] The translation starting region can be derived from the transcription starting region or the structural gene. To facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector used can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants, an antibiotic marker with resistance, or an anti-chemical reagent marker gene, etc. It is also possible not to add any selectable marker gene and directly screen the transformed plants under stress.

[0026] More specifically, the recombinant expression vector is the pORE-Cas9 / gRNA-NtSKOR1B gene knockout vector.

[0027] The present invention also provides a tobacco variety, which is cultivated by the above method.

[0028] Beneficial effects

[0029] The present invention controls the salt tolerance of tobacco by regulating the expression level of the NtSKOR1B gene, and obtains transgenic crops with different expression levels of the NtSKOR1B gene and different salt tolerances through genetic engineering means;

[0030] Knocking out the NtSKOR1B gene can improve the tolerance of tobacco to high salt;

[0031] The present invention meets the requirements of the development of modern agriculture and has important practical value and market prospects for researching the salt tolerance mechanism of tobacco, increasing the utilization of new functions of tobacco, and improving the utilization of saline-alkali land. Brief description of the drawings

[0032] The drawings incorporated in the specification and constituting a part of the specification illustrate the embodiments of the present invention and, together with the description thereof, are used to explain the principles of the present invention.

[0033] Figure 1 Phylogenetic tree analysis and characteristic sequence alignment of Shaker family members of Arabidopsis thaliana, rice and tobacco, (a): Shaker K of tobacco, Arabidopsis thaliana and rice + Phylogenetic analysis of channels: The amino acid sequences of Arabidopsis thaliana and rice are from TAIR (https: / / www.arabidopsis.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) respectively. The red circle represents NTSKOR1B. A phylogenetic tree was generated by the Neighbor-Joining method using MEGA6 software, (b): The characteristic signature sequence TXXTXGYG of the selective K+ channel is highlighted by a black box, and the red circle represents NtSKOR1B.

[0034] Figure 2PCR identification of tobacco ProNtSKOR1B::GUS transgenic plants, where: M, Marker DL5000; +, pBI101-NtSKOR1Bpro plasmid; -, ZHONGYAN100.

[0035] Figure 3 Analysis of the expression pattern of NTSKOR1B under salt treatment conditions. A: Relative expression level of the qRT-PCR NtSKOR1B gene in tobacco seedlings under salt treatment conditions; B-D: GUS staining at 0 h, 12 h, and 30 h after salt treatment; E-H: GUS chemical staining of leaves, leaf veins, stems, and roots at 30 h after salt treatment.

[0036] Figure 4 Gene editing positions and protein translations of two NtSKOR1B gene knockout mutants, with black triangles representing truncated proteins.

[0037] Figure 5 Phenotypic analysis of NtSKOR1B knockout materials under salt stress. A: Phenotypes of the wild type and two knockout materials under control and salt treatment conditions. B-K: Represent the changes in leaf length, leaf width, biomass accumulation, aboveground potassium content, aboveground sodium content, aboveground potassium / sodium ratio, underground potassium content, underground sodium content, and underground potassium / sodium ratio of the wild type and two knockout materials under control and salt treatment conditions, respectively. One-way ANOVA with LSD test (*p < 0.05) was used to analyze statistical significance. Detailed implementation manners

[0038] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions, and values set forth in these embodiments do not limit the scope of the present invention.

[0039] Example 1. Construction of NtSKOR1B knockout materials

[0040] 1. Using the LOC104103384 gene sequence (GenBank No. XM_009611282.3) as a template, specific primers NtSKOR1B-1F / NtSKOR1B-1R were designed, and PCR amplification was performed using common tobacco cDNA as a template, named the NtSKOR1B gene.

[0041] The nucleotide sequence of the NtSKOR1B gene is shown as follows:

[0042]

[0043]

[0044] The amino acid sequence of the NtSKOR1B protein encoded by the NtSKOR1B gene is as follows:

[0045]

[0046] NtSKOR1B-1F: (SEQ ID NO: 3)

[0047] NtSKOR1B-1R: (SEQ ID NO: 4)

[0048] 2. Determine the knockout site according to the obtained NtSKOR1B sequence (SEQ ID NO: 1), design the knockout primers NtSKOR1B-Crispr-58F / NtSKOR1B-Crispr-58R, and construct the pORE-Cas9 / gRNA-NtSKOR1B gene knockout vector.

[0049] NtSKOR1B-Crispr-58F: (SEQ ID NO: 5)

[0050] NtSKOR1B-Crispr-58R: (SEQ ID NO: 6)

[0051] Example 2. Screening and identification of NtSKOR1B knockout materials

[0052] 1. Screening of NtSKOR1B knockout materials. Using the Agrobacterium-mediated leaf disc method, transfer the pORE-Cas9 / gRNA-NtSKOR1B gene knockout vector constructed in Example 1 into Nicotiana tabacum cv. Zhongyan 100 to obtain the C0 generation of transgenic materials. Use the primer pair NtSKOR1B-Crispr-9734F / NtSKOR1B-Crispr-10293R to screen out 9 edited tobacco plants.

[0053] NtSKOR1B-Crispr-9734F: (SEQ ID NO: 7)

[0054] NtSKOR1B-Crispr-10293R: (SEQ ID NO: 8)

[0055] 2. Homozygous identification of the NtSKOR1B knockout material. Nine C1 generation lines were planted, the DNA of transgenic plants was extracted, and PCR amplification and sequencing were performed using specific detection primers NtSKOR1B-Crispr-9734F and NtSKOR1B-Crispr-10293R to obtain two homozygous knockout materials with different editing methods. Among them, the editing method of C2 was the insertion of T at position 911, and the editing method of C6 was a continuous deletion of 11 bases starting from position 989, resulting in translation termination. The editing method is shown in Figure 4 。

[0056] Example 3. Construction of the NTSKOR1B promoter material

[0057] The genomic DNA of Nicotiana tabacum variety Zhongyan 100 was extracted by the CTAB method. Using the promoter sequence upstream of the start codon ATG of the LOC104103384 gene sequence (GenBank No. XM_009611282.3) as a template, primers NtSKOR1Bpro-1F / NtSKOR1Bpro-1R were designed for PCR amplification to obtain an NtSKOR1B promoter sequence with a length of 2439 bp. Using NtSKOR1Bpro-2F / NtSKOR1Bpro-2R as primers for amplification, a binary vector pBI101-NtSKOR1Bpro driven by the NtSKOR1B promoter was obtained.

[0058] NtSKOR1Bpro-1F: (SEQ ID NO: 9)

[0059] NtSKOR1Bpro-1R: (SEQ ID NO: 10)

[0060] NtSKOR1Bpro-2F: (SEQ ID NO: 11)

[0061] NtSKOR1Bpro-2R: (SEQ ID NO: 12)

[0062] Example 4. Identification of the NTSKOR1B promoter material

[0063] Using the Agrobacterium-mediated leaf disc method, the binary vector pBI101-NtSKOR1Bpro constructed in Example 2 was transferred into Nicotiana tabacum variety Zhongyan 100 to obtain transgenic materials (ProNtSKOR1B::GUS). Positive identification was performed on the T0 generation materials ( Figure 2) The identification primers were NtSKOR1Bpro-3F / NtSKOR1Bpro-1R. The T1 generation seeds of the positive plants were harvested and sown on 1 / 2 MS plates containing 50 mg / L kanamycin sulfate. After resistance screening, lines with a resistance ratio of 3:1 after strict self-crossing were selected, and resistant T2 homozygous materials were selected for histochemical staining.

[0064] NtSKOR1Bpro-3F: (SEQ ID NO: 13)

[0065] Example 5. Cultivation of tobacco materials, salt stress treatment and sampling

[0066] The plant material used in this example was the common tobacco (Nicotiana tabacum) variety Zhongyan 100 (ZY100). Tobacco plants were cultivated in a greenhouse. The greenhouse conditions were: 16 hours of light / 8 hours of darkness, temperature 23 - 25 °C, relative humidity 70%. First, tobacco seeds were sown in the soil. Twenty days after seed germination, the seedlings were transplanted onto floating trays for hydroponics (1 / 2 Hoagland nutrient solution). After 6 days of acclimation culture, salt treatment (1 / 2 Hoagland nutrient solution plus 100 mM NaCl) was carried out, and the control treatment was still 1 / 2 Hoagland nutrient solution. The nutrient solution was changed every 3 days.

[0067] Sampling for NtSKOR1B qRT-PCR analysis: After 6 days of hydroponics of ZY100, samples of roots, stems and leaves were taken respectively after culturing for 0 h, 6 h, 12 h, 30 h, 60 h, 6 d under salt treatment conditions (100 mM NaCl), and were quickly frozen in liquid nitrogen and stored at -80 °C. Total RNA was extracted from plant tissues using the RNeasyPlus Mini kit (Qiagen, catalog number / ID: 74134). qRT-PCR: RNA was reverse transcribed into cDNA using the Evo M-MLV Mix Kit with gDNA Clean for qPCR (Hunan Aikerui Bioengineering Co., Ltd., catalog number / ID: AG11728, Changsha, China), and amplification was carried out using a 96 fluorescence quantitative instrument (F. Hoffmann-La Roche Ltd, Switzerland) and SYBR Green Pro Taq HS qPCR Premix (Hunan Aikerui Bioengineering Co., Ltd., catalog number / ID: AG11728, Changsha, China). The primers were: NtSKOR1B-Crispr-9734F / NtSKOR1B-Crispr-10293R, and the 2 -ΔC ′ t method was used for relative gene expression analysis.

[0068] NtSKOR1B-Crispr-9734F: (SEQ ID NO: 7)

[0069] NtSKOR1B-Crispr-10293R: (SEQ ID NO: 8)

[0070] Sampling for GUS chemical staining test: Sow the NtSKOR1B promoter material (ProNtSKOR1B::GUS). After 20 days of germination, after 6 days of hydroponic culture, samples of roots, stems, and leaves were taken at 0 h, 12 h, and 30 h under salt treatment for GUS staining (Beijing Leagene Biotechnology Co., Ltd., Catalog No. / ID: DP0013, Beijing, China), and observed after chemical staining.

[0071] Sampling for determination of physiological indexes: After 6 days of hydroponic culture of tobacco seedlings, salt treatment was carried out. On the 11th day of salt treatment, young tobacco plants were collected, the length and width of the third leaf position were measured, different tissues were selected (leaves: all leaves without the main vein; stems; roots), blanched at 105 °C for 30 minutes and then dried to a constant weight at 80 °C, and the biomass was weighed. The potassium and sodium ion contents were determined by atomic absorption method.

[0072] The qRT-PCR results showed that the expression level of NtSKOR1B in roots under salt treatment was significantly up-regulated with the increase of salt treatment time. The expression level after 30 h of salt treatment was 9.98 times that of the 0 h treatment. The expression levels of NtSKOR1B in stems and leaves were lower than those in roots, but also showed different degrees of up-regulation with the increase of salt treatment time.

[0073] The GUS staining results showed that no obvious GUS activity was detected in tobacco seedlings without salt treatment. After 12 h of salt treatment, obvious GUS activity began to be detected in roots, stems, and some leaf veins. After 30 h of salt treatment, strong GUS activity was detected in roots, stems, and leaf veins. The results showed that under normal culture conditions, the expression level of NtSKOR1B in tobacco seedlings was low. After salt treatment, the gene was highly expressed in the cortex of roots, vascular tissues of stems, and leaf veins ( Figure 3 ), and the expression level was significantly up-regulated with the increase of salt treatment time.

[0074] Such as Figure 5As shown, under salt conditions, the NtSKOR1B knockout lines had higher biomass accumulation, leaf length, and leaf width than the wild type, with increases of 34.06%-39.24%, 15.87%-21.63%, and 14.89%-17.02% respectively; knocking out NtSKOR1B increased the potassium ion content in the above-ground and root parts of tobacco seedlings, with increases of 22.64%-25.47% and 18.06%-21.02% respectively compared to the wild type, and the difference in potassium ion content in the above-ground part was significant; knocking out NtSKOR1B decreased the sodium ion content in the above-ground and root parts of the plant, with decreases of 9.83%-11.48% and 7.52%-7.95% respectively compared to the wild type, and the difference in sodium ion content in the above-ground part was significant; knocking out NtSKOR1B significantly increased the potassium-sodium ratio of the plant. Under normal conditions, there were no significant differences in the above physiological indexes and ion contents.

[0075] In summary, the gene related to the potassium ion channel in tobacco mined by the present invention can negatively regulate the salt tolerance of tobacco. By inhibiting the expression of this potassium ion channel gene, it helps to cultivate crops with high salt tolerance and improve saline soil.

[0076] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

1. NtSKOR1B Use of a gene in cultivating transgenic plants with different degrees of salt tolerance, characterized in that: The NtSKOR1B gene negatively regulates the tolerance of tobacco to salt stress. Knocking out NtSKOR1B the gene can improve the tolerance of tobacco seedlings to salt stress. The NtSKOR1B nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The transgenic plant is tobacco.

3. A method for cultivating transgenic plants with different degrees of salt tolerance, characterized in that: Using the CRISPR / Cas9-mediated gene editing technology, construct a CRISPR / Cas9 gene editing vector for knocking out the NtSKOR1B gene described in claim 1, and obtain NtSKOR1B a transgenic plant with the edited gene through genetic transformation.

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