Tobacco potassium transporter NtKUP7 gene and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a gene for regulating and controlling the potassium content of tobacco leaves, in particular to a tobacco potassium transporter NtKUP7 gene and application thereof. The gene is a potassium transporter, and the base sequence of the potassium transporter is shown as SEQ ID NO. 1; wherein nucleotides from the 2092nd site to the 2442nd site are specific nucleic acid fragments. According to the invention, real-time PCR (polymerase chain reaction) shows that the gene has the highest expression quantity at the root of the tobacco. After a gene transient silencing technology (VIGS) is adopted or the NtKUP7 gene is knocked out, the potassium ion content of tobacco leaves is remarkably reduced compared with that of a control group. According to the invention, the transport function of the NtKUP7 gene on the potassium content of the tobacco leaves is defined, a new evidence is provided for analyzing a molecular regulation mechanism of the potassium ion content of the tobacco leaves, a new thought is provided for regulating and cultivating high-potassium-content tobacco leaves through gene expression, and a new gene resource is provided for improving the potassium content of the tobacco leaves through a molecular means.

Description

Technical Field

[0001] This invention relates to genes related to potassium ion absorption and transport in tobacco, belonging to the field of genetic engineering technology, and specifically to a potassium transporter that can regulate the potassium ion content of tobacco leaves. NtKUP7 Genes and their applications. Background Technology

[0002] Potassium is an essential macronutrient for plant growth and development, playing a crucial role in various physiological processes, including water absorption, transpiration, enzyme activation, and photosynthesis. Tobacco ( Nicotiana tabacum L. Tobacco is a potassium-loving crop with a high potassium requirement. High potassium content in tobacco leaves results in better field maturity, a deep orange-yellow leaf color, higher reducing sugar content, lower total nitrogen content, richer aroma, and better elasticity and resilience. Potassium not only significantly improves the color and combustibility of tobacco leaves but also increases their toughness and softness. Therefore, a higher potassium content in tobacco leaves can improve both yield and quality. Tobacco primarily absorbs potassium from the soil through its roots. + K + Accumulated in leaves. Current research on potassium ion absorption and transport in plants mainly focuses on potassium ion channel proteins (AKT, GORK, KAT, TPK, etc.) and transport proteins (HKT, KUP / HAK / KT, CPAs, etc.). Although studies have reported on potassium ion absorption and transport in tobacco... BUY / HAK / KT Whole genome identification of the family, but only a few genes such as NtHAK1 , NtHAK5 and NtKT12 Reports of cloning have been published. Summary of the Invention

[0003] The purpose of this invention is to provide a tobacco potassium transporter. NtKUP7 Genes, thus laying the foundation for breeding varieties that regulate the potassium ion content of tobacco leaves.

[0004] The objective of this invention is achieved through the following technical solution: A tobacco potassium transporter NtKUP7 A gene consists of 2565 base pairs. ， This gene originates from tobacco ( Nicotiana tobacco Its base sequence is shown in SEQ ID NO.1; where nucleotides 2092-2442 are specific nucleic acid fragments.

[0005] Tobacco potassium transporter NtKUP7The protein encoded by the gene consists of 854 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0006] The tobacco potassium transporter NtKUP7 The protein encoded by the gene is used in tobacco to regulate potassium ion content.

[0007] The tobacco potassium transporter NtKUP7 The application of this gene in tobacco plants resulted in a significant decrease in potassium ion content in the leaves after the gene was silenced.

[0008] For silencing tobacco potassium transporters NtKUP7 The transient gene silencing VIGS vector was constructed using virus-induced gene silencing (VIGS) technology, with the aforementioned tobacco potassium transporter... NtKUP7 Using a gene-specific nucleotide fragment as a guide sequence, the specific nucleic acid fragment is ligated into the transient expression vector TRV2, which is then transformed into *E. coli* DH5α to construct the transient silencing vector TRV2-. NtKUP7 .

[0009] The use of transiently silencing tobacco potassium transporters NtKUP7 Application of the VIGS gene vector in tobacco for silencing the tobacco potassium transporter NtKUP7 Genes that silence the tobacco potassium transporter NtKUP7 After gene therapy, the potassium ion content in plant leaves decreased significantly.

[0010] Using the aforementioned potassium transporter for silencing tobacco NtKUP7 The VIGS vector constructed from genes NtKUP7 The method for transiently silencing plants is characterized by utilizing virus-induced gene silencing technology, specifically the transient silencing vector TRV2- NtKUP7 After conversion of Nicotiana benthamiana, tobacco potassium transporters were screened and identified. NtKUP7 Transiently silenced genes in plants, compared to non-silenced plants, NtKUP7 Potassium ion content decreased significantly in gene-silenced plants.

[0011] The potassium ion content in the leaves of plants with the NtKUP7 potassium transporter gene silenced was significantly lower than that in the control, indicating that NtKUP7 may be a key regulatory factor for the accumulation of potassium ions in tobacco. This provides an important genetic resource for improving the potassium content of tobacco leaves and breeding high-quality new tobacco varieties through molecular biology.

[0012] This invention discovers the tobacco potassium transporter using real-time PCR. NtKUP7 The gene was expressed at the highest level in the roots of tobacco. A silencer was constructed using virus-induced gene silencing (VIGS) technology. NtKUP7The VIGS vector of the gene was successfully transformed to obtain repressor. NtKUP7 Silent plants expressed in *Nicotiana benthamiana* exhibited a specific phenotype with significantly lower potassium ion content compared to control plants. Analysis of potassium ion content in *Nicotiana benthamiana* revealed a significant reduction in potassium ion content in gene-silenced plants, decreasing by at least 49.5%, indicating that silencing this gene can significantly reduce potassium ion content in plants.

[0013] In summary, it can be seen that gene silencing technology or gene knockout can be used to achieve this. NtKUP7 The gene can produce gene-silenced plants with reduced potassium ion content, indicating that... NtKUP7 It is a positive regulator of potassium ion content in tobacco, providing a new genetic resource for increasing potassium content in tobacco leaves through molecular means. Attached Figure Description

[0014] Figure 1 For different tissues and organs NtKUP7 The relative expression level; Figure 2 For silent plants NtKUP7 The relative expression level of genes; Figure 3 The potassium ion content in tobacco leaves with virus-induced gene silencing after drying and control tobacco leaves. Detailed Implementation

[0015] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the basic situation of some biological materials, experimental reagents, experimental instruments and other items involved in the following embodiments will be briefly introduced as follows. Biomaterials:

[0016] Tobacco material: Tobacco Benedict ( Nicotiana benthamiana ), a commercial tobacco variety; Transient expression vector: TRV2, purchased from China Plasmid Vector Bacterial Cell Gene Preservation Center; Gene sequencing and primer synthesis were provided by Shanghai Sangon Biotech. Experimental reagents:

[0017] LA Taq enzyme, PstI restriction endonuclease, plasmid extraction kit, gel extraction kit, etc., were purchased from Takara. The In-Fusion one-step cloning kit was purchased from clontech. The RNA extraction kit was purchased from GeneAnswer. The reverse transcription kit and RT-PCR kit were purchased from Roche. Peptone, yeast extract, etc., were purchased from Oxoid. The following is a brief description of some reagent formulations and preparation methods: (1) LB liquid medium (1L): 10 g bacterial peptone, 10 g sodium chloride (NaCl), 5 g yeast extract, autoclaved at high temperature; (2) YEB liquid culture medium (1L): 5 g beef extract, 5 g bacterial peptone, 5 g sucrose, 1 g yeast extract, 2 ml 1M magnesium sulfate (MgSO4), autoclaved at high temperature; (3) 1M 2-(N-morpholine) ethanesulfonic acid (MES) stock solution: Dissolve MES in ddH2O, filter and sterilize, store at -20℃ for later use; (4) 200 mM Acetosyringone stock solution: Dissolve acetosyringone in dimethyl sulfoxide (DSMO) and store at -20℃ for later use; (5) MMA (1L): 20 g sucrose, 5 g MS salts (Duchefa Biochemie), 1.95 g MES, 1 ml acetylsuccinone (200 mM), pH=5.6; Experimental apparatus: Tgradient PCR instrument, a product of Biometra. LightCycler 96 real-time quantitative PCR instrument, a product of Roche. Example 1

[0018] This embodiment mainly focuses on the tobacco potassium transporter. NtKUP7 The process of obtaining genes is briefly described below.

[0019] Using cultivated tobacco leaves as samples, total RNA was extracted from tobacco leaves using an RNA extraction kit and reverse transcribed into cDNA for later use. By using homology comparison, referencing Arabidopsis thaliana AtKUP7 Based on the gene sequence and the known partial gene sequence of tobacco, the amplification primer sequences are designed as follows: F: 5'-CGCGAGCTCGGTACC ATGGTGAATGTGGGATTGG -3', R: 5'-GCTCACCATGGATCC TCACACCATATATGTCATGCC-3'; Using the prepared cDNA as a template, PCR amplification was performed using the primers described above. The amplified product was then purified and sequenced to obtain the tobacco potassium transporter. NtKUP7 The gene sequence, whose base sequence is shown in SEQ ID No.1, consists of 2565 bases, of which nucleotides 2092-2442 are specific nucleic acid fragments.

[0020] After translation of the gene sequence, the encoded protein sequence is shown in SEQ ID No.2, which includes 854 amino acids. Further comparative analysis showed that the protein contains highly homologous and conserved sequences. Example 2

[0021] In order to detect NtKUP7 To investigate the expression specificity of the gene in different tissues, samples were taken from the roots, stems, and leaves of tobacco plants during their vigorous growth phase and from the flowers during their full bloom phase. RNA was extracted and reverse transcribed into cDNA for the corresponding organs. Real-time PCR was then used to detect the expression of the gene in different tissues. Specific quantitative primer sequences were designed as follows: F: 5'-ATGGAGATGATGGTTCGACG-3' R: 5'-ATCAATGCGAGGGCCAG-3' The results showed that ( Figure 1 Tobacco potassium transporter NtKUP7 The gene is expressed in all tissues of tobacco, with the highest expression level in the roots. Example 3

[0022] Using the tobacco potassium transporter obtained in Example 1 NtKUP7 The inventors further constructed a vector for transient gene silencing, and the relevant construction process is briefly described below.

[0023] A relatively specific nucleic acid fragment from this gene (nucleotide sequence 2092-2442 of SEQ ID NO.1) was selected as the guide sequence for VIGS. The primer sequence is designed as follows, and this sequence was amplified. The primer sequence design for constructing the VIGS transient silencing vector is as follows: NtKUP7 -F:5'-AGATCGACGACAAGACCCTGCAGCTCGAGAAGTTCATTCGCAG-3', NtKUP7 -R:5'-TTCTGAGGAGAAGAGCCCTGCAGCCAGCCTTCAGCTCTTCTG-3'; Amplification length: 350bp.

[0024] The amplified fragments were ligated into the TRV2 vector using the in-fusion method (ligation at 50°C for 15 min) and transformed into E. coli DH5α. ， Build and obtain TRV2- NtKUP7 Carrier.

[0025] After successful sequencing, the recombinant plasmid was transformed into Agrobacterium GV3101 using the freeze-thaw method. After culturing at 30°C for 48 hours, single colonies were picked, cultured in liquid, and then verified by bacterial PCR to ensure that the target fragment was transformed correctly. Example 4

[0026] Using the VIGS transient silencing vector constructed in Example 3, the inventors further conducted cultivation experiments with *Nicotiana benthamiana* as an example to silence the tobacco potassium transporter in the plant. NtKUP7 The gene and related experimental procedures are briefly described below.

[0027] Sow tobacco seeds in seedling pots and cultivate seedlings. Two weeks after germination, transplant the seedlings into plastic pots (10cm×10cm). Carry out daily fertilization and watering under the conditions of 22℃, 16h light / 8h dark. After 4-5 weeks of growth, select 12 pots of tobacco seedlings with uniform growth for later use.

[0028] The contents of TRV1, TRV2, TRV2-PDS (positive control), and TRV2- NtKUP7 Single colonies of Agrobacterium were inoculated into YEB (5 mL) medium (kanamycin, 50 μg / mL), and cultured overnight at 28℃ and 250 r / min with shaking for about 48 h before being transferred to 50 mL of YEB. Incubate overnight at 28°C with shaking. Centrifuge at 4000 rpm for 8 min to collect Agrobacterium bacteria into 50 mL centrifuge tubes. Adjust the OD value of the bacterial culture to approximately 1.0 using a mixed solution containing 10 mmol / L 2-N-morpholinyl ethanesulfonic acid (MES), 250 μmol / L acetosyringone (As), and 10 mmol / L MgCl2. [The text then abruptly shifts to a seemingly unrelated topic:] ...in the presence of TRV2, TRV2-... PDS TRV2- NtKUP7 Add an equal volume of MMA suspension of Agrobacterium to the MMA suspension containing TRV1 Agrobacterium, mix well, and let stand at room temperature for 3-6 hours.

[0029] For inoculation, select approximately 4-5 leaves of uniform growth. Using a 1 mL needleless sterile syringe, inject Agrobacterium suspension containing different TRV recombinant plasmids into the fully expanded leaves from the back of the leaves using a pressure filtration method, ensuring the bacterial suspension fills the entire leaf. Incubate at 22℃ and 75% humidity. Four pots were inoculated with plants containing the TRV2-PDS positive control, while the remaining plants contained the TRV2 empty vector and the TRV2-PDS ... NtKUP7 Six pots of each type of plant were inoculated.

[0030] Six weeks after inoculation, the levels of the virus in silent plants and control plants were measured. NtKUP7 Relative gene expression levels and potassium ion content.

[0031] In silent plants and control plants NtKUP7 The relative gene expression levels were analyzed by qRT-PCR, and the results are as follows: Figure 2 As shown, the target gene NtKUP7 was significantly reduced in the silenced plants, reaching only 30% of that in the control.

[0032] The potassium ion content of the obtained silent plants and control plants was detected. The detection method is as follows: Take samples of the leaves of the above-mentioned plants, taking 3-4 leaves from each plant, wrapping them in aluminum foil, and drying them overnight in an oven at 90°C. The dried sample was crushed using a grinder. 0.05g of tobacco leaves (accurate to 0.0001g) was weighed, 15ml of 5% acetic acid solution was added, and the sample was placed in a constant temperature shaker (30℃ shaker) and shaken for 30min. After filtration through filter paper, the potassium ion content was determined using a continuous flow analyzer.

[0033] The results are as follows Figure 3 The results show that TRV2- NtKUP7 The potassium ion content in virus-induced gene-silenced plants was approximately 50.5% of that in control plants. That is, after gene silencing, the potassium ion content in the plants decreased by about 49.5%.

[0034] SEQ ID NO.1 SEQ ID NO.2

[0035] MVNVGLEKDRENNGLASMDSIESRWVFQDEYDSDINSVDHHTVNGGDDGSTPRNVLDLDSEDEDDNAVRKLIRTGPRIDSFDVEALEVPGAQKNDFDDVSAGRKILLAFQTLGVVFGDVGTSPLYTFSVMFSKAPVNGNEDVLGALSLVLYTLILIPLIVKYVLIVLWANDDGEGGTFALYSLLCRHAKVNLLPNQLPSDARISGFRLKVPSPELERSLRIKERLEASLTLKKLLLMLVLAGTAMVIADGVVTPAMSVMSAVGGLRVGVSGIKQDQVVMISVAFLVILFSVQKYGTSKVGLFVGPALFIWFCSLGGIGIYNLINYDSRVWRAFNPVHIYYYFKRNPTKAWYSLGGCLLCATGSEAMFADLCYFSVRSVQLTFVFLVLPCLLLGYLGQAAYLMENHADTTQAFSSVPSGAFWPVFLIA NVAALIASRAMTTATFSCIKQSTALGCFPRLKIIHTSRKFMGQIYIPVMNWFLLALSLVMCSISSIYEIGNAYGIAELGVMMMTTILVTIVMLLIWQINIIVVLSFVVIFLGLELMFFSSVLWSVGDGSWIILVFAVVLFFVMYIWNYGSKLKYETEVKQKMSMDLLRELGPNLGTIRAPGIGLLYNELAKGIPAIFGHFLTTLPAVHSMIIFVCIKYIPVPVVPQNERFLFRRVCPRSYHIFRCVARYGYKDVRKENHQMFEQLLIESLEKFIRRDAQERSLESDGNGESDSEEHAFSRVLVAPNGSVYSLGVPLADFRDTGKAVMEESTSEELKAGPSSESLLSNADQSFEKELSFLRKAKESGVVYLLGHGNIRARKSSWFIKKLFINYFYAFLRKNCRREIASLSVPHSHLMQVGMTYMV

Claims

1. A tobacco potassium transporter NtKUP7 The gene, whose CDS sequence consists of 2565 bases. ， Its base sequence is shown in SEQ ID NO.1; where nucleotides 2092-2442 are specific nucleic acid fragments.

2. The tobacco potassium transporter according to claim 1 NtKUP7 The protein encoded by the gene is characterized by, It contains 854 amino acids, and its amino acid sequence is shown in SEQ ID NO.

2.

3. The tobacco potassium transporter according to claim 2 NtKUP7 The application of the protein encoded by the gene in tobacco is characterized by, Used to regulate the potassium ion content of tobacco leaves.

4. The tobacco potassium transporter according to claim 1 NtKUP7 The application of genes in tobacco is characterized by, Silencing this gene reduced the potassium ion content in tobacco leaves.

5. For silencing the tobacco potassium transporter of claim 1 NtKUP7 Transient gene silencing (VIGS) vectors, characterized in that, The tobacco potassium transporter was obtained through the following construction method: NtKUP7 A specific nucleotide fragment of the gene was ligated into the transient expression vector TRV2, and then transformed into *E. coli* DH5α to construct the transient silencing vector TRV2-. NtKUP7.

6. The potassium transporter for silencing tobacco as described in claim 5 NtKUP7 The application of the VIGS gene vector in tobacco is characterized by, For silencing tobacco potassium transporters NtKUP7 Gene.

7. The method of using the potassium transporter for silencing tobacco as described in claim 5 NtKUP7 The VIGS vector constructed from genes NtKUP7 The method for transiently silencing plants is characterized by, Using virus-induced gene silencing technology, the transient silencing vector TRV2- NtKUP7 After conversion of Nicotiana benthamiana, tobacco potassium transporters were screened and identified. NtKUP7 Transiently silenced genes in plants, compared to non-silenced plants, NtKUP7 Potassium ion content was significantly reduced in gene-silenced plants.

8. The claim 7 NtKUP7 The application of transient silencing or RNAi transgenic plants in tobacco potassium ion content regulation breeding is characterized by... The potassium ion content in the plant leaves decreased.

Images