A tobacco NtSWEET12i protein, a coding gene thereof, a recombinant vector and application thereof in improving drought resistance and saline-alkali tolerance of tobacco

Abstract

The application belongs to the technical field of plant genetic engineering, and discloses a tobacco NtSWEET12i protein, a coding gene thereof, a recombinant carrier and application of the tobacco NtSWEET12i protein in improving drought resistance and saline-alkali tolerance of tobacco. The tobacco NtSWEET12i protein is one of the following sequences: 1) a protein with an amino acid sequence as shown in SEQ ID NO. 1; 2) a fusion protein formed by connecting an amino-terminal end or a carboxyl-terminal end of an amino acid sequence as shown in SEQ ID NO. 1 with a protein tag; and 3) a protein with more than 95% identity with the protein with the amino acid sequence as shown in SEQ ID NO. 1 and the same function. The coding gene of the above protein is one of the following sequences: (1) a gene sequence with a nucleotide sequence as shown in SEQ ID NO. 2; and (2) a gene sequence capable of hybridizing with the gene defined in (1) under stringent conditions and encoding the protein NtSWEET12i. The gene regulates the stress resistance performance such as drought resistance and saline-alkali tolerance of a plant body, and has important significance in cultivating a tobacco strain with stronger stress resistance and further improving the yield of tobacco.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a tobacco NtSWEET12i protein, its encoding gene, recombinant vector, and its application in improving the drought resistance and salt tolerance of tobacco. Background Technology

[0002] With global climate change, abiotic stresses, particularly drought and salinity stress, are adversely affecting crop growth and development, thus threatening global food security. Identifying abiotic stress-related genes in plants is crucial for improving their tolerance to drought and salinity stress. With the application of plant biotechnology in plant breeding, the regulatory network of plant resistance genes is becoming increasingly clear. However, the transcriptional regulatory mechanisms in response to abiotic stresses remain unclear and require further investigation.

[0003] Tobacco, a dicotyledonous plant, is an annual or limited perennial herbaceous plant belonging to the genus Nicotiana in the Solanaceae family. As an important model plant in plant science, it not only has a good foundation for crop molecular genetic technology and is widely used to study gene functional diversity and create new germplasm, but also has certain economic value as an economic crop. However, tobacco growth is easily affected by extreme environments, including abiotic environmental stresses such as drought and salinity, which can lead to a reduction in its yield.

[0004] Therefore, it is of great significance to study and discover new tobacco genes related to salinity and drought stress, and to breed tobacco varieties with stronger stress resistance.

[0005] Therefore, the existing technology needs further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a tobacco NtSWEET12i protein, its encoding gene, a recombinant vector, and its application in improving the drought resistance and salt tolerance of tobacco. By upregulating the expression level of this gene in plants, its drought resistance and salt tolerance can be improved, and it can be applied to the breeding of tobacco varieties with stronger stress resistance.

[0007] In a first aspect, this application provides a tobacco NtSWEET12i protein, which is one of the following sequences:

[0008] 1) A protein with an amino acid sequence as shown in SEQ ID NO.1;

[0009] 2) Fusion proteins formed by attaching a protein tag to the amino-terminus or carboxyl-terminus of the amino acid sequence shown in SEQ ID NO.1;

[0010] 3) Proteins that share more than 95% identity with the amino acid sequence shown in SEQ ID NO.1 and have the same function.

[0011] The SEQ ID NO.1 consists of 291 amino acid residues and is derived from tobacco (Nicotiana tabacum L.).

[0012] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] To facilitate the purification or detection of the above proteins, a protein tag can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1.

[0014] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. Protein tags include, but are not limited to: GST (glutathione thiotransferase) protein tag, His6 protein tag (His-tag), MBP (maltose-binding protein) protein tag, Flag protein tag, SUMO protein tag, HA protein tag, Myc protein tag, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag protein tag, etc.

[0015] The aforementioned amino acid sequences include those shown in SEQ ID NO: 1, as well as homologous sequences with 95% or more homology to the protein sequence. These sequences can be obtained by mutating the sequence shown in SEQ ID NO: 1 using existing techniques. The protein activity of these homologous sequences is the same as that of the protein with the sequence SEQ ID NO: 1.

[0016] Secondly, this application also provides a gene encoding the above-mentioned tobacco NtSWEET12i protein, which is one of the following sequences:

[0017] (1) A gene sequence with a nucleotide sequence as shown in SEQ ID No. 2;

[0018] (2) Hybridize with the gene specified in (1) under strict conditions, and the gene sequence encoding the protein NtSWEET12i.

[0019] SEQ ID NO.2 consists of 876 nucleotides, and its open reading frame (ORF) is from position 1 to position 876 from the 5′ end, encoding a protein with the amino acid sequence shown in SEQ ID NO.1.

[0020] The stringent conditions were: hybridization in a solution of 2×SSC and 0.1% SDS at 68°C, followed by two washes of 5 min each, and then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by two washes of 15 min each.

[0021] The aforementioned gene sequence includes not only the sequence shown in SEQ ID NO: 2, but also homologous sequences with 95% or more homology to the gene sequence. These sequences can be obtained by mutating the sequence shown in SEQ ID NO: 2 using existing techniques. The proteins expressed by these homologous sequences have the same activity as the proteins expressed by the sequence SEQ ID NO: 2.

[0022] Furthermore, any nucleotides that have been artificially modified and have 95% or more of the same encoding nucleotide sequence as the protein MsRGP1 isolated in this invention, as long as the encoded protein function and activity are substantially the same, are all derived from the nucleotide sequence of this invention and are equivalent to the sequence of this invention.

[0023] This application aims to screen for the NtSWEET12i protein in tobacco that responds to drought and salinity stress. On one hand, an overexpression vector pCAMBIA super1300-NtSWEET12i-GFP was constructed and transformed into Arabidopsis thaliana using Agrobacterium-mediated transformation. After screening and identification, tobacco plants transiently overexpressing NtSWEET12i were obtained. Experiments showed that the drought and salinity tolerance of tobacco plants overexpressing this protein was significantly improved. The content of total soluble sugar, the content of stress-related hormone ABA, proline, POD activity, SOD activity, reactive oxygen species content, and malondialdehyde content in the plant were also regulated by this gene.

[0024] Therefore, the NtSWEET12i protein is a new and important gene that affects the drought and salt tolerance of plants. It can be used to cultivate new plant materials with stronger stress resistance, and its application value is high.

[0025] Thirdly, this application also provides an expression cassette that expresses a DNA molecule of the protein NtSWEET12i in a host cell. The DNA molecule may include not only a promoter that initiates transcription of the NtSWEET12i gene, but also a terminator that terminates transcription of the NtSWEET12i gene.

[0026] Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-associated protein 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007)). 10099169.7), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0027] Fourthly, this application also provides a vector carrying the gene encoding the aforementioned tobacco NtSWEET12i protein.

[0028] Recombinant vectors containing the NtSWEET12i encoding gene expression cassette can be constructed using plant expression vectors. These plant expression vectors can be Gateway system vectors or binary Agrobacterium vectors, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA super1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant vectors using NtSWEET12i, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0029] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0030] In a specific embodiment of the present invention, the recombinant vector is the recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP. The recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP is obtained by replacing the fragment between the Xba I and Pst I restriction sites of the vector pCAMBIAsuper1300-GFP with the DNA molecule shown in SEQ ID NO.2, while keeping other sequences unchanged.

[0031] Fifthly, this application also provides a recombinant microorganism, which is transformed with the aforementioned recombinant vector.

[0032] The recombinant microorganism may specifically be yeast, bacteria, algae, or fungi. Further optionally, the bacteria may be Agrobacterium GV3101 strain.

[0033] Sixthly, this application also provides the application of the aforementioned NtSWEET12i protein, the encoding gene of the aforementioned NtSWEET12i protein, the aforementioned expression cassette, the aforementioned recombinant vector, or the aforementioned recombinant microorganism in regulating plant drought resistance and salt tolerance.

[0034] Seventhly, this application also provides the application of the aforementioned NtSWEET12i protein, the aforementioned NtSWEET12i protein encoding gene, the aforementioned expression cassette, the aforementioned recombinant vector, or the aforementioned recombinant microorganism in regulating the content of total soluble sugars, stress-related hormone ABA, proline, POD activity, SOD activity, reactive oxygen species, and malondialdehyde in plants.

[0035] In this application, the high drought and salt tolerance mentioned above is mainly reflected in increasing the total soluble sugar content, stress-related hormone ABA content, proline content, POD activity, and SOD activity of plants, while reducing the content of reactive oxygen species and malondialdehyde.

[0036] Optionally, the plant is a dicotyledonous or monocotyledonous plant. Preferably, in this application, the plant is tobacco.

[0037] Optionally, in the application, the drought resistance and salt tolerance of plants are improved by upregulating the expression level of the NtSWEET12i protein in plants.

[0038] In the above applications, the specific application in plant breeding can be to hybridize plants containing the protein or the protein-coding gene NtSWEET12i with other plants to carry out plant breeding.

[0039] Sixthly, this application also provides a method for cultivating drought-resistant and salt-tolerant tobacco, the method being as follows:

[0040] The NtSWEET12i plant expression vector was constructed, transformed by Agrobacterium-mediated transformation, and used to infect wild-type plants. Through cultivation and screening, NtSWEET12i expression / overexpression plants were obtained, which are drought-resistant and salt-tolerant plants.

[0041] Alternatively, the plant may be tobacco.

[0042] In the above methods, the expression or overexpression method involves introducing the gene encoding the protein NtSWEET12i into the target plant. In the above methods, the gene encoding the protein NtSWEET12i can be introduced into the target plant using a plant expression vector carrying the NtSWEET12i gene of this invention.

[0043] Plant expression vectors carrying the gene NtSWEET12i of this invention can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant cells or tissues can be cultured into plants.

[0044] In the above method, the nucleotide sequence of the gene encoding the protein NtSWEET12i is the DNA molecule shown in SEQ ID NO.2.

[0045] In a specific embodiment of the present invention, the plant expression vector carrying the gene NtSWEET12i of the present invention can be pCAMBIA super1300-NtSWEET12i-GFP. Specifically, pCAMBIA super1300-NtSWEET12i-GFP is obtained by inserting the DNA molecule shown in SEQ ID NO.2 into the pCAMBIAsuper1300-GFP vector using restriction endonucleases Xba I and Pst I.

[0046] The present invention has the following beneficial effects:

[0047] 1. This invention is the first to discover that the NtSWEET12i protein participates in the plant's response to drought and salt-alkali stress, positively regulating the plant's drought and salt-alkali resistance. It can improve the plant's drought and salt-alkali resistance by upregulating the expression level of this gene in the plant.

[0048] In this application, the gene encoding the NtSWEET12i protein was introduced into tobacco using different methods, resulting in transgenic tobacco plants transiently overexpressing NtSWEET12i and transgenic tobacco plants with virus-induced gene silence (VIGS) of NtSWEET12i. These two types of transgenic plants were subjected to drought and salt-alkali stress treatments. The results showed that, compared to the control, the overexpressing transgenic lines exhibited enhanced drought and salt-alkali tolerance, while the gene-silenced transgenic lines showed decreased drought and salt-alkali tolerance. Specifically, this was reflected in increases / decreases in the plant's total soluble sugar content, the content of stress-related hormones ABA, proline, POD activity, and SOD activity, while simultaneously decreasing / increasing reactive oxygen species content and malondialdehyde content.

[0049] Therefore, the NtSWEET12i gene and its encoded protein provided by this invention play an important role in the process of drought and salt tolerance in plants. Through overexpression technology, it has important application value in improving the drought and salt tolerance of plants and has broad application space and market prospects in the agricultural field.

[0050] 2. The NtSWEET12i gene provides new gene resources for improving the drought and salt tolerance of tobacco cultivars and for breeding germplasm resources, and provides new application value for promoting the quality improvement, efficiency enhancement and transformation and upgrading of the tobacco industry. Attached Figure Description

[0051] Figure 1 For NtSWEET12i gene sequence analysis, expression analysis and subcellular localization. (A) Expression analysis of NtSWEET12i in tobacco after treatment with mannitol, NaCl, NaHCO3 and ABA for 48 h. Data are expressed as mean ± SD (n = 3). Tobacco Ntactin was used as an internal reference gene. * (or different lowercase letters) and ** (or different uppercase letters) indicate the significance of differences at P < 0.05 and P < 0.01, respectively, using Student's test (or Ordinary one-way ANOVA multiple comparison).

[0052] Figure 2 The image shows the genome structure of NtSWEET12i, with boxes representing exons and lines representing introns.

[0053] Figure 3 To achieve subcellular localization of NtSWEET12i, a fusion construct of NtSWEET12i-GFP and the membrane marker PIP2-mCherry was transformed into the epidermal cells of *Nicotiana benthamiana* leaves. bar = 25 μm;

[0054] Figure 4 To identify the drought and salt tolerance of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants in pots; CK was the control, OE-1 and OE-2 were transient overexpression, and VIGS-1 and VIGS-2 were VIGS-silenced NtSWEET12i transgenic tobacco lines.

[0055] Figure 5 Determination of total soluble sugar content in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants;

[0056] Figure 6 The ABA content of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was determined.

[0057] Figure 7The proline content of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was determined.

[0058] Figure 8 The POD activity of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was determined.

[0059] Figure 9 The SOD activity of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was measured.

[0060] Figure 10 The hydrogen peroxide content was determined in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants.

[0061] Figure 11 Determination of malondialdehyde (MDA) content in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants;

[0062] Among them, CK was the control, OE-1 and OE-2 were overexpression, and VIGS-1 and VIGS-2 were VIGS-silenced NtSWEET12i transgenic tobacco lines.

[0063] Figure 12 To analyze the expression of relevant genes in the leaves of NtSWEET12i transgenic plants and CK plants under untreated or mannitol, sodium chloride and sodium bicarbonate stress. Detailed Implementation

[0064] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0065] Example 1: Obtaining the NtSWEET12i gene

[0066] 1.1 Experimental Methods

[0067] (1) Obtaining cDNA template

[0068] Total RNA was extracted from the tobacco cultivar *Symplocos rubra* using a plant total RNA extraction kit. This total RNA was then processed using PrimeScript. TMThe RT reagent kit with gDNA Eraser was used to reverse transcribe first-strand cDNA.

[0069] (2) Using the cDNA obtained in step 1 as a template, primers NtSWEET12i-F and NtSWEET12i-R were designed based on the sequences provided at http: / / lifenglab.hzau.edu.cn / Nicomics / . PCR amplification was performed to obtain a CDS amplification product of approximately 876 bp, which was then sequenced. The primer sequences are as follows:

[0070] NtSWEET12i-F: 5′-AATCCTATTTCACAGCCAAGTA-3′

[0071] NtSWEET12i-R: 5′-CACTGCCAATTCCAGAAGTGAA-3′

[0072] 1.2 Experimental Results and Analysis

[0073] According to the sequencing results, the nucleotide sequence of CDS is shown in SEQ ID NO.2, which is 876 bp in length. The gene shown in this sequence is named NtSWEET12i gene, and the protein it encodes is named NtSWEET12i protein, with the amino acid sequence shown in SEQ ID NO.1.

[0074] Example 2: Gene, cellular localization, and expression analysis of NtSWEET12i in tobacco

[0075] 2.1 Gene structure analysis of NtSWEET12i

[0076] (1) Experimental methods

[0077] Based on the genome and coding sequences of NtSWEET12i, exon and intron structure analysis was performed using TBtools-II software and the GSDS2.0 online website (https: / / gsds.gao-lab.org / ).

[0078] (2) Experimental results and analysis

[0079] The results are as follows Figure 2 As shown, the NtSWEET12i gene contains 6 exons and 5 introns.

[0080] 2.2 Subcellular localization analysis of NtSWEET12i

[0081] 1. Experimental Methods

[0082] The subcellular localization of NtSWEET12i was detected by transient co-expression of PIPII-mCherry and NtSWEET12i-GFP proteins in tobacco leaves. PIPII-mCherry is a membrane marker gene, exhibiting red fluorescence in subcellular localization analysis. Its co-expression with NtSWEET12i facilitated the subcellular localization of the NtSWEET12i protein.

[0083] The specific method is as follows:

[0084] After constructing PCAMBIA super1300-NtSWEET12i-GFP according to the method in Example 3, PIPII-mCherr, pCAMBIA super1300-GFP, and PCAMBIA super1300-NtSWEET12i-GFP were transformed into Agrobacterium (manufacturer: Weidi Biotechnology; catalog number: CAT#:AC1003), respectively. The Agrobacterium was then transformed using tobacco transient transformation technology according to… Figure 3 The combination shown was used to inject and infect tobacco leaves. After 48 h, the fluorescence expression was observed using a confocal microscope (LSM880, Zeiss, GER).

[0085] The combined injection groups were: Combination 1: injection of PIPII-mCherry and pCAMBIA super1300-GFP; Combination 2: injection of PIPII-mCherry and pCAMBIA super1300-NtSWEET12i-GFP.

[0086] 2. Experimental Results and Analysis

[0087] Fluorescence signal analysis showed that NtSWEET12i was localized to the cell membrane (see...). Figure 3 ).

[0088] 2.3 Expression analysis of NtSWEET12i in wild-type tobacco

[0089] (1) Experimental method:

[0090] Wild-type 4-leaf stage *Syngonium spp.* plants were treated with Hogrange solutions containing 200 mM mannitol, 200 mM sodium chloride, 100 mM sodium bicarbonate, and 0.1 mM ABA (sprayed) for 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. The expression profile of NtSWEET12i was quantitatively analyzed by qRT-PCR, with the NtACTIN gene of tobacco as an internal control.

[0091] (2) Experimental results and analysis

[0092] The results are as follows Figure 1 As shown, NtSWEET12i expression was upregulated by mannitol, sodium chloride, sodium bicarbonate and ABA (abscisic acid), indicating that mannitol, sodium chloride, sodium bicarbonate or ABA in the environment can induce an increase in the expression of NtSWEET12i protein in tobacco, which may be related to the plant's stress resistance.

[0093] Example 3 Construction of transgenic tobacco lines OE-1 and OE-2 with transient overexpression of NtSWEET12i

[0094] 3.1 Construction of recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP

[0095] (1) A double-stranded DNA molecule with the sequence shown in SEQ ID NO.2 was artificially synthesized. Using the double-stranded DNA molecule as a template, PCR amplification was performed using primers OE-F-BamHI and OE-R-BamHI. The amplification product was recovered and the amplification product was 906 bp in length.

[0096] OE-F-BamHI:

[0097] 5'-CCCAAGCTTGTCGACGGATCCATGTCTGGTCACTGGGCTTTG-3'

[0098] (SEQ ID NO.3)

[0099] OE-R-BamHI:

[0100] 5'-GCCCTTGCTCACCATGGATCCGGCTTTCACAGTTTGCAGCTT -3' (SEQ ID NO.4)

[0101] (The underlined area above indicates the recognition site of the restriction endonuclease BamHI.)

[0102] (2) The vector pCAMBIA super1300-GFP and the above amplification products were digested with the restriction endonuclease BamHI, and the vector backbone 1 of about 10783 bp was recovered. The above amplification products were digested in the same way and recovered.

[0103] (3) The enzyme-digested fragment 2 was ligated to the vector backbone 1 to obtain the recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP, and then sequenced.

[0104] Experimental results and analysis:

[0105] According to the sequencing results, the structure of the recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP is as follows: the small fragment between the restriction endonuclease BamHI recognition sequences of the recombinant plasmid pCAMBIA super1300-GFP is replaced with an 876bp DNA molecule as shown in SEQ ID NO.2, so that it can subsequently express the NtSWEET12i protein shown in SEQ ID NO.1.

[0106] 3.2 Transformation

[0107] The pCAMBIA super1300-NtSWEET12i-GFP plasmid was transformed into Agrobacterium (manufacturer: Weidi Biotechnology; catalog number: CAT#:AC1003), and Agrobacterium was injected into tobacco leaves using tobacco transient transformation technology.

[0108] 3.3 Screening of overexpressing plants

[0109] After 48 hours of infection with Agrobacterium, a small portion of leaves from the tobacco plants were taken for NtSWEET12i expression analysis. Plants OE-1 and OE-2, which showed significantly increased NtSWEET12i expression, were selected as transgenic tobacco lines that transiently overexpressed NtSWEET12i for subsequent experiments.

[0110] Example 4: Construction and screening of VIGS-1 and VIGS-2 transgenic tobacco lines with VIGS silenced NtSWEET12i

[0111] 4.1 Construction of recombinant plasmid pTRV2-NtSWEET12i

[0112] Suitable sequences were selected from the non-conserved domains of the NtSWEET12i gene. Primers were designed using Primer 5. Using the CDS of NtSWEET12i as a template, amplification was performed using primers NtSWEET12i-VIGS-F and NtSWEET12i-VIGS-R. The amplified products and VIGS vector were digested with enzymes and then ligated to construct the pTRV2-NtSWEET12i recombinant plasmid.

[0113] The primer sequences are as follows:

[0114] NtSWEET12i-VIGS-F:

[0115] 5'-GTGAGTAAGGTTACCGAATTCTGGGTGGCTTTGGTGCTATT -3' (SEQ ID NO.5)

[0116] NtSWEET12i-VIGS-R:

[0117] 5'-CGTGAGCTCGGTACCGGATCCACCACAATCACTGCTGGCTT-3' (SEQ ID NO.6)

[0118] (2) Plasmid transformation

[0119] The recombinant plasmid pTRV2-NtSWEET12i was transformed into Agrobacterium (manufacturer: Weidi Biotechnology; catalog number CAT#:AC1003), and the Agrobacterium was injected into tobacco leaves. This method uses existing tobacco transient conversion technology.

[0120] (3) Screening of VIGS-1 and VIGS-2 in genetically modified tobacco

[0121] After 48 hours of treatment with Agrobacterium-infected tobacco plants, a small portion of leaves from the plants were taken for NtSWEET12i expression analysis. Plants VIGS-1 and VIGS-2, whose NtSWEET12i expression was significantly reduced, were selected as VIGS-silenced NtSWEET12i transgenic tobacco lines for subsequent experiments.

[0122] Example 5: Drought and Salt Tolerance Tests of NtSWEET12i Transgenic Tobacco Plants

[0123] 5.1 Experimental Methods

[0124] The aforementioned transiently overexpressing NtSWEET12i transgenic tobacco lines OE-1 and OE-2, and the VIGS-silenced NtSWEET12i transgenic tobacco lines VIGS-1 and VIGS-2, and Nicotiana benthamiana (CK) were planted in flowerpots with a vermiculite to nutrient soil ratio of 1:1. After transplanting, they were subjected to stress treatment by irrigating with 200 mM mannitol, 200 Mm sodium chloride, or 100 Mm sodium bicarbonate. After 48 hours of treatment, samples were taken from each line to observe the degree of wilting of the tobacco plants.

[0125] The processing for each group is as follows:

[0126] a. Control group, i.e. no stress treatment: normal watering for 48 h, and observation of plant growth;

[0127] Treatment group b was subjected to irrigation with 200 mM mannitol, 200 Mm sodium chloride, or 100 Mm sodium bicarbonate, respectively. Plant growth was observed 48 hours after treatment.

[0128] 5.2 Experimental Results and Analysis

[0129] The results are as follows Figure 4As shown, there was no significant difference in the degree of wilting between the control group CK and NtSWEET12i transgenic tobacco plants; however, the leaves of the treatment group VIGS transgenic tobacco line (NtSWEET12i silenced) were severely wilted, the leaves of the CK plants were moderately wilted, and the leaves of the overexpressing transgenic plants were mildly wilted.

[0130] The results of this experiment show that overexpression of the NtSWEET12i gene can significantly improve the resistance of tobacco to drought and saline-alkali stress.

[0131] Example 6: Determination of physiological and biochemical indicators of NtSWEET12i transgenic tobacco plants

[0132] 6.1 Determination of total soluble sugar content

[0133] Sugars are not only a carbon source, energy source, and structural material for plant cells, but also a signaling molecule that plays an important role in plant growth, development, and responses to abiotic stress. Under stress conditions, plants increase their soluble sugar content to help maintain water balance, increase cell osmotic pressure, and protect cell membranes.

[0134] (1) Test method:

[0135] The soluble total sugar content of tobacco plants was determined using a soluble total sugar content assay kit (Jiangsu Aidisheng Biotechnology Co., Ltd.). The tobacco plants used were the control and treatment groups described in Example 5 above. The plants included NtSWEET12i overexpressing transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants. The experiment was repeated three times, and the average value was taken.

[0136] (2) Experimental results and analysis

[0137] The results are as follows Figure 5 As shown, the control group was without stress, while the treatment group was under stress. These results indicate that the total soluble sugar content of plants overexpressing NtSWEET12i transgenic tobacco was significantly higher than that of the control group (CK), while the total soluble sugar content of plants with VIGS-silenced NtSWEET12i transgenic tobacco was significantly lower than that of the control group (CK).

[0138] 6.2 Determination of ABA content

[0139] Abscisic acid (ABA) plays a crucial role in plant responses to abiotic stress. ABA can enhance salt tolerance, alleviate osmotic and ion stress caused by excessive salt, maintain water balance, induce the accumulation of proline (an osmotic regulator), maintain cell membrane stability, and increase the activity of protective enzymes. Under drought stress, ABA significantly reduces leaf water evaporation, decreases leaf cell membrane permeability, increases the content of soluble protein in leaf cells, induces the formation of protective enzymes in biomembrane systems, reduces membrane lipid peroxidation, enhances antioxidant capacity, and improves the plant's drought and salt tolerance.

[0140] (1) Test method:

[0141] The ABA content of tobacco plants was detected using an ABA content kit (Jiangsu Aidisheng Biotechnology Co., Ltd.). The tobacco plants used were the control and treatment groups described in Step 5 above. The tobacco plants included NtSWEET12i overexpressing transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants. The experiment was repeated three times, and the results were averaged.

[0142] (2) Experimental results and analysis

[0143] The results are as follows Figure 6 As shown, the control group was without stress, while the treatment group was under stress. The results showed that the ABA content of the NtSWEET12i transgenic tobacco plants was significantly higher than that of the CK, while the ABA content of the VIGS-silenced NtSWEET12i transgenic tobacco plants was significantly lower than that of the CK.

[0144] 6.3 Determination of proline content

[0145] Under normal conditions, plants contain very low levels of free proline. However, when faced with stresses such as drought, low temperature, and salinity, free proline accumulates in large quantities, and the accumulation index is related to the plant's stress resistance. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

[0146] (1) Test method:

[0147] The proline (PRO) content in tobacco plants was detected using a proline (PRO) content kit (Jiangsu Adison Biotechnology Co., Ltd.). The tobacco plants used were those from the control and treatment groups described in Example 5 above. The tobacco plants included those overexpressing and VIGS-silenced NtSWEET12i transgenic tobacco lines and those of Nicotiana benthamiana (CK). The experiment was repeated three times, and the results were averaged.

[0148] (2) Experimental results and analysis

[0149] The results are as follows Figure 7As shown, the control group was without stress, while the treatment group was under stress. The results showed that the proline content of the NtSWEET12i transgenic tobacco plants was significantly higher than that of the control group (CK), while the proline content of the VIGS-silenced NtSWEET12i transgenic tobacco plants was significantly lower than that of the control group (CK).

[0150] 6.4 POD activity assay

[0151] POD activity can serve as a biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of stress damage suffered by the plant.

[0152] (1) Test method:

[0153] The POD activity of tobacco plants was detected using a peroxidase (POD) kit (Jiangsu Aidisheng Biotechnology Co., Ltd.). The tobacco plants were the control and treatment groups described in Example 5 above, including NtSWEET12i transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and plants of Nicotiana benthamiana (CK).

[0154] (2) Experimental results and analysis

[0155] The results are as follows Figure 8 As shown, the control group was without stress, while the treatment group was under stress. The results showed that the POD activity of the NtSWEET12i transgenic tobacco plants was significantly higher than that of the CK, while the POD activity of the VIGS-silenced NtSWEET12i transgenic tobacco plants was significantly lower than that of the CK.

[0156] 6.5 SOD Activity Assay

[0157] Superoxide dismutase (SOD) activity can serve as a biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of damage the plant suffers from adverse conditions.

[0158] (1) Test method:

[0159] Superoxide dismutase (SOD) activity in tobacco plants was detected using a superoxide dismutase (SOD) kit (Jiangsu Adison Biotechnology Co., Ltd.). The tobacco plants used were the control and treatment groups described in Example 5 above. The plants included NtSWEET12i overexpressing transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants. The experiment was repeated three times, and the average value was taken.

[0160] (2) Experimental results and analysis

[0161] The results are as follows Figure 9As shown, the control group was without stress, while the treatment group was under stress. The results showed that the SOD activity of the NtSWEET12i transgenic tobacco plants was significantly higher than that of the CK, while the SOD activity of the VIGS-silenced NtSWEET12i transgenic tobacco plants was significantly lower than that of the CK.

[0162] 6.6 Determination of H2O2 content

[0163] When plants are under stress or aging, the increased metabolism of reactive oxygen species (ROS) leads to the accumulation of H2O2. H2O2 can directly or indirectly oxidize intracellular macromolecules such as nucleic acids and proteins, and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H2O2 content, the greater the degree of damage the plant suffers from environmental stress.

[0164] (1) Test method:

[0165] A hydrogen peroxide (H2O2) reagent kit (Jiangsu Aidisheng Biotechnology Co., Ltd.) was used to detect the H2O2 accumulation in tobacco plants. The tobacco plants were the control and treatment groups described in Example 5 above. The tobacco plants included NtSWEET12i transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants. The experiment was repeated three times, and the results were averaged.

[0166] (2) Experimental results and analysis

[0167] The results are as follows Figure 10 As shown, the control group was without stress, while the treatment group was under stress. The results showed that the H2O2 content of the NtSWEET12i transgenic tobacco plants was significantly lower than that of the CK, while the H2O2 content of the VIGS-silenced NtSWEET12i transgenic tobacco plants was significantly higher than that of the CK.

[0168] 6.7 Determination of malondialdehyde content

[0169] When plants are subjected to abiotic stress, they produce large amounts of superoxide free radicals, which cause membrane lipid peroxidation, producing malondialdehyde (MDA). Excessive accumulation of MDA can lead to cross-linking and polymerization of biomolecules such as proteins and nucleic acids, resulting in alterations in the structure and function of the cell membrane. Therefore, membrane lipid peroxidation is an important marker of damage to plant cell membranes.

[0170] (1) Test method:

[0171] The accumulation of MDA in tobacco plants was detected using a malondialdehyde (MDA) kit (Jiangsu Aidisheng Biotechnology Co., Ltd.). The tobacco plants used were the control and treatment groups from Example 5 above. The plants included NtSWEET12i overexpressing transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants. The experiment was repeated three times, and the average value was taken.

[0172] (2) Experimental results and analysis

[0173] The results are as follows Figure 11 As shown, the control group was without stress, while the treatment group was under stress. The results showed that the MDA content of plants overexpressing NtSWEET12i transgenic tobacco was significantly lower than that of CK, while the MDA content of plants VIGS-silenced NtSWEET12i transgenic tobacco was significantly higher than that of CK.

[0174] 6.8 Expression analysis of stress resistance-related genes

[0175] (1) Experimental method:

[0176] The aforementioned transiently overexpressing NtSWEET12i transgenic tobacco lines OE-1 and OE-2, and the VIGS-silenced NtSWEET12i transgenic tobacco lines VIGS-1 and VIGS-2 were planted in flowerpots with a vermiculite-to-nutrient soil ratio of 1:1. After transplanting, they were subjected to stress treatment by irrigation with 200 mM mannitol, 200 Mm sodium chloride, or 100 Mm sodium bicarbonate, respectively. The untreated group served as the control group. After 48 hours of simultaneous treatment, samples were taken from each line, and multiple genes (NtSWEET12i, NtNCED3, NtABI1, NtSOD, NtAPX3, NIP5CS, NtRD26, and NtRD29) related to the ABA signaling pathway, proline biosynthesis, and ROS scavenging system of each tobacco line were analyzed by qRT-PCR.

[0177] (2) Experimental results and analysis

[0178] like Figure 12As shown, under drought and saline-alkali conditions, compared with the control (CK), key genes related to ABA biosynthesis (NtNCED3), ABA signaling (NtRD26 and NtRD29), proline biosynthesis (NtP5CS), and ROS scavenging (NtSOD and NtAPX3) were significantly upregulated in NtSWEET12i-OE plants; while in NtSWEET12i-VIGS plants, key genes related to ABA biosynthesis (NtNCED3), ABA signaling (NtRD26 and NtRD29), proline biosynthesis (NtP5CS), and ROS scavenging (NtSOD and NtAPX3) were significantly downregulated.

[0179] This shows that NtSWEET2i is a positive regulator of abiotic resistance, while NtABI1 is a negative regulator of abiotic resistance. Therefore, the expression level of NtABI1 in NtSWEET2i transgenic lines exhibits an opposite pattern.

[0180] The above results indicate that overexpression of the NtSWEET12i gene can improve the drought and salt tolerance of tobacco.

[0181] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims. This invention can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without the need for unnecessary experiments. Although specific embodiments are given in this invention, it should be understood that further improvements can be made to the invention. In summary, according to the principles of this invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A NtSWEET12i protein, a gene encoding the NtSWEET12i protein, an expression cassette, a recombinant vector or a recombinant microorganism for use in improving drought resistance and saline-alkali tolerance of tobacco; the amino acid sequence of the NtSWEET12i protein is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding the NtSWEET12i protein is shown as SEQ ID No.2; the expression cassette contains a DNA molecule for expressing the NtSWEET12i protein in a host cell, which includes a promoter for initiating transcription of the NtSWEET12i gene and a terminator for terminating transcription of the NtSWEET12i gene; the recombinant vector carries the gene encoding the tobacco NtSWEET12i protein; the recombinant microorganism transforms the recombinant vector.

2. Use according to claim 1, characterized in that, The drought resistance and saline-alkali tolerance of tobacco are improved by up-regulating the expression amount of the NtSWEET12i protein in tobacco. 3.A NtSWEET12i protein, a gene encoding the NtSWEET12i protein, an expression cassette, a recombinant vector or a recombinant microorganism for use in preparing a product for improving drought resistance and saline-alkali tolerance of tobacco; the amino acid sequence of the NtSWEET12i protein is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding the NtSWEET12i protein is shown as SEQ ID No.2; the expression cassette contains a DNA molecule for expressing the NtSWEET12i protein in a host cell, which includes a promoter for initiating transcription of the NtSWEET12i gene and a terminator for terminating transcription of the NtSWEET12i gene; the recombinant vector carries the gene encoding the tobacco NtSWEET12i protein; the recombinant microorganism transforms the recombinant vector.

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