Tobacco NtSWEET12i protein, coding gene thereof, recombinant vector and application of tobacco NtSWEET12i protein in improvement of drought resistance and saline-alkaline tolerance of tobacco

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a tobacco NtSWEET12i protein, a coding gene thereof, a recombinant vector and application of the tobacco NtSWEET12i protein in improvement of drought resistance and saline-alkaline tolerance of tobacco, and 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 a protein tag to the amino terminal or carboxyl terminal of the amino acid sequence as shown in SEQ ID NO.1; and 3) a protein which has the same function with the protein with the amino acid sequence as shown in SEQ ID NO.1, wherein the identity of the protein and the protein with the amino acid sequence as shown in SEQ ID NO.1 reaches 95% or above. The coding gene of the protein is one of the following sequences: (1) a nucleotide sequence, such as a gene sequence as shown in SEQ ID No.2; and (2) a gene sequence which is hybridized with the gene limited by (1) under strict conditions and is used for coding the protein NtSWEET12i. The gene can regulate the stress resistance such as drought resistance, saline-alkaline tolerance and the like of plants, and has important significance in cultivation of tobacco strains with higher stress tolerance and further improvement of tobacco yield.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a tobacco NtSWEET12i protein, a coding gene, a recombinant vector and an application thereof in improving tobacco drought resistance and salt-alkali resistance. Background Art

[0002] With global climate change, abiotic stresses, especially drought and salinity stresses, have adverse effects on crop growth and development, thus threatening global food security. Identifying genes related to adverse stresses in plants is crucial for improving drought and salinity stress tolerance in plants. With the application of plant biotechnology in plant breeding, the regulatory network of plant resistance genes has gradually become clear. However, the transcriptional regulatory mechanism in response to abiotic stress in plants remains unclear and requires further study.

[0003] Among them, tobacco, a dicotyledonous plant, is an annual or limited perennial herb of the genus Nicotiana in the Solanaceae family. As an important model plant in plant science, it not only has a good foundation in 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, tobacco growth is easily affected by extreme environments, including abiotic environmental stresses such as drought and salinity, which will lead to a decrease in its yield.

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

[0005] Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a tobacco NtSWEET12i protein, its encoding gene, a recombinant vector and its use in improving the drought resistance and salt-alkali tolerance of tobacco. The drought resistance and salt-alkali tolerance of tobacco can be improved by upregulating the expression level of the gene in the plant, and the protein can be applied to the cultivation of tobacco lines with stronger stress tolerance.

[0007] In a first aspect, the present application provides a tobacco NtSWEET12i protein, which is one of the following sequences: 1) A protein having an amino acid sequence as shown in SEQ ID NO.1; 2) A fusion protein formed by connecting a protein tag to the amino terminal or carboxyl terminal of the amino acid sequence shown in SEQ ID NO.1; 3) A protein having an amino acid sequence identity of more than 95% with the protein shown in SEQ ID NO. 1 and having the same function.

[0008] The SEQ ID NO. 1 consists of 291 amino acid residues and is derived from tobacco (Nicotiana tabacum L.).

[0009] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In order to facilitate purification or detection of the above protein, a protein tag may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1.

[0011] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag includes but is not limited to: GST (glutathione sulfhydryl transferase) 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 (monomeric red fluorescent protein) or AviTag protein tag and other protein tags.

[0012] The above amino acid sequence includes the sequence shown in SEQ ID NO: 1, and also includes homologous sequences with 95% or more identity to the protein sequence, which can be obtained by mutating the sequence shown in SEQ ID NO: 1 using existing technology methods. The protein activity of these homologous sequences is the same as the protein activity of SEQ ID NO: 1.

[0013] In a second aspect, the present application also provides a gene encoding the above tobacco NtSWEET12i protein, which is one of the following sequences: (1) The nucleotide sequence is the gene sequence shown in SEQ ID No. 2; (2) A gene sequence that hybridizes with the gene defined in (1) under stringent conditions and encodes the protein NtSWEET12i.

[0014] Among them, SEQ ID NO.2 consists of 876 nucleotides, and its open reading frame (ORF) is from the 1st to the 876th position from the 5′ end, encoding a protein with an amino acid sequence as shown in SEQ ID NO.1.

[0015] The stringent conditions are hybridization and washing the membrane twice at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and then hybridization and washing the membrane twice at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 min each time.

[0016] The above gene sequence includes not only the sequence shown in SEQ ID NO: 2, but also homologous sequences with 95% or more identity to the gene sequence, which can be obtained by mutating the sequence shown in SEQ ID NO: 2 using existing technology methods. The protein activity expressed by these homologous sequences is the same as the protein activity expressed by the sequence of SEQ ID NO: 2.

[0017] In addition, those artificially modified nucleotides that have 95% or more identity with the nucleotide sequence encoding the protein MsRGP1 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the function and activity of the encoded protein are basically the same.

[0018] The present application screened the NtSWEET12i protein that responds to drought and saline-alkali stress from tobacco. On the one hand, an overexpression vector pCAMBIA super1300-NtSWEET12i-GFP was constructed, and Arabidopsis thaliana was transformed using the Agrobacterium-mediated method. After screening and identification, NtSWEET12i transiently overexpressing tobacco plants were obtained. Experiments have shown that the drought and saline resistance of tobacco overexpressing this protein is significantly improved, and the total soluble sugar content, stress-related hormone ABA content, proline content, POD activity, SOD activity, reactive oxygen content and malondialdehyde content in the body are also regulated by this gene.

[0019] Therefore, NtSWEET12i protein is a new important gene that affects the drought and salt-alkali resistance of plants. It can be used to cultivate new plant materials with stronger stress resistance, and its application value is high.

[0020] In a third aspect, the present application also provides an expression cassette, which expresses a DNA molecule of the protein NtSWEET12i in a host cell. The DNA molecule may include not only a promoter for initiating transcription of the NtSWEET12i gene, but also a terminator for terminating transcription of the NtSWEET12i gene.

[0021] Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present 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, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related protein 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or a LAP promoter (both inducible by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); a seed-specific promoter, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 10099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, 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 incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline 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 et al. (1991) Mol. Gen. Genet, 262:141; (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).

[0022] In a fourth aspect, the present application also provides a vector carrying the gene encoding the aforementioned tobacco NtSWEET12i protein.

[0023] A recombinant vector containing the NtSWEET12i encoding gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA super1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When NtSWEET12i is used to construct a recombinant vector, any enhancing, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of 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. The translation initiation region can come from the transcription initiation region or the structural gene.

[0024] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0025] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP. The recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP is a recombinant vector 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.

[0026] In a fifth aspect, the present application also provides a recombinant microorganism transformed with the aforementioned recombinant vector.

[0027] The recombinant microorganism can specifically be yeast, bacteria, algae and fungi. Further optionally, the bacteria is Agrobacterium GV3101 strain.

[0028] In a sixth aspect, the present application also provides the use of the aforementioned NtSWEET12i protein, the aforementioned coding gene of the NtSWEET12i protein, the aforementioned expression cassette, the aforementioned recombinant vector or the aforementioned recombinant microorganism in regulating plant drought resistance and salt-alkali tolerance.

[0029] In the seventh aspect, the present application also provides the use 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 the total soluble sugar content of plants, the content of stress-related hormone ABA, the proline content, POD activity, SOD activity, reactive oxygen content and malondialdehyde content.

[0030] In the present application, the high drought and salt-alkali resistance mentioned in the aforementioned part is mainly reflected in increasing the total soluble sugar content of plants, the content of stress-related hormone ABA, the content of proline, the activity of POD and the activity of SOD, while reducing the content of active oxygen and malondialdehyde.

[0031] Optionally, the plant is a dicotyledon or a monocotyledon. Preferably, in the application, the plant is tobacco.

[0032] Optionally, in the application, the expression level of NtSWEET12i protein in the plant is upregulated to improve the drought resistance and salt-alkali tolerance of the plant.

[0033] Among the above applications, the application in plant breeding can specifically be hybridizing plants containing the protein or the protein encoding gene NtSWEET12i with other plants to carry out plant breeding.

[0034] In a sixth aspect, the present application also provides a method for cultivating drought-resistant and salt-alkali-tolerant tobacco, the cultivation method comprising: The NtSWEET12i plant expression vector is constructed, transformed by Agrobacterium-mediated transformation, and infected into wild-type plants. NtSWEET12i expression / overexpression plants are obtained by cultivation and screening, which are drought-resistant and salt-alkali-tolerant plants.

[0035] Optionally, the plant is tobacco.

[0036] In the above method, the method of expression or overexpression is to introduce the gene encoding protein NtSWEET12i into the target plant. In the above method, the gene encoding protein NtSWEET12i can be introduced into the target plant by introducing the plant expression vector carrying the NtSWEET12i gene of the present invention into the target plant.

[0037] The plant expression vector carrying the gene NtSWEET12i of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the transformed plant cells or tissues can be cultivated into plants.

[0038] In the above method, the nucleotide sequence of the gene encoding the protein NtSWEET12i is the DNA molecule shown in SEQ ID NO.2.

[0039] 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.

[0040] The present invention has the following beneficial effects: 1. The present invention discovered for the first time that the NtSWEET12i protein is involved in the plant's response to drought resistance and salt-alkali stress, positively regulates the plant's drought resistance and salt-alkali tolerance, and can improve its drought resistance and salt-alkali tolerance by upregulating the expression level of the gene in the plant.

[0041] In this application, the coding gene of NtSWEET12i protein was introduced into tobacco by different methods, and transgenic tobacco plants transiently overexpressing NtSWEET12i and transgenic tobacco plants with virus-induced gene silencing (VIGS) NtSWEET12i were obtained respectively. The above two types of transgenic plants were subjected to drought and saline-alkali stress treatment, and it was found that: compared with the control, the drought and saline resistance of the overexpressing transgenic strains was enhanced, while the drought and saline resistance of the gene-silenced transgenic strains was reduced, which was specifically reflected in the increase / decrease of the total soluble sugar content of plants, the content of stress-related hormone ABA, the proline content, the POD activity and the SOD activity, and at the same time, the active oxygen content and the malondialdehyde content were reduced / increased.

[0042] Therefore, the NtSWEET12i gene provided by the present invention and the protein encoded by it play an important role in the process of plant drought and salt-alkali resistance. Through overexpression technology, it has important application value in improving plant drought and salt-alkali resistance, and has broad application space and market prospects in the agricultural field.

[0043] 2. The NtSWEET12i gene provides new genetic resources for improving the drought and salt-alkali resistance of tobacco cultivars and for the selection of germplasm resources, and provides new application value for promoting the quality improvement, efficiency enhancement and transformation and upgrading of the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Sequence analysis, expression analysis and subcellular localization of NtSWEET12i gene. (A) Expression analysis of NtSWEET12i in tobacco after 48h of treatment with mannitol, NaCl, NaHCO3 and ABA. Data are expressed as mean ± SD (n = 3). Tobacco Ntactin was used as the internal reference gene. * (or different lowercase letters) and ** (or different uppercase letters) indicate significant differences at P < 0.05 and P < 0.01 using Student's t-test (or Ordinary one-way ANOVA multiple comparison), respectively. Figure 2 is the genomic structure of NtSWEET12i, boxes represent exons, and lines represent introns; Figure 3 To investigate the subcellular localization of NtSWEET12i, epidermal cells of Nicotiana benthamiana leaves were transformed with a fusion construct of NtSWEET12i-GFP and the membrane marker PIP2-mCherry. bar = 25 μm; Figure 4 Pot culture identification of drought and salt tolerance of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; CK is the control, OE-1 and OE-2 are transient overexpression, VIGS-1 and VIGS-2 are VIGS silenced NtSWEET12i transgenic tobacco lines; Figure 5 To determine the total soluble sugar content of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; Figure 6 Determination of ABA content in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; Figure 7 Determination of proline content in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; Figure 8 POD activity assay of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; Fig. 9 The SOD activity of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was measured; Fig.10Determination of hydrogen peroxide content in NtSWEET12i transgenic tobacco plants and wild-type tobacco plants; Fig.11 The malondialdehyde content of NtSWEET12i transgenic tobacco plants and wild-type tobacco plants was determined; Among them, CK is the control, OE-1 and OE-2 are overexpression, and VIGS-1 and VIGS-2 are VIGS-silenced NtSWEET12i transgenic tobacco lines; Fig.12 Expression analysis of related genes in leaves of NtSWEET12i transgenic plants and CK plants under untreated or mannitol, sodium chloride and sodium bicarbonate stress. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work 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. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0046] Example 1 Acquisition of NtSWEET12i gene 1.1 Experimental Methods (1) Obtaining cDNA template The total RNA of tobacco variety Honghua Dajinyuan was extracted using a plant total RNA extraction kit and the total RNA was purified by PrimeScript TM RT reagent Kit with gDNA Eraser kit is used to reverse transcribe the first-strand cDNA.

[0047] (2) Using the cDNA obtained in step 1 as a template, design primers NtSWEET12i-F and NtSWEET12i-R according to the sequence provided on the website http: / / lifenglab.hzau.edu.cn / Nicomics / . Perform PCR amplification to obtain a CDS amplification product of approximately 876 bp and sequence it. The primer sequences are as follows: NtSWEET12i-F: 5′-AATCCTATTTCACAGCCAAGTA-3′ NtSWEET12i-R: 5′-CACTGCCAATTCCAGAAGTGAA-3′ 1.2 Experimental Results and Analysis According to the sequencing results, the nucleotide sequence of CDS is shown in SEQ ID NO.2, which is 876 bp long. The gene shown in the sequence is named NtSWEET12i gene, and the protein encoded by it is named NtSWEET12i protein. The amino acid sequence is shown in SEQ ID NO.1.

[0048] Example 2 Analysis of the gene, cellular localization and expression of NtSWEET12i in tobacco 2.1 Analysis of the gene structure of NtSWEET12i (1) Experimental methods Based on the above-mentioned genomic sequence and coding sequence of NtSWEET12i, exon and intron structure analysis was performed using TBtools-II software and GSDS2.0 online website (https: / / gsds.gao-lab.org / ).

[0049] (2) Experimental results and analysis The results are as follows Figure 2 As shown, the NtSWEET12i gene contains 6 exons and 5 introns.

[0050] 2.2 Subcellular localization analysis of NtSWEET12i 1. Experimental methods The subcellular localization of NtSWEET12i was detected by transiently co-expressing PIPII-mCherry and NtSWEET12i-GFP proteins in leaves of Nicotiana benthamiana. PIPII-mCherry is a membrane marker gene, which is a red fluorescence in subcellular localization analysis. By co-expressing it with NtSWEET12i, the subcellular localization of NtSWEET12i protein was performed.

[0051] The specific method is: After constructing PCAMBIA super1300-NtSWEET12i-GFP according to the method of Example 3, PIPII-mCherr, pCAMBIA super1300-GFP and PCAMBIA super1300-NtSWEET12i-GFP were respectively transformed into Agrobacterium (manufacturer: Weidi Biotechnology; catalog number: CAT#: AC1003), and the Agrobacterium was transformed into the tobacco transient transformation technique according to Figure 3 The indicated combinations were injected into tobacco leaves, and after 48 h, the fluorescence expression was observed using a confocal microscope (LSM880, Zeiss, GER).

[0052] Among them, the combined injection groups are as follows: Combination 1: injection of PIPII-mCherry and pCAMBIA super1300-GFP; Combination 2: injection of PIPII-mCherry and pCAMBIA super1300-NtSWEET12i-GFP 2. Experimental results and analysis Fluorescence signal analysis showed that NtSWEET12i was localized in the cell membrane (see Figure 3 ).

[0053] 2.3 Expression analysis of NtSWEET12i in wild-type tobacco (1) Experimental methods: The wild-type 4-leaf stage S. safflower Dajinyuan plants were treated in Hoagland's solution containing 200 mM mannitol, 200 mM sodium chloride, 100 mM sodium bicarbonate and 0.1 mM ABA (spraying) for 0h, 0.5h, 1h, 3h, 6h, 12h, 24h and 48h, and samples were taken. The expression profile of NtSWEET12i was quantitatively analyzed by qRT-PCR, and the NtACTIN gene of tobacco was used as an internal reference.

[0054] (2) Experimental results and analysis The results are as follows Figure 1 As shown, NtSWEET12i was upregulated by mannitol, sodium chloride, sodium bicarbonate and ABA (abscisic acid), which indicates that mannitol, sodium chloride, sodium bicarbonate or ABA in the environment can induce the increase of NtSWEET12i protein expression in tobacco, and this protein may be related to the plant's stress resistance.

[0055] Example 3 Construction of transgenic tobacco lines OE-1 and OE-2 transiently overexpressing NtSWEET12i 3.1 Construction of recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP (1) A double-stranded DNA molecule of NtSWEET12i with a sequence as shown in SEQ ID NO. 2 was artificially synthesized, and the double-stranded DNA molecule was used as a template to perform PCR amplification with primers OE-F-BamHI and primer OE-R-BamHI, and the amplified product was recovered. The amplified product was 906 bp in length.

[0056] OE-F-BamHI: 5'-CCCAAGCTTGTCGACGGATCCATGTCTGGTCACTGGGCTTTG-3' (SEQ ID NO.3) OE-R-BamHI: 5'-GCCCTTGCTCACCATGGATCCGGCTTTCACAGTTTGCAGCTT -3' (SEQ ID NO.4) (The underlined part is the recognition site of restriction endonuclease BamHI) (2) The vector pCAMBIA super1300-GFP and the above amplified product were digested with restriction endonuclease BamHI to recover the approximately 10783 bp vector backbone 1. The above amplified product was digested with the same restriction endonuclease and recovered.

[0057] (3) The enzyme-cut fragment 2 was connected to the vector backbone 1 to obtain the recombinant plasmid pCAMBIA super1300-NtSWEET12i-GFP, and then sequenced and analyzed.

[0058] Experimental results and analysis: 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 a 876 bp DNA molecule as shown in SEQ ID NO.2, so that it subsequently expresses the NtSWEET12i protein shown in SEQ ID NO.1.

[0059] 3.2 Conversion The pCAMBIA super1300-NtSWEET12i-GFP plasmid was transferred into Agrobacterium (manufacturer: Weidi Biotechnology; product number: CAT#: AC1003), and the Agrobacterium was injected to infect tobacco leaves using tobacco transient transformation technology.

[0060] 3.3 Screening of overexpression plants After waiting for 48 hours after Agrobacterium infection, a small portion of leaves from the plants were taken for NtSWEET12i expression analysis, and plants OE-1 and OE-2 with significantly increased NtSWEET12i expression were selected as transiently overexpressing NtSWEET12i transgenic tobacco lines for subsequent experiments.

[0061] Example 4 Construction and Screening of VIGS-1 and VIGS-2 Transgenic Tobacco Lines Silencing NtSWEET12i 4.1 Construction of recombinant plasmid pTRV2-NtSWEET12i A suitable sequence was selected in the non-conserved domain of the NtSWEET12i gene, primers were designed using Primer 5, the CDS of NtSWEET12i was used as a template, and amplification was performed with primers NtSWEET12i-VIGS-F and NtSWEET12i-VIGS-R. The amplified product and the VIGS vector were digested and then ligated to construct the pTRV2-NtSWEET12i recombinant plasmid.

[0062] The primer sequences are as follows: NtSWEET12i-VIGS-F: 5'-GTGAGTAAGGTTACCGAATTCTGGGTGGCTTTGGTGCTATT -3' (SEQ ID NO.5) NtSWEET12i-VIGS-R: 5'-CGTGAGCTCGGTACCGGATCCACCACAATCACTGCTGGCTT-3' (SEQ ID NO.6) (2) Plasmid transformation The recombinant plasmid pTRV2-NtSWEET12i was transferred into Agrobacterium (manufacturer: Weidi Biotechnology; CAT#: AC1003), and the Agrobacterium was injected to infect tobacco leaves. This method uses the existing tobacco transient transformation technology.

[0063] (3) Screening of transgenic tobacco VIGS-1 and VIGS-2 After the tobacco plants were infected with Agrobacterium for 48 hours, a small portion of the leaves were taken from the plants for analysis of the expression level of NtSWEET12i. Plants VIGS-1 and VIGS-2 with significantly reduced NtSWEET12i expression were selected as VIGS-silenced NtSWEET12i transgenic tobacco lines for subsequent experiments.

[0064] Example 5 Drought and salt tolerance test of NtSWEET12i transgenic tobacco plants 5.1 Experimental Methods The aforementioned transiently overexpressing NtSWEET12i transgenic tobacco lines OE-1, OE-2 and VIGS-silenced NtSWEET12i transgenic tobacco lines VIGS-1, VIGS-2 and Nicotiana benthamiana (CK) were planted in pots with a ratio of vermiculite to nutrient soil of 1:1. After transplantation, they were irrigated with 200 mM mannitol, 200 Mm sodium chloride or 100 Mm sodium bicarbonate for stress treatment. After 48 h of treatment, samples were taken from each line to observe the degree of wilting of the tobacco plants.

[0065] The treatments for each group were as follows: a Control group, i.e. no stress treatment: normal watering for 48 h, and observation of plant growth; b Treatment groups were irrigated with 200 mM mannitol, 200 Mm sodium chloride or 100 Mm sodium bicarbonate stress, and the plant growth was observed after 48 h of treatment.

[0066] 5.2 Experimental Results and Analysis The results are as follows Figure 4 As shown in the figure, there was no significant difference in the degree of wilting between the control group CK and NtSWEET12i transgenic tobacco plants; while the leaves of the VIGS transgenic tobacco line (NtSWEET12i silenced) in the treatment group wilted severely, the leaves of the CK plants wilted moderately, and the leaves of the overexpressing transgenic plants wilted slightly.

[0067] The experimental results showed that overexpression of the NtSWEET12i gene can significantly improve tobacco's resistance to drought and saline-alkali stress.

[0068] Example 6 Determination of Physiological and Biochemical Indexes of NtSWEET12i Transgenic Tobacco Plants 6.1 Determination of total soluble sugar content Sugar is not only a carbon source, energy source and structural material for plant cells, but also a signal molecule that plays an important role in plant growth and development and response to adverse stress. Under adverse conditions, plants increase the content of soluble sugars to help themselves maintain water balance, increase cell osmotic pressure and protect cell membranes.

[0069] (1) Test method: The soluble total sugar content of tobacco plants was detected using a soluble total sugar content kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants were the tobacco plants of the control group and the treatment group in Example 5 above, and the tobacco plants were 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.

[0070] (2) Experimental results and analysis The results are as follows Figure 5 As shown, the control group is without stress, and the treatment group is under stress. The results show that the total soluble sugar content of the plants overexpressing NtSWEET12i transgenic tobacco lines is significantly higher than that of CK, and the total soluble sugar content of the plants of VIGS-silenced NtSWEET12i transgenic tobacco lines is significantly lower than that of CK.

[0071] 6.2 Determination of ABA content Abscisic acid (ABA) plays an important role in plant stress response. ABA can improve plant salt tolerance, alleviate osmotic stress and ion stress caused by excessive salt, maintain water balance, induce the accumulation of proline, an osmotic regulator in plants, maintain the stability of cell membrane structure, and increase the activity of protective enzymes. During drought stress, ABA can significantly reduce leaf water evaporation, reduce leaf cell membrane permeability, increase the soluble protein content of leaf cells, induce the formation of protective enzymes in the biomembrane system, reduce the degree of membrane lipid peroxidation, enhance antioxidant capacity, and improve the drought and salt-alkali resistance of plants.

[0072] (1) Test method: The ABA content of tobacco plants was detected using an ABA content kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants were the tobacco plants of the control group and the treatment group in the above-mentioned embodiment 5. The tobacco plants were plants of 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.

[0073] (2) Experimental results and analysis The results are as follows Figure 6 As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the ABA content of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly higher than that of CK, and the ABA content of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly lower than that of CK.

[0074] 6.3 Determination of proline content Under normal conditions, the free proline content of plants is very low, but when encountering stresses such as drought, low temperature, and salt, the free amino acid will accumulate in large quantities, and the accumulation index is related to the stress resistance of the plant. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

[0075] (1) Test method: The proline content of tobacco plants was detected using a proline (PRO) content kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants were the tobacco plants of the control group and the treatment group in Example 5 above. The tobacco plants were plants of the NtSWEET12i transgenic tobacco lines that overexpressed and silenced VIGS and plants of Nicotiana benthamiana (CK). The experiment was repeated three times, and the results were averaged.

[0076] (2) Experimental results and analysis The results are as follows Figure 7As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the proline content of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly higher than that of CK, and the proline content of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly lower than that of CK.

[0077] 6.4 POD activity assay POD activity can be used as a biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of damage to the plant caused by adversity.

[0078] (1) Test method: The POD activity of tobacco plants was detected using a peroxidase (POD) kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants were the tobacco plants of the control group and the treatment group in Example 5, and the tobacco plants were NtSWEET12i overexpressing transgenic tobacco lines, VIGS-silenced NtSWEET12i transgenic tobacco lines, and Nicotiana benthamiana (CK) plants.

[0079] (2) Experimental results and analysis The results are as follows Figure 8 As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the POD activity of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly higher than that of CK, and the POD activity of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly lower than that of CK.

[0080] 6.5 SOD activity assay SOD activity can be used as a biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of damage to the plant caused by adversity.

[0081] (1) Test method: The SOD activity of tobacco plants was detected using a superoxide dismutase (SOD) kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants were the tobacco plants of the control group and the treatment group in Example 5 above, and the tobacco plants were 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.

[0082] (2) Experimental results and analysis The results are as follows Fig. 9 As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the SOD activity of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly higher than that of CK, and the SOD activity of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly lower than that of CK.

[0083] 6.6 Determination of H2O2 content When plants are in adverse conditions or aging, H2O2 accumulates due to the enhanced metabolism of active oxygen in the body. H2O2 can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H2O2 content, the greater the degree of damage to plants caused by adverse conditions.

[0084] (1) Test method: A hydrogen peroxide (H2O2) kit (Jiangsu Addison Biotechnology Co., Ltd.) was used to detect the H2O2 accumulation of tobacco plants. The tobacco plants were the tobacco plants of the control group and the treatment group in Example 5. The tobacco plants were 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.

[0085] (2) Experimental results and analysis The results are as follows Fig.10 As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the H2O2 content of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly lower than that of CK, and the H2O2 content of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly higher than that of CK.

[0086] 6.7 Determination of malondialdehyde content When plants are subjected to adverse stress, they will produce a large number of superoxide free radicals, which will cause membrane lipid peroxidation and produce malondialdehyde. Excessive accumulation of malondialdehyde will cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, leading to changes in the structure and function of cell membranes. Therefore, membrane lipid peroxidation is an important sign of plant cell membrane damage.

[0087] (1) Test method: The MDA accumulation of tobacco plants was detected using a malondialdehyde (MDA) kit (Jiangsu Addison Biotechnology Co., Ltd.). The tobacco plants used were the tobacco plants of the control group and the treatment group in Example 5. The tobacco plants were 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.

[0088] (2) Experimental results and analysis The results are as follows Fig.11As shown, the control group was without stress, and the treatment group was under stress treatment. The results showed that the MDA content of plants overexpressing NtSWEET12i transgenic tobacco lines was significantly lower than that of CK, and the MDA content of plants of VIGS-silenced NtSWEET12i transgenic tobacco lines was significantly higher than that of CK.

[0089] 6.8 Expression analysis of stress resistance-related genes (1) Experimental methods: The aforementioned transiently overexpressing NtSWEET12i transgenic tobacco lines OE-1 and OE-2 and VIGS-silenced NtSWEET12i transgenic tobacco lines VIGS-1 and VIGS-2 were planted in pots with a ratio of vermiculite to nutrient soil of 1:1. After transplantation, they were irrigated with 200 mM mannitol, 200 Mm sodium chloride or 100 Mm sodium bicarbonate for stress treatment. The untreated group was used as the control group. After 48 h of treatment, each line was sampled, and multiple genes related to the ABA signaling pathway, proline biosynthesis and ROS scavenging system (NtSWEET12i, NtNCED3, NtABI1, NtSOD, NtAPX3, NIP5CS, NtRD26 and NtRD29) of each tobacco line were analyzed by qRT-PCR.

[0090] (2) Experimental results and analysis like Fig.12 As shown in the figure, under drought and saline conditions, compared with CK, key genes related to ABA biosynthesis (NtNCED3), ABA signaling (NtRD26 and NtRD29), proline biosynthesis (NtP5CS) and ROS scavenging (NtSOD and NtAPX3) were significantly up-regulated in NtSWEET12i-OE plants; while key genes related to ABA biosynthesis (NtNCED3), ABA signaling (NtRD26 and NtRD29), proline biosynthesis (NtP5CS) and ROS scavenging (NtSOD and NtAPX3) were significantly down-regulated in NtSWEET12i-VIGS plants.

[0091] It can be seen from this that NtSWEET2i acts as a positive regulatory factor for stress resistance, and NtABI1 acts as a negative regulatory factor for stress resistance. Therefore, the expression level of NtABI1 in the NtSWEET2i transgenic line shows an opposite pattern.

[0092] The above results showed that overexpression of NtSWEET12i gene could improve the drought and salt-alkali resistance of tobacco.

[0093] It is understandable that, for those of ordinary skill in the art, equivalent replacements or changes can be made according to the technical solutions and the inventive concept of the present invention, and all these changes or replacements should belong to the protection scope of the claims attached to the present invention. Without departing from the purpose and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes that are out of the scope disclosed in this application and are made with conventional techniques known in the art. According to the scope of the following attached claims, some basic features can be applied.

Claims

1. A NtSWEET12i protein, characterized in that is one of the following sequences: 1) A protein having an amino acid sequence as shown in SEQ ID NO. 1; 2) A fusion protein formed by connecting a protein tag to the amino or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 1; 3) A protein that has an amino acid sequence identity of at least 95% with the protein shown in SEQ ID NO. 1 and has the same function.

2. A gene encoding the tobacco NtSWEET12i protein according to claim 1, characterized in that: is one of the following sequences: (1) The nucleotide sequence is the gene sequence shown in SEQ ID No. 2; (2) A gene sequence that hybridizes with the gene defined in (1) under stringent conditions and encodes the protein NtSWEET12i.

3. An expression cassette, characterized in that The DNA molecule for expressing protein NtSWEET12i in a host cell may include not only a promoter for initiating transcription of the NtSWEET12i gene, but also a terminator for terminating transcription of the NtSWEET12i gene.

4. A recombinant vector, characterized in that A vector carrying the gene encoding the tobacco NtSWEET12i protein as claimed in claim 2.

5. A recombinant microorganism, characterized in that The recombinant vector according to claim 4 is transformed.

6. Use of the NtSWEET12i protein according to claim 1, the gene encoding the NtSWEET12i protein according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the recombinant microorganism according to claim 5 in regulating plant drought resistance and salt-alkali tolerance.

7. Use of the NtSWEET12i protein according to claim 1, the gene encoding the NtSWEET12i protein according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the recombinant microorganism according to claim 5 in regulating plant total soluble sugar content, stress-related hormone ABA content, proline content, POD activity, SOD activity, reactive oxygen content, and malondialdehyde content.

8. The use according to claim 6 or 7, characterized in that The applications include: improving or reducing the drought resistance and salt-alkali tolerance of plants, and preparing products that improve the drought resistance and salt-alkali tolerance of plants.

9. The use according to claim 6 or 7, characterized in that By upregulating the expression of NtSWEET12i protein in plants, the drought resistance and salt-alkali tolerance of plants can be improved.

10. A method for cultivating drought-resistant and salt-alkali-tolerant plants, characterized in that: The cultivation method is: A plant NtSWEET12i expression vector is constructed, transformed through Agrobacterium-mediated transformation, and infected into wild-type plants. NtSWEET12i expression / overexpression plants are obtained through cultivation and screening, which are drought-resistant and salt-alkali-tolerant plants.

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