Use of the ntNRAMP2 protein, methods for increasing sensitivity of tobacco to cadmium stress and methods for obtaining tobacco plants resistant to cadmium stress

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of NtNRAMP2 protein, a method for improving tobacco cadmium stress resistance and a method for obtaining tobacco plants with cadmium stress resistance. The application provides application of NtNRAMP2 protein in creating tobacco with cadmium stress resistance or breeding tobacco with cadmium stress resistance, and an amino acid sequence of the NtNRAMP2 protein is shown as SEQ ID NO:2. The NtNRAMP2 protein is synthesized by NtNRAMP2 gene, and it is proved by transgenic and functional identification that the NtNRAMP2 natural resistance macrophage protein synthesized by the NtNRAMP2 gene is closely related to tobacco cadmium absorption regulation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of NtNRAMP2 protein, methods to improve the sensitivity of tobacco to cadmium stress, and methods to obtain cadmium-resistant tobacco plants. Background Technology

[0002] In recent years, with industrial development and the extensive use of chemical fertilizers and pesticides in agriculture, heavy metal pollution in soil has become increasingly serious. In my country, cadmium (Cd) is a major heavy metal pollutant in agricultural production. After being ingested by organisms, Cd can remain in the organism for many years and exhibits extremely high toxicity and biological tolerance. In addition, Cd is highly mobile, and its toxic effects on organisms are characterized by insidiousness and irreversibility.

[0003] Heavy metal transport-related proteins in plants can be divided into uptake transport proteins and excretion proteins. The natural resistance associated macrophage proteins (NRAMPs) family is a class of transmembrane transport proteins involved in the transport of various divalent metal cations. Members of this family were first discovered in mice and are involved in the uptake, transport, or intracellular regionalization of metal ions such as Fe, Mn, Cd, and Pb. Meanwhile, NRAMP proteins are widely distributed in plants and participate in the uptake and transport of heavy metals such as cadmium. Therefore, cloning plant ion transport protein genes and analyzing their biological functions is of significant biological importance for studying Cd uptake in plants.

[0004] Tobacco, as one of my country's important economic crops, is prone to cadmium (Cd) accumulation, primarily in its leaves. When tobacco leaves are processed into tobacco shreds, Cd enters the human body through the smoke, and long-term accumulation may cause adverse organ reactions and trigger various diseases. Currently, tobacco cadmium stress-tolerant materials and genetic resources are limited. No reports have been found regarding the function and research of the tobacco NtNRAMP2 protein. Further research is needed to identify functional genes involved in cadmium absorption and to explore effective methods to expand the application of these genes in improving tobacco cadmium tolerance. This will provide new technical insights for enriching disease-resistant gene resources and for molecular-assisted breeding and the creation of disease-resistant materials. Summary of the Invention

[0005] The purpose of this invention is to provide an application of NtNRAMP2 protein in the creation or breeding of cadmium-resistant tobacco, wherein the NtNRAMP2 protein can improve the plant's resistance to cadmium stress.

[0006] This invention provides the application of NtNRAMP2 protein in the creation of cadmium-resistant tobacco or the selection of cadmium-resistant tobacco. The amino acid sequence of NtNRAMP2 protein is shown in SEQ ID NO:2.

[0007] This invention provides the application of the NtNRAMP2 protein encoding gene NtNRAMP2 in the creation of cadmium-stress-resistant tobacco or the breeding of cadmium-resistant tobacco, the nucleotide sequence of the encoding gene NtNRAMP2 is shown in SEQ ID NO:1.

[0008] Preferably, the cadmium concentration is ≥25 μmol / L.

[0009] Preferably, the tobacco includes tobacco variety K326.

[0010] This invention provides a method for improving the sensitivity of tobacco to cadmium stress, comprising: applying ethylene or ABA to tobacco plants to increase the expression level of the genes described in the above technical solution.

[0011] Preferably, the working concentration of ABA is 4–8 mmol / L.

[0012] Preferably, the ethylene is applied in the form of an ethephon aqueous solution, wherein the working concentration of ethephon in the ethephon aqueous solution is 1–10 mmol / L.

[0013] Preferably, the method of application includes spraying.

[0014] This invention provides a method for obtaining cadmium-tolerant tobacco plants, comprising constructing an overexpression vector for the NtNRAMP2 protein encoding gene of tobacco, and transfecting the overexpression vector into tobacco plants to obtain cadmium-tolerant tobacco plants; the nucleotide sequence of the encoding gene NtNRAMP2 is shown in SEQ ID NO:1.

[0015] Preferably, the concentration of cadmium is ≥25 μmol / L.

[0016] The beneficial effects of this invention: This invention provides the application of NtNRAMP2 protein in the creation or breeding of cadmium-resistant tobacco. The amino acid sequence of the NtNRAMP2 protein is shown in SEQ ID NO:2. The NtNRAMP2 protein of this invention is encoded and synthesized by the NtNRAMP2 gene. Transgenic and functional identification have confirmed that the NtNRAMP2 natural resistant macrophage protein synthesized by the NtNRAMP2 gene of this invention is closely related to the regulation of cadmium absorption in tobacco. High expression of the NtNRAMP2 gene can promote the absorption of cadmium by tobacco plants. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1This is a gel electrophoresis image of the NtNRAMP2 gene from Example 1; Figure 1 In Chinese, UTR refers to the untranslated region;

[0019] Figure 2 This is a maximum likelihood phylogenetic tree and gene domain analysis diagram of NtNRAMP2 homologous genes in different species in Example 1;

[0020] Figure 3 The expression patterns of the NtNRAMP2 gene in tobacco induced by cadmium concentration gradients are shown in Examples 2-5 and Comparative Example 1.

[0021] Figure 4 The expression patterns of the NtNRAMP2 gene in tobacco induced by cadmium time gradient are shown in Examples 6-9 and Comparative Example 2.

[0022] Figure 5 This is a diagram illustrating the ABA-induced expression pattern of the NtNRAMP2 gene in tobacco, as shown in Example 10.

[0023] Figure 6 This is a diagram illustrating the ethylene-induced expression pattern of the NtNRAMP2 gene in tobacco, as shown in Example 10.

[0024] Figure 7 This is a subcellular localization map of the NtNRAMP2 gene in tobacco, as shown in Example 12.

[0025] Figure 8 This is a schematic diagram of the culture process of transgenic tobacco plants overexpressing the NtNRAMP2 gene in Example 12.

[0026] Figure 9 This is a DNA positive identification image of tobacco plants overexpressing the NtNRAMP2 gene in Example 12;

[0027] Figure 10 This is a graph showing the expression level of the NtNRAMP2 gene in tobacco plants that overexpress NtNRAMP2 in Example 12;

[0028] Figure 11 This is a graph showing the expression level of the NtNRAMP2 gene in tobacco plants overexpressing NtNRAMP2 under cadmium stress in Example 12.

[0029] Figure 12 A graph showing the absorption of Cd by tobacco plants overexpressing the NtNRAMP2 gene. Detailed Implementation

[0030] This invention provides the application of NtNRAMP2 protein in the creation of cadmium-resistant tobacco or the breeding of cadmium-resistant tobacco, wherein the amino acid sequence of NtNRAMP2 protein is shown in SEQ ID NO:2.

[0031] This invention provides a key protein for cadmium absorption, NtNRAMP2 (i.e., the tobacco heavy metal transporter NRAMP), which not only enriches the cadmium absorption gene resource but also lays the foundation for breeding and creating cadmium-resistant germplasm materials. The NtNRAMP2 protein is a novel NRAMP-based naturally resistant macrophage protein that plays a role in cadmium absorption, providing technical support for green and sustainable methods to influence new germplasm resources for cadmium absorption in tobacco.

[0032] The present invention also provides the application of the gene NtNRAMP2 encoding the tobacco NtNRAMP2 protein described in the above technical solution in the creation of cadmium-stress-resistant tobacco or the breeding of cadmium-resistant tobacco, wherein the nucleotide sequence of the encoding gene NtNRAMP2 is shown in SEQ ID NO:1.

[0033] The NtNRAMP2 gene described in this invention is a novel gene encoding the NtNRAMP2 natural resistance macrophage protein, which is closely related to the regulation of cadmium uptake in tobacco. Based on the function of the NtNRAMP2 gene and combined with existing technologies, cadmium-resistant tobacco plants or germplasm materials can be obtained. The NtNRAMP2 gene described in this invention can be used for molecular-assisted breeding to select cadmium-resistant materials and create new genetically cadmium-resistant materials, achieving the goal of green and sustainable control of cadmium stress. Overexpression of the NtNRAMP2 gene can improve the sensitivity of tobacco plants to cadmium stress.

[0034] In this invention, the cadmium concentration is preferably ≥25 μmol / L, more preferably 25 μmol / L to 75 μmol / L, and even more preferably 50 μmol / L.

[0035] In this invention, the tobacco preferably includes the K326 tobacco variety.

[0036] The gene NtNRAMP2 encoding the NtNRAMP2 protein described in this invention is obtained by PCR amplification using amplification primers, which include an upstream primer NtNramp2-F and a downstream primer NtNramp2-R.

[0037] This invention designs upstream primer NtNramp2-F and downstream primer NtNramp2-R based on the predicted mRNA sequence of the NtNramp2 gene from common tobacco (NCBI accession number: LOC107807465). Using cDNA from the roots of tobacco K326 seedlings as a template, PCR amplification is performed to obtain the gene NtNRAMP2 encoding the tobacco NtNRAMP2 protein. In this invention, the preferred nucleotide sequence of the upstream primer NtNramp2-F is shown in SEQ ID NO:3, and the preferred nucleotide sequence of the downstream primer NtNramp2-R is shown in SEQ ID NO:4.

[0038] In this invention, the amplification system for PCR amplification using cDNA from the roots of tobacco K326 seedlings as a template is preferably 50 μL, comprising: 25.0 μL of KOD One™ PCR Master Mix, 1 μL of upstream primer (primer concentration 10 μmol / L), 1 μL of downstream primer (primer concentration 10 μmol / L), 1 μL of template (template concentration 50 ng / μL), and sterile ddH2O to a final volume of 50 μL; the PCR amplification program preferably includes: denaturation at 98℃ for 10 s, annealing at 53℃ for 5 s, extension at 68℃ for 10 s, and 35 cycles.

[0039] This invention provides a method for improving the sensitivity of tobacco plants to cadmium stress. The exogenous hormone ethylene or ABA (abscisic acid) is applied to the tobacco plants to increase the expression levels of the genes described in the above-mentioned scheme. In this invention, the working concentration of ABA is preferably 4–8 mmol / L, more preferably 4–6 mmol / L, and even more preferably 4 mmol / L. The ethylene used in this invention is applied in the form of an ethephon agent, and the mass concentration of the ethephon agent is preferably 1–10 mmol / L, and even more preferably 4 mmol / L.

[0040] This invention increases the expression level of the NtNRAMP2 gene, which encodes the NtNRAMP2 protein, in tobacco plants by spraying them with exogenous hormones ethylene or ABA. Overexpression of the NtNRAMP2 gene enhances the sensitivity of tobacco plants to cadmium stress. The expression level of the NtNRAMP2 gene is preferably determined by real-time quantitative PCR (RT-PCR). The preferred RT-PCR amplification system includes: 10 μL of 2×SYBR Premix Wiz Taq™; 0.4 μL of upstream primer Primer 1 (primer concentration 10 μM); 0.4 μL of downstream primer Primer 2 (primer concentration 10 μM); 2 μL of template cDNA (200 ng / μL); and sterile ddH2O to a final volume of 20 μL. The preferred RT-PCR amplification procedure includes: Step 1: pre-denaturation, 95℃, 30 seconds; Step 2: 95℃, 10 seconds; Step 3: 60℃, 30 seconds; Step 2 and Step 3 are repeated, and each repetition of Step 2 and Step 3 constitutes one cycle, for a total of 40 cycles.

[0041] This invention provides a method for breeding and creating germplasm resources that affect cadmium uptake in tobacco. An overexpression vector for the NtNRAMP2 protein encoding gene is constructed, and the overexpression vector is transferred into tobacco plants to obtain cadmium-tolerant tobacco plants. The nucleotide sequence of the NtNRAMP2 encoding gene is shown in SEQ ID NO:1.

[0042] The overexpression vector of the present invention includes a 35S:NtNramp2 overexpression vector; the 35S:NtNramp2 overexpression vector includes the original vector and the gene described in the above technical solution; the original vector includes pCambia1300.

[0043] This invention does not impose any particular limitation on the construction method of the 35S:NtNramp2 overexpression vector; conventional methods can be used. The preferred method for constructing the 35S:NtNramp2 overexpression vector of this invention includes the following steps: amplifying the gene described in the above technical solution using amplification primers to obtain an amplification product; ligating the amplification product into the original vector to obtain the 35S:NtNramp2 overexpression vector.

[0044] After obtaining the overexpression vector, the preferred method for transferring the overexpression vector into tobacco plants according to the present invention includes Agrobacterium-mediated transformation, preferably utilizing Agrobacterium-mediated plant genetic transformation. The Agrobacterium-mediated transformation method described in this invention can employ conventional methods and is not particularly limited.

[0045] The cadmium concentration of the cadmium stress resistant material described in this invention is preferably ≥25 μmol / L, more preferably 25-75 μmol / L, and even more preferably 50 μmol / L.

[0046] This invention cloned a natural resistant macrophage protein, named NtNRAMP2, and investigated its role in the cadmium uptake pathway in tobacco. Furthermore, using genetic engineering techniques, the NtNRAMP2 gene was genetically transformed into tobacco explants to obtain transgenic tobacco plants overexpressing the gene. This invention achieves genetic improvement of tobacco varieties through the overexpression of the NtNRAMP2 gene.

[0047] This invention discloses for the first time a natural resistant macrophage protein, NtNRAMP2, that plays a role in cadmium uptake. The NtNRAMP2 protein is encoded by the NtNRAMP2 gene, and overexpression of the NtNRAMP2 gene enhances the sensitivity of tobacco to cadmium stress. This invention provides a novel solution for improving cadmium tolerance at the molecular level and can provide technical support for the creation of new cadmium-tolerant germplasm.

[0048] This invention utilizes RNA-seq (transcriptome sequencing) and RT-qPCR techniques to discover that the expression of the tobacco NtNRAMP2 gene is significantly upregulated under ethylene and ABA induction. Furthermore, the expression level in overexpressing plants under cadmium stress is significantly higher than that in wild-type plants, preliminarily indicating that the tobacco NtNRAMP2 gene is associated with cadmium uptake in tobacco. Therefore, this invention clarifies the function of the NtNRAMP2 gene in the process of cadmium uptake in tobacco through overexpression and other techniques.

[0049] This invention provides a method for breeding and creating germplasm resources that affect cadmium uptake in tobacco. The core of this method lies in overexpressing the NtNRAMP2 gene, which provides new insights and ideas for the development of cadmium stress tolerance in tobacco.

[0050] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1: Cloning of the tobacco NtNRAMP2 gene

[0052] 1. Obtaining tobacco seedlings

[0053] After tobacco seedlings were raised using Hogrange solution, they were transferred to nutrient soil for cultivation to obtain tobacco seedlings. Specifically, plump K326 tobacco seeds were selected and scattered on absorbent cotton in a petri dish. 1 / 4 of the Hogrange solution was added to keep the absorbent cotton moist. The seeds were then cultivated in a light incubator at 25℃ for one week, with a photoperiod of 16 hours of light followed by 8 hours of darkness. Alternating light and dark cultivation was used to induce seed germination. The germinated K326 seedlings were then transplanted into nutrient soil mixed with vermiculite and cultivated in a greenhouse at 25℃ for about one month with a photoperiod of 16 hours of light followed by 8 hours of darkness to obtain tobacco seedlings.

[0054] The composition of 1 / 4 Hoagland medium is as follows: calcium nitrate tetrahydrate 945 mg / L; potassium nitrate 607 mg / L; magnesium sulfate heptahydrate 493 mg / L; sodium hydrogen phosphate dihydrate 156 mg / L; boric acid 14.3 mg / L; manganese sulfate tetrahydrate 9.05 mg / L; zinc sulfate heptahydrate 1.1 mg / L; copper sulfate pentahydrate 0.4 mg / L; sodium molybdate monohydrate 0.1 mg / L; disodium EDTA 75.4 mg / L; ferrous sulfate heptahydrate 55.6 mg / L. The same applies below.

[0055] 2. Obtain the NtNRAMP2 target gene

[0056] (1) Synthesize the cDNA of the NtNRAMP2 gene.

[0057] The gene for the natural resistance macrophage protein NtNRAMP2 was cloned from tobacco using gene cloning methods, and its gene sequence information was analyzed. Specifically, 50-100 mg of tobacco seedling roots from step (1) were thoroughly ground with liquid nitrogen, and total RNA was extracted using the KK Ultrafast Plant Total RNA Extraction Kit (catalog number: ZP405K) purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd. Using this RNA as a template, [the following steps were performed]. III. Root cDNA was obtained by reverse transcription using the 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Nanjing Novizan Biotechnology Co., Ltd.).

[0058] (2) PCR amplification

[0059] The NtNRAMP2 gene was cloned from the cDNA of tobacco seedlings using a gene cloning method. Based on the Arabidopsis thaliana AtNramp2 protein sequence, the predicted NtNramp2 mRNA sequence (accession number: LOC107807465) of common tobacco was obtained by comparing it with the NCBI (National Center for Biotechnology Information) database. Primers NtNramp2-F and NtNramp2-R were designed based on this sequence. The primer sequences are shown in Table 1.

[0060] Table 1 Primer sequences

[0061]

[0062]

[0063] Underlined: Enzyme cleavage site.

[0064] Using the specific primer NtNramp2 in Table 1, cDNA from the roots of tobacco K326 seedlings obtained in step (1) was used as a template for PCR amplification using KOD One™ PCR Master Mix. The PCR amplification reaction system (50.0 μL) consisted of: 25.0 μL KOD One™ PCR Master Mix, 1.0 μL cDNA template (total RNA concentration after reverse inversion 50 ng / μL), 1.0 μL each of primers NtNramp2-F (10 μmol / L) and NtNramp2-R (10 μmol / L), and 22.0 μL ddH2O. The PCR amplification program was: 98℃ denaturation for 10 s, 53℃ annealing for 5 s, 68℃ extension for 10 s, for 35 cycles. The coding region sequence (i.e., CDS sequence) of the NtNramp2 gene was obtained. The PCR products were detected by 1% agarose gel electrophoresis. Figure 1 . Figure 1 This is a schematic diagram of CDS sequence amplification of the tobacco NtNramp2 gene. Figure 1 In this diagram, M represents the DL2000 marker. The CDS sequence obtained through alignment using DNAMAN software is consistent with the sequence predicted in the NCBI database. The coding region sequence of the NtNramp2 gene (i.e., the CDS sequence) is shown in SEQ ID NO.1.

[0065] SEQ ID NO.1 (NtNarmp2 gene coding region sequence (CDS)):

[0066]

[0067] Will Figure 1 The correctly sized bands were excised from the gel, recovered according to the DNA fragment recovery kit instructions, ligated into a T vector, and then sequenced, analyzed, and spliced ​​to obtain the full-length sequence of the NtNramp2 gene. Through these steps, the CDS sequence of this natural resistant macrophage protein in tobacco was obtained, and its protein-coding sequence was deduced. The amino acid sequence encoded by the NtNRAMP2 gene is shown in SEQ ID NO.2.

[0068] SEQ ID NO.2 (Result of amino acid sequence encoded by NtNarmp2 gene):

[0069] * (In the above amino acid sequence, * indicates a stop codon.)

[0070] 3. NtNramp2 gene sequence information analysis

[0071] After cloning the NtNramp2 gene in step 2, gene sequence information analysis was performed. The amino acid sequence of the NtNramp2 gene was compared with other reported amino acid sequences of the Nramp family found in NCBI (National Center for Biotechnology Information). A phylogenetic tree of tobacco NtNramp2 and other species was constructed using MEGA 7.0 with the neighbor-joining method (NJ). Conservation domain analysis was performed using CD-search (https: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi) and MEME (https: / / meme-suite.org / meme / tools / meme), and visualization was performed using TBtools software. CD-search parameters: E value < 0.01; MEME parameters: motif number set to 10, other parameters left as default. Figure 2 This is a schematic diagram showing the conserved domains and phylogenetic tree of tobacco NtNramp2 protein and Nramp homologous proteins in other plants. Figure 2 The green diagram on the right represents the structural domains possessed by the Nramp2 protein in different species; Figure 2 The positions and lengths of the different colored motifs and domains on the left side appearing on the horizontal lines indicate their positions and lengths on the protein. Figure 2 In this context, "motif" represents a motif. Figure 2StNramp2 represents Solanum tuberosum (protein sequence number: XP_006362075.1) of potato; SlNramp2 represents Solanum lycopersicum (protein sequence number: NP_001308166.1) of tomato; NtNramp2 represents Nicotiana tabacum (protein sequence number: XP_016487349.1) of tobacco; CaNramp2 represents Capsicum annuum (protein sequence number: XP_016568839.1) of chili pepper; AhNramp2 represents Arachis hypogaea (protein sequence number: XP_025613549.1) of peanut; MsNramp2 represents Medicago sativa (protein sequence number: QJU69414.1) of alfalfa; GmNramp2 represents Glycine in soybean. max (protein sequence number: XP_003527729.1); VvNramp2 represents grape (Vitis vinifera) (protein sequence number: XP_002285122.1); PtrNramp2 represents poplar (Populus trichocarpa) (protein sequence number: XP_002302424.2); CsNramp3 represents cucumber (Cucumis sativus) (protein sequence number: XP_004151871.1); CsubNramp represents Cocconyxa subellipsoidea C-169 (protein sequence number: XP_005647718.1); GrNramp2 represents cotton (Gossypium raimondii) (protein sequence number: XP_012446255.1); HvNramp2 represents barley (Hordeum). vulgare (protein sequence number: XP_044982509.1); AtNramp2 represents Arabidopsis thaliana (protein sequence number: NP_175157.1); ZmNramp2 represents Zea mays (protein sequence number: NP_001150280.1); OsNramp2 represents Oryza sativa Japonica Group (protein sequence number: XP_015632573.1); LmNramp2 represents Lolium rigidum (protein sequence number: XP_047089775.1).

[0072] according to Figure 2It was found that NtRAMP2 had the lowest homology with *Coccomyxa subellipsoidea*, at 44.49%. Among monocots, NtNRAMP2 showed homology of 63.00%, 62.41%, 63.10%, and 62.18% with rice, barley (*Hordeum vulgare*), maize (*Zea mays*), and ryegrass, respectively. NtRAMP2 showed significantly higher homology with dicots than with monocots, with the highest homology being with pepper (*Capsicum annuum*) (92.48%), tomato (92.99%), and potato (94.28%). A phylogenetic tree constructed using MEGA 7.0 software showed that tobacco NtNarmp2 was most closely related to pepper, tomato, and potato in evolution (…). Figure 2 This indicates that the gene is highly conserved during evolution.

[0073] Example 2: Cadmium Concentration Gradient Treatment

[0074] To determine the expression level of the NtNramp2 gene in response to cadmium stress and to further study the application of the tobacco NtNramp2 gene in cadmium uptake, seedlings were treated with cadmium.

[0075] Cd concentration gradient treatment: Plump K326 tobacco seeds were sown on defatted cotton in petri dishes, and 1 / 4 Hogrange solution was added to keep the cotton moist. The dishes were then cultured for one week in a 25℃ light incubator with a photoperiod of 16 hours of light followed by 8 hours of darkness. Alternating light and dark conditions were used to induce seed germination. Germinated K326 seedlings were transplanted into nutrient soil mixed with vermiculite and cultured for 20 days in a greenhouse at 25℃ with a photoperiod of 16 hours of light followed by 8 hours of darkness. Sterile seedlings with uniform growth after 20 days of germination were selected, and CdCl2 was added to 1 / 2 MS solid medium to a final Cd concentration of 25 μmol / L. The sterile seedlings were then spread on 1 / 2 MS solid medium with a CdCl2 concentration of 25 μmol / L. After 14 days of treatment, tobacco roots were collected and frozen in liquid nitrogen for later use. Each CdCl2 concentration treatment was performed in triplicate.

[0076] Example 3

[0077] Same as Example 2, except that CdCl2 was added to 1 / 2MS solid culture medium until the final Cd concentration was 50 μmol / L.

[0078] Example 4

[0079] Same as Example 2, except that CdCl2 was added to 1 / 2MS solid culture medium until the final Cd concentration was 75 μmol / L.

[0080] Example 5

[0081] Same as Example 2, except that CdCl2 was added to 1 / 2MS solid culture medium until the final Cd concentration was 100 μmol / L.

[0082] Comparative Example 1

[0083] Same as Example 2, except that CdCl2 was added to 1 / 2MS solid culture medium until the final Cd concentration was 0 μmol / L.

[0084] Total RNA was extracted from tobacco plants treated with cadmium in Examples 2-5 and Comparative Example 1, and this RNA was used as a template for... III. Reverse transcription into cDNA was performed using the 1st Strand cDNA Synthesis Kit (+gDNA wiper). The actin gene from tobacco was used as an internal control, and gene-specific primers qNtNramp2 (Table 1) were designed. NtNramp2 expression levels were detected using quantitative real-time PCR (qRT-PCR) on a Step One Plus instrument. In this embodiment, a relative quantification method (2...) was employed. -ΔΔCt The relative expression levels of the gene were calculated, and data processing was performed using Microsoft Excel 2016. Examples 2-5 and Comparative Example 1 were each repeated three times, with one sample taken from each replicate. Three biological replicates were performed for each sample when detecting NtNramp2 expression. The relative expression level of the target gene was calculated; the NtRAMP2 gene expression level in the untreated tobacco plant of Comparative Example 1 was set to 1.

[0085] See results Figure 3 And Table 2, Figure 3 The data in this paper represent the expression analysis of seedlings under different concentrations of cadmium induction treatment. Actin was used as an internal reference gene. The results showed that the NtNarmp2 gene can respond to cadmium induction. Each set of data represents the mean ± standard deviation of three replicate experiments. Figure 3 The horizontal axis represents cadmium treatment concentration, and the vertical axis represents relative expression levels. Figure 3"0h" in the figure represents the result of Comparative Example 1. The expression level was set to 1 at a concentration of 0 μmol / L. As the Cd concentration increased, the expression of the NtNarmp2 gene first increased and then decreased. Compared with Comparative Example 1, the expression level of the NtNarmp2 gene did not change at a Cd concentration of 25 μmol / L. When the Cd concentration reached 50 μmol / L, the expression level of the NtNarmp2 gene reached its maximum, which was 1.27 times that of Comparative Example 1. When the Cd concentration increased to 75 μmol / L, the expression level of the NtNarmp2 gene was 1.2 times that of Comparative Example 1. However, when the Cd concentration reached 100 μmol / L, the expression level of the NtNarmp2 gene was not significantly different from that of Comparative Example 1. This indicates that the NtNarmp2 gene is not sensitive to low concentrations of cadmium, while higher concentrations of Cd can induce the expression of the NtNarmp2 gene. Excessively high Cd concentrations may cause cell damage, resulting in a decrease in gene expression.

[0086] Table 2. Relative expression levels of the NtNarmp2 gene in tobacco plants treated with cadmium in Examples 2-5 and Comparative Example 1.

[0087]

[0088]

[0089] Note: Different letters indicate significant differences (P<0.05), the same applies below.

[0090] Example 6: Cadmium Time Gradient Treatment

[0091] Cd time gradient treatment: Plump K326 tobacco seeds were sown on defatted cotton in petri dishes, and 1 / 4 Hogrange solution was added to keep the cotton moist. The dishes were then cultured for one week in a 25℃ light incubator with a photoperiod of 16 hours of light followed by 8 hours of darkness. Alternating light and dark conditions were used to induce seed germination. Germinated K326 seedlings were transplanted into nutrient soil mixed with vermiculite and cultured for 60 days in a greenhouse at 25℃ with a photoperiod of 16 hours of light followed by 8 hours of darkness. Sixty-day-old germinating plants with uniform growth were selected and uniformly irrigated once with 1 / 4 Hogrange solution (Cd final concentration 50 μmol / L). Two hours after irrigation, roots were collected and frozen in liquid nitrogen for later use. This treatment resulted in three seedlings.

[0092] Example 7

[0093] Same as Example 6, except that the tobacco plants were uniformly irrigated once with 1 / 4 Hoagland culture solution with a final Cd concentration of 50 μmol / L, and the roots of the tobacco plants were taken 6 hours after irrigation.

[0094] Example 8

[0095] Same as Example 6, except that the tobacco plants were uniformly irrigated once with 1 / 4 Hoagland culture solution with a final Cd concentration of 50 μmol / L, and the roots of the tobacco plants were taken 12 hours after irrigation.

[0096] Example 9

[0097] Same as Example 6, except that the tobacco plants were uniformly irrigated once with 1 / 4 Hoagland culture solution with a final Cd concentration of 50 μmol / L, and the roots of the tobacco plants were taken 24 hours after irrigation.

[0098] Comparative Example 2

[0099] Same as Example 6, the only difference being that the tobacco plants were irrigated once with 1 / 4 Hoagland medium without added Cd. Comparative Example 2 was considered as the control group after 0 hours of induction.

[0100] Total RNA was extracted from tobacco plants treated with cadmium in Examples 6-9 and Comparative Example 2 to determine the expression level of the NtNarmp2 gene. The methods for RNA extraction and determination of NtNarmp2 gene expression were the same as in Example 2. The results are shown below. Figure 4 And Table 3. Figure 4 The data in this paper presents the expression analysis of seedlings under cadmium-induced treatment at different time points, with Actin used as an internal reference gene. According to... Figure 4 It can be seen that, Figure 4 The horizontal axis represents the treatment time when the final Cd concentration was 50 μmol / L, and the vertical axis represents the relative expression level. Figure 4 In this context, "0h" represents the result of Comparative Example 2, where the 0h-induced expression level is set to 1. Figure 4 Each data point represents the mean ± SD (standard deviation) of three replicate experiments. After 2 hours of Cd stress treatment, the expression level of the NtNarmp2 gene increased slightly, but the change was not significant. After 6 hours of treatment, the expression level of the NtNarmp2 gene reached its maximum, 1.81 times that of the control. With increasing time, the expression level of the NtNarmp2 gene decreased, but remained significantly higher than that of the control group (P < 0.05). These results indicate that the NtNarmp2 gene can respond to cadmium induction.

[0101] Table 3. Total RNA determination of NtNarmp2 gene expression in tobacco plants after cadmium treatment in Examples 6-9 and Comparative Example 2.

[0102] Cd processing time relative expression level 0 <![CDATA[1±0.004 a ]]> 2h <![CDATA[1.057±0.053 a <!-- 9 -->]]> 6h <![CDATA[1.857±0.138 c ]]> 12h <![CDATA[1.542±0.056 b ]]> 24h <![CDATA[1.467±0.207 b ]]>

[0103] Example 10 Cadmium Stress and Hormone Treatment

[0104] To determine the expression level of the NtNramp2 gene in response to ethylene and ABA induction, and to further investigate how the tobacco gene NtNramp2 functions in cadmium uptake, seedlings were treated with ethylene and ABA.

[0105] 1. ABA hormone treatment

[0106] Tobacco plants with uniform growth and 60 days after germination were selected, and the cultivation of the tobacco plants was the same as in Example 6. Fifteen tobacco plants were taken and numbered 1, 2, 3, 4, 5, 6, ... 15. Tobacco plants numbered 1 to 12 were sprayed with 20 mL of ABA solution with a molar concentration of 4 mmol / L, serving as the treatment group. Tobacco plants numbered 13, 14, and 15 were sprayed with 20 mL of water, serving as the control group. All 15 tobacco plants were cultivated in a greenhouse.

[0107] Roots from three tobacco plants (numbered 1, 2, and 3) were collected at 2 hours of greenhouse cultivation; roots from three tobacco plants (numbered 4, 5, and 6) were collected at 4 hours; roots from three tobacco plants (numbered 7, 8, and 9) were collected at 8 hours; and roots from three tobacco plants (numbered 10, 11, and 12) were collected at 24 hours. Total RNA was extracted from the roots after ABA treatment, and this RNA was used as a template for... III. Reverse transcription into cDNA was performed using the 1stStrand cDNA Synthesis Kit (+gDNA wiper).

[0108] Tobacco samples from the control group were collected 24 hours after water spraying. Roots from tobacco plants numbered 13, 14, and 15 in the control group were used to extract total RNA after water treatment. This RNA was then used as a template for... III. The 1st Strand cDNA Synthesis Kit (+gDNA wiper) was used for reverse transcription into cDNA.

[0109] Using cDNA from the treatment group and the control group as templates, and the actin gene of tobacco as an internal reference (sequence shown in Table 1), gene-specific primer qNtNramp2 (sequence shown in Table 1) was designed and three biological replicates were performed. The expression level of NtNramp2 was detected by quantitative real-time PCR (qRT-PCR) on a Step One Plus instrument.

[0110] The preferred system for RT-PCR amplification includes: 10 μL of 2×SYBR Premix Wiz Taq™; 0.4 μL of upstream primer Primer 1 (primer concentration 10 μM); 0.4 μL of downstream primer Primer 2 (primer concentration 10 μM); 2 μL of template cDNA (200 ng / μL); and sterile ddH2O to a final volume of 20 μL. The preferred RT-PCR amplification program includes: Step 1: Pre-denaturation, 95℃, 30 s; Step 2: 95℃, 10 s; Step 3: 60℃, 30 s; repeat Step 2 and Step 3 40 times.

[0111] The relative quantitative method (2) was adopted. -ΔΔCt The relative expression levels of genes were calculated, and data processing was performed using Microsoft Excel 2016. The relative expression levels of the target genes were calculated; the expression level of the untreated control group was set to 1. Results are shown below. Figure 5 And Table 4.

[0112] Table 4. Expression levels of NtNramp2 in tobacco roots at different treatment times under ABA treatment.

[0113] Processing time 0 (Control Group) 2 4 8 24 NtNramp2 expression level <![CDATA[1±0.002 a ]]> <![CDATA[1.38±0.164 b ]]> <![CDATA[1.30±0.086 b ]]> <![CDATA[1.57±0.203 c ]]> <![CDATA[1.41±0.087 d ]]>

[0114] Figure 5 In the diagram, the horizontal axis represents the treatment time using ABA, and the vertical axis represents the relative expression level of the NtNramp2 gene. Figure 5 "0h" in the figure represents the result of the control group. The expression level was set to 1 at 0h. After 2h of ABA treatment, the expression level of NtNarmp2 gene was significantly upregulated (P<0.05) and reached its maximum at 8h, which was 1.58 times that of the control. This indicates that the expression of NtNarmp2 gene is regulated by the ABA signaling pathway and responds more rapidly to ABA signals.

[0115] 2. Ethylene hormone treatment

[0116] Tobacco plants with uniform growth and 60 days after germination were selected, and the cultivation of the tobacco plants was the same as in Example 6.

[0117] Fifteen tobacco plants were selected and numbered 1, 2, 3, 4, 5, 6...15. Plants numbered 1-12 were sprayed with 20 mL of ethephon aqueous solution at a molar concentration of 4 mmol / L, serving as the treatment group. Plants numbered 13, 14, and 15 were sprayed with 20 mL of water, serving as the control group. All 15 tobacco plants were cultivated in a greenhouse.

[0118] Roots from three tobacco plants (numbered 1, 2, and 3) were collected at 2 hours of greenhouse cultivation; roots from three tobacco plants (numbered 4, 5, and 6) were collected at 4 hours; roots from three tobacco plants (numbered 7, 8, and 9) were collected at 8 hours; and roots from three tobacco plants (numbered 10, 11, and 12) were collected at 24 hours. Total RNA was extracted from the roots after ABA treatment, and this RNA was used as a template for... III. Reverse transcription into cDNA was performed using the 1stStrand cDNA Synthesis Kit (+gDNA wiper).

[0119] Tobacco samples from the control group were collected 24 hours after water spraying. Roots from tobacco plants numbered 13, 14, and 15 in the control group were used to extract total RNA after water treatment. This RNA was then used as a template for... III. The 1st Strand cDNA Synthesis Kit (+gDNA wiper) was used for reverse transcription into cDNA.

[0120] Using cDNA from the treatment group and the control group as templates, and the actin gene from tobacco as an internal control (sequence shown in Table 1), a gene-specific primer qNtNramp2 (sequence shown in Table 1) was designed, and three biological replicates were performed. The expression level of NtNramp2 was detected using quantitative real-time PCR (qRT-PCR) on a Step One Plus instrument. The parameters for qRT-PCR were the same as in "1. ABA hormone treatment".

[0121] The relative quantitative method (2) was adopted. -ΔΔCt The relative expression levels of genes were calculated, and data processing was performed using Microsoft Excel 2016. The relative expression level of the target gene NtNramp2 was calculated; the expression level of the untreated control group was set to 1. Results are shown in […]. Figure 6 See Table 5.

[0122] Table 5. Expression levels of NtNramp2 in tobacco roots after ethylene treatment for different durations.

[0123] Processing time 0 (Control Group) 2 4 8 24 NtNramp2 expression level <![CDATA[1±0.002 b ]]> <![CDATA[1.05±0.119 b ]]> <![CDATA[1.04±0.122 b ]]> <![CDATA[1.42±0.174 c ]]> <![CDATA[0.77±0.118 a ]]>

[0124] Figure 6 In the diagram, the horizontal axis represents the treatment time with ethephon, and the vertical axis represents the relative expression level of the NtNramp2 gene. Figure 6The "0h" in the figure represents the control group result. With a 0h induction value of 1, there was no change in expression levels at 2h and 4h under exogenous ethylene treatment. At 8h, the NtNarmp2 gene expression level reached its maximum, 1.43 times that of the control group, indicating that NtNarmp2 gene expression is regulated by the ethylene signaling pathway and responds more rapidly to ABA signals. (The ethylene and ABA treatments were applied to different batches of tobacco plants.)

[0125] Example 11 Construction of plant expression vector

[0126] The NtNRAMP2 gene cloned in Example 1 was operably constructed into the expression regulatory sequence to form a plant expression vector containing the NtNRAMP2 gene. There are two types of plant expression vectors: one is a 35S:GFP-NtNramp2 subcellular localization vector; the other is a 35S:NtNramp2 overexpression vector.

[0127] 1. Construction of the 35S:GFP-NtNramp2 subcellular localization vector

[0128] The specific process for constructing the 35S:GFP-NtNramp2 subcellular localization vector is as follows: Homologous recombination primers were designed, and the NtNarmp2 gene was amplified using the NtNarmp2-T vector as a template and N2-GFPF and N2-GFP R (Table 1) as primers. Subsequently, after enzyme digestion (the amplified gene fragment and the pMDC43 vector were double-digested with restriction endonucleases AscⅠ and SpeⅠ, respectively, and NtNramp2 was ligated to the pMDC43 vector according to the ligation kit instructions), recovery, and ligation transformation, the full-length NtNarmp2 sequence was inserted forward into the downstream of the CaMV35S promoter of the pMDC43 vector. After complete sequencing, the 35S:GFP-NtNramp2 subcellular localization vector was obtained.

[0129] 2. Construction of 35S:NtNramp2 overexpression vector

[0130] Construction of the 35S:NtNramp2 overexpression vector: Homologous recombination primers were designed. Using the NtNarmp2-T vector as a template, and N2-1300 F and N2-1300R (Table 1) as primers, the full-length sequence of the NtNarmp2 gene was amplified by PCR. Subsequently, after enzyme digestion (the amplified gene fragment and the pCambia1300 vector were double-digested with restriction endonucleases AscⅠ and SpeⅠ, respectively, and NtNramp2 was ligated to the pCambia1300 vector according to the ligation kit instructions), recovery, and ligation transformation, the full-length sequence of NtNarmp2 was inserted forward downstream of the CaMV35S promoter of the pCambia1300 vector. After complete sequencing, the overexpression vector 35S:NtNramp2 was obtained.

[0131] Recombinant plasmids containing the 35S:GFP-NtNramp2 vector, recombinant plasmids containing the 35S:NtNramp2 vector, and pMDC43 empty vector plasmids, all verified by sequencing, were transformed into Agrobacterium competent cells using the heat shock method. After colony PCR identification, the Agrobacterium-positive cells containing the 35S:GFP-NtNramp2 recombinant plasmid, the Agrobacterium-positive cells containing the 35S:NtNramp2 recombinant plasmid, and the Agrobacterium-positive cells containing the pMDC43 empty vector were used as the 35S-GFP empty control.

[0132] Example 12 Subcellular localization of NtNramp2 in tobacco and acquisition of transgenic tobacco plants

[0133] (1) Subcellular localization of tobacco NtNramp2

[0134] The *Agrobacterium* positive strain containing 35S:GFP-NtNramp2 obtained in Example 11 was streaked and activated. Single clones were selected and cultured in LB liquid medium at 28°C for 12 hours on a shaker to obtain a bacterial suspension. The bacterial suspension was then expanded in 50 mL centrifuge tubes. The cultured product was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the *Agrobacterium* was resuspended in infection solution (10 mM MES / KOH, 10 mM MgCl2, 150 μM acetylsyleugenol), and the OD value was adjusted to approximately 0.4. The bacterial resuspension was then placed at room temperature in the dark for 3 hours. The prepared infection solution was slowly injected into the leaves of *Tobacco Bengal* plants that were 6-8 weeks old and growing well using a syringe injection method, allowing the infection solution to penetrate into the tobacco leaves. The plants were cultured in the dark for 12 hours, followed by light culture. After 72 hours of infection, the tobacco leaves were cut into 0.5 cm pieces, placed on clean glass slides, covered with a glass slide, and observed using a confocal microscope. The observation results are as follows. Figure 7 As shown, NtNramp2 is located in the cell membrane.

[0135] (2) Obtaining transgenic tobacco plants

[0136] The Agrobacterium strain containing 35S:NtNramp2 obtained in Example 3 was genetically transformed into tobacco tissue to obtain transgenic tobacco strains, numbered 1, 2, 3, 4, 5, 6, 18, 19, 20, 23, and 24.

[0137] The specific process of genetic transformation of tobacco tissue with Agrobacterium strain containing 35S:NtNramp2 was as follows: Green tobacco leaves were taken and disinfected in a clean bench with 75% alcohol for 1 min, followed by 0.1% mercuric chloride for 8 min. After rinsing twice with sterile water and drying with sterile filter paper, the leaves were cut into 0.5 cm leaf discs and placed on induction medium with the back facing up. The discs were then incubated in the dark at 28℃ for 3 days. Agrobacterium with the expression vector, stored at -80℃, was revived and expanded in 50 mL of LB broth medium supplemented with kanamycin (final concentration 50 mg / L) for resistance. The expansion culture was carried out at 180 rpm at 28℃ for 6 h. When the OD of the Agrobacterium culture... 600 Centrifuge at 5000 rpm for 10 min when the bacterial concentration is between 0.4 and 0.8. Discard the supernatant, add 15 mL of MS liquid medium to resuspend the bacteria, and centrifuge again at 5000 rpm for 10 min. Discard the supernatant. Add 50 mL of infection medium to the centrifuged product for activation and let stand for 30 min. Place the sterilized and cut leaves into Erlenmeyer flasks, ensuring the Agrobacterium tumefaciens solution completely submerges the leaves, and incubate for 20 min, shaking the flask every 10 min. After incubation, remove the leaves, blot dry on sterile filter paper, and place the incubated leaves into induction medium. Incubate in the dark at 28°C for 3 days. Wash the incubated leaves twice with sterile water and cephalosporin solution (cephalosporin concentration: [missing information]). Wash the leaves twice with 100 mg / L water, then wash them three times with sterile water. Blot the leaves dry with sterile filter paper, place them on subculture medium, and grow them in a 25°C light incubator. After one week, wash them with sterile water and transfer them to selection medium for culture, observing their growth at any time. When the resistant shoots differentiated from the callus tissue reach 2 cm in length, transfer the shoots to rooting medium to induce rooting. Once they have grown longer roots, harden them off to obtain transgenic plants. After one week, transplant them into soil and cultivate them in a greenhouse. Figure 8 This is a schematic diagram of the tobacco tissue culture process. It takes 40 to 60 days from explant infection to induction of transgenic rooting.

[0138] MS liquid culture medium consists of 4.45 g / L MS powder (Murashige, T and F Skoog.1962) (catalog number: m519) + 30 g / L sucrose, with a pH of 5.8.

[0139] The MS solid medium consists of 4.45 g / L MS powder + 30 g / L sucrose + 10 g / L agar, with a pH of 5.8.

[0140] The infection medium consisted of Ms liquid medium and 0.1 mmol / L acetylsuccinone.

[0141] The induction medium consisted of Ms solid medium, 0.5 mg / L IAA, and 2 mg / L 6-BA.

[0142] The subculture medium consisted of: Ms solid medium + 0.5 mg / L IAA + 2 mg / L 6-BA + 100 mg / L cephalosporin.

[0143] The screening medium consisted of: Ms solid medium + 0.5 mg / L IAA + 2 mg / L 6-BA + 100 mg / L cephalosporin + 9 mg / L hygromycin.

[0144] The rooting medium consisted of: 1 / 2 Ms solid medium (except for agar, all other components were reduced by half) + 0.5 mg / L IAA + 100 mg / L cephalosporin.

[0145] (3) Identification of genetically modified tobacco

[0146] The identification of transgenic tobacco plants numbered 1, 2, 3, 4, 5, 6, 18, 19, 20, 23, and 24 was specifically as follows: DNA was extracted from leaves of the transgenic tobacco plants obtained in step (2) and wild-type (K326) tobacco plants, and then PCR verification was performed. The identification primers are shown in Table 1. Figure 9 The image shows a positive identification diagram of tobacco plants overexpressing the NtNramp2 gene. Figure 9 In the table, CK represents non-transgenic tobacco lines as a template negative control; 1, 2, 3, 4, 5, 6, 18, 19, 20, 23, and 24 represent transgenic tobacco plants; b represents water as a template blank water control; 1300 represents the pCambia1300 NtNramp2 vector (i.e., the pCambia1300 vector carrying the target gene NtNramp2), and the pCambia1300 NtNramp2 vector serves as a template positive control. The primers used are shown in Table 1 (Jian1300-R). Figure 9 Tobacco lines 5, 6, 18, 19, 20, and 23 all showed positive transgenes, resulting in transgenic tobacco lines OE-5, OE-6, OE-18, OE-19, OE-20, and OE-23. OE represents overexpressing transgenic lines, and 5, 6, 18, 19, 20, and 23 represent different overexpressing line numbers.

[0147] The results of NtNramp2 gene expression level analysis in plants overexpressing the NtNramp2 gene are shown in the figure. Figure 10And Table 6, Figure 10 This is a graph showing the expression level of the overexpressed NtNramp2 gene in plants. Figure 10 The horizontal axis represents the plant line and the vertical axis represents the relative expression level. The expression levels of the NtNramp2 gene in the OE-5, OE-6, and OE-18 lines, which showed consistent growth and overexpression of NtNramp2, as well as in the wild-type K326 plant, were analyzed. Quantitative primers are shown in Table 1. The results showed that the NtNramp2 gene expression level was significantly upregulated in the overexpressing plants.

[0148] Table 6. NtNramp2 gene expression levels in different transgenic tobacco strains.

[0149] strain Ck OE-5 OE-6 OE-18 relative expression level <![CDATA[1.00±0.132 a ]]> <![CDATA[1.35±0.087 b ]]> <![CDATA[1.62±0.073 c ]]> <![CDATA[1.31±0.083 d ]]>

[0150] Figure 11 Table 7 shows the NtNramp2 gene expression levels in OE-5 and OE-6 plants overexpressing the NtNramp2 gene after cadmium stress (50 μmol / L). Figure 11 In the figure, the horizontal axis represents the strain and different treatments of the same strain, and the vertical axis represents the relative expression level. The expression level of 0 μmol / L induction was set to 1 for each strain. After Cd stress treatment, the expression level of NtNarmp2 gene increased. Actin was used as an internal reference gene. The results showed that overexpression of NtNarmp2 gene can respond to cadmium induction. Figure 11 Each set of data represents the mean ± SD (standard deviation) of three repeated experiments.

[0151] Table 7. Results of NtNramp2 gene expression analysis in tobacco lines under different cadmium stresses.

[0152] strain OE-5 OE-6 Cadmium concentration 0 μmol / L <![CDATA[1.00±0.082 a ]]> <![CDATA[1.48±0.127 a ]]> cadmium concentration 50 μmol / L <![CDATA[1.00±0.081 b ]]> <![CDATA[1.23±0.115 b ]]>

[0153] The Cd content in the roots of CK, OE-5, and OE-6 under 50 μmol / L Cd treatment is shown in the figure. Figure 12 See Table 8.

[0154] Table 8. Cd content (mg / Kg) in the roots of CK, OE-5, and OE-6 plants treated with 50 μmol / L Cd.

[0155]

[0156]

[0157] according to Figure 12 As shown in Table 7, high expression of the NtNRAMP2 gene in this invention can promote the absorption of cadmium by tobacco plants.

[0158] In summary, this invention cloned a natural resistant macrophage protein, named NtNRAMP2. Using genetic engineering, the NtNRAMP2 gene was genetically transformed into tobacco explants to obtain transgenic tobacco plants that overexpressed the gene. The expression level of the NtNRAMP2 gene was significantly upregulated when the cadmium concentration increased, and the high expression of the NtNRAMP2 gene could promote the absorption of cadmium by tobacco plants.

[0159] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the sensitivity of tobacco to cadmium stress, characterized in that, include: Applying ethylene or ABA to tobacco plants enhances the coding genes. NtNRAMP2 The expression level of the encoded gene; NtNRAMP2 The nucleotide sequence is shown in SEQ ID NO:1; The working concentration of ABA is 4-8 mmol / L; the working time of ABA is 2-8 h; The ethylene is applied in the form of an ethephon aqueous solution, wherein the working concentration of ethephon in the ethephon aqueous solution is 1~10 mmol / L; and the working time of the ethephon is 8 h.

2. The method according to claim 1, characterized in that, The method of application includes spraying.

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