CN117025627A - Tobacco chloride channel protein NtCLC13, and coding gene and application thereof - Google Patents

Abstract

The invention discloses tobacco chloride channel protein NtCLC13, and a coding gene and application thereof, and belongs to the technical field of plant genetic engineering. The coding gene NtCLC13 of the tobacco chloride channel protein NtCLC13 is obtained through cloning, and subcellular localization analysis is utilized to find that the tobacco chloride channel protein NtCLC13 is localized in cytoplasm. And transferring the constructed gene editing vector into a tobacco plant by using a CRISPR/Cas9 technology through an agrobacterium transformation method, and successfully constructing the NtCLC13 gene knockout tobacco plant. The chloridion content of the leaves of the knocked-out plant is obviously reduced, which makes it possible to cultivate new varieties of low-chlorine tobacco, and provides a new direction for improving the condition of higher chloridion content of flue-cured tobacco in partial tobacco areas in China. Meanwhile, the gene network for regulating and controlling chloride ions in tobacco is enriched, and the method has important significance for regulating and controlling the content of the chloride ions in the tobacco and further understanding the molecular regulation and control of chloride ion transport.

Description

Tobacco chloride channel protein NtCLC13 and its encoding genes and applications

Technical field

The invention relates to tobacco chloride ion channel protein NtCLC13 and its encoding gene and application, and belongs to the technical field of plant genetic engineering.

Background technique

Existing research generally believes that the chloride ion content in tobacco leaves is preferably 0.3 to 0.8. When the content reaches 1%, it will affect the smoldering fire-holding property. When it is further higher than 1%, black ash flameout will occur; on the other hand, , when the chloride ion content in tobacco leaves is too high, it will cause excessive starch accumulation, the leaves will be thick and brittle, and they are highly hygroscopic, making the color easy to darken during storage and producing bad odor. In short, the chloride ion content in tobacco leaves has an important direct impact on the quality of tobacco leaves. At present, the chloride ion content in tobacco leaves in some tobacco areas such as Henan and Yunnan is relatively high, which reduces the quality and yield of tobacco leaves. This has become an urgent problem for growers and researchers.

Traditional improvement methods mostly start with cultivation technology, stabilizing and improving tobacco leaf quality by optimizing field management measures and improving modulation and fermentation technology. But overall, these measures have not fundamentally changed the situation of low quality and low industrial practicality of these tobacco leaves.

With the rapid development of genomics, especially gene editing technology, people have a deeper and deeper understanding of the relationship between chloride ion accumulation in tobacco leaves and related genes. Based on existing research, it is already known that there are 7 members of the chloride channel protein (CLC) protein family in Arabidopsis. Studies have shown that AtCLCa plays a key role in driving nitrate into the vacuole, and AtCLCb does not have this function; other AtCLCs are distributed in other cell compartments. AtCLCd and AtCLCf may play an acidifying role in the transport Golgi vesicle network; AtCLCe may be involved in the anion permeability of the thylakoid membrane; AtCLCg has the same function as AtCLCc. Involved in plant salt stress. In 2007, Anne Marmagne et al. discovered when studying Arabidopsis CLCf that (Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively, Journal of Experimental Botany, 2007, 58( 12):3385-3393), AtCLCf localizes to the Golgi membrane and functionally complements yeast gef1 mutants disrupted in the single CLC gene encoding Golgi-associated proteins. But in general, due to the diversification of functions of CLC family proteins, and the functions of CLC family proteins in different species are not completely consistent. As far as tobacco variety improvement is concerned, in-depth research on the functions of CLC family proteins in tobacco may lay a theoretical and application basis for tobacco variety improvement and even other plant improvements. Therefore, discovering the members of the CLC family proteins in tobacco and their related functions is of great significance for regulating the chloride ion content in tobacco and further understanding the molecular regulation of chloride ion transport.

Contents of the invention

In order to solve the above problems, the first purpose of the present invention is to provide a tobacco chloride ion channel protein encoding gene NtCLC13 gene, which can encode the tobacco chloride ion channel protein NtCLC13 and thereby regulate chloride ion transport in tobacco.

The second object of the present invention is to provide a tobacco chloride ion channel protein NtCLC13. The present invention proves through experiments that the NtCLC13 protein participates in the absorption and transport of chloride ions in tobacco, and can regulate the content of chloride ions in tobacco by regulating the expression of the protein. .

The third object of the present invention is to provide the application of the tobacco chloride channel protein encoding gene NtCLC13 gene in regulating tobacco chloride ion transport. Experiments have proved that regulating the expression of the NtCLC13 gene can affect the content of chloride ions in tobacco and enrich the chlorine content in tobacco. Ion-regulated gene networks.

The fourth object of the present invention is to provide the application of the tobacco chloride channel protein encoding gene NtCLC13 gene in obtaining low-chlorine tobacco varieties. Through CRISPR/Cas9 gene editing technology, the NtCLC13 gene in tobacco plants is knocked out and the knockout strain is found. The chloride ion content in the leaves was significantly reduced, and low-chlorine tobacco varieties were obtained.

In order to achieve the above object, the technical solution adopted by the tobacco chloride ion channel protein encoding gene NtCLC13 gene of the present invention is:

A tobacco chloride channel protein encoding gene NtCLC13 gene is characterized in that its nucleotide sequence is:

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

(2) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO. 1 is substituted and/or deleted and/or one or more nucleotides are added and expresses the same functional protein.

The beneficial effect of the above technical solution is that by designing specific primers, the present invention clones the tobacco chloride ion channel protein encoding gene NtCLC13 gene. By inhibiting its expression in tobacco, the transport of chloride ions to the shoots is reduced, indicating that the NtCLC13 gene is involved. Processes regulating chloride ion transport in tobacco.

In order to achieve the above objectives, the technical solution adopted by the tobacco chloride ion channel protein NtCLC13 of the present invention is:

A tobacco chloride ion channel protein NtCLC13, its amino acid sequence is:

(1) The amino acid sequence shown in SEQ ID NO.2;

(2) A derivative protein in which the amino acid sequence shown in SEQ ID NO. 2 is substituted and/or deleted and/or one or more amino acid residues are added and has the same function.

The beneficial effect of the above technical solution is that the NtCLC13 protein is encoded and expressed by the NtCLC13 gene. It has been verified that the tobacco chloride ion channel protein NtCLC13 is closely related to the absorption and transport of chloride ions in tobacco, inhibiting its expression, and transporting chloride ions from the roots to the shoots. The ability is reduced, and the chloride ion content in tobacco leaves is significantly reduced.

In order to achieve the above purpose, the technical solution adopted in the application of the tobacco chloride ion channel protein encoding gene NtCLC13 gene in regulating tobacco chloride ion transport in the present invention is:

Application of the tobacco chloride channel protein encoding gene NtCLC13 gene in regulating tobacco chloride ion transport.

The beneficial effect of the above technical solution is that the NtCLC13 gene knockout tobacco plants obtained by the present invention through CRISPR/Cas9 technology, the NtCLC13 gene knockout strain is almost the same as the control group in the normal medium. After adding salt stress, NtCLC13 Compared with the control group, the growth of the gene knockout strain was significantly inhibited and the root length was shortened, indicating that the NtCLC13 gene is involved in tobacco salt stress response.

As a further improvement, by inhibiting the expression of the NtCLC13 gene, the chloride ion content in tobacco leaves was significantly reduced.

The beneficial effect of the above technical solution is that: through further testing, it was found that the chloride ion content in the leaves of the NtCLC13 gene knockout strain decreased significantly, indicating that the expression of the NtCLC13 gene was inhibited and the ability of chloride ions to be transported from the roots to the shoots was reduced.

In order to achieve the above purpose, the technical solution adopted in the application of the tobacco chloride ion channel protein encoding gene NtCLC13 gene in obtaining low-chlorine tobacco varieties of the present invention is:

The application of the tobacco chloride channel protein encoding gene NtCLC13 gene in obtaining low-chlorine tobacco varieties. By inhibiting the expression of the NtCLC13 gene, the chloride ions in tobacco leaves are reduced, and low-chlorine tobacco varieties are obtained.

The beneficial effect of the above technical solution is that: the present invention uses CRISPR/Cas9 technology to knock out the NtCLC13 gene in tobacco, inhibit its expression, and obtain NtCLC13 gene knockout tobacco plants. The detection found that chloride ions were present in the leaves of the obtained NtCLC13 gene knockout strain. The significantly reduced content can lay a good foundation for cultivating new tobacco varieties with low chlorine content, while also providing technical support for stabilizing tobacco leaf quality and improving cigarette quality.

As a further improvement, the method of inhibiting the expression of the NtCLC13 gene is to construct a gene editing vector to knock out the tobacco chloride channel protein encoding gene NtCLC13 gene.

The beneficial effect of the above technical solution is that compared with traditional gene interference, CRISPR/Cas9 technology has the advantages of high knockout efficiency and easy operation.

As a further improvement, the gene editing vector is obtained by the following method: connecting the double-stranded DNA of the target site to the gene editing empty vector and sequencing for identification.

As a further improvement, the sgRNA sequence corresponding to the double-stranded DNA of the target site is: TCTCGGCGATAGTGCTCCAC.

The beneficial effect of the above technical solution is that the use of the above sgRNA can effectively knock out the NtCLC13 gene in tobacco, successfully construct NtCLC13 gene knockout tobacco plants, and lay the foundation for studying the function of the NtCLC13 gene.

As a further improvement, the low-chlorine tobacco variety is obtained by the following method: transforming the gene editing vector into Agrobacterium as the infection solution, then transforming tobacco, and obtaining a tobacco variety with a significantly reduced chloride ion content in the leaves through screening and identification.

Description of the drawings

Figure 1 is a subcellular localization map of tobacco NtCLC13 protein in Example 3 of the present invention;

Figure 2 is a schematic diagram of target site selection for NtCLC13 gene knockout in Example 3 of the present invention;

Figure 3 is a diagram showing the sequencing results of the knockout target site of the NtCLC13 gene T ₀ generation gene editing plant in Example 3 of the present invention;

Figure 4 shows the phenotype of NtCLC13 gene-edited plants under salt stress in Example 3 of the present invention;

Figure 5 is a graph showing the chloride ion content in the leaves of NtCLC13 gene-edited plants in Example 3 of the present invention (** represents p<0.01).

Detailed ways

The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in each of the examples, experimental examples and comparative examples can be obtained from commercial sources.

biomaterials:

Tobacco varieties: K326 and China Tobacco 100, the seeds used are provided by the National Tobacco Gene Research Center.

Vector: pCS1300 was donated from Wuhan Tianwen Biotechnology Co., Ltd.; the CRISPR/Cas9 vector was provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University.

Strains: Trans5α chemically competent cells, purchased from Beijing Quanshijin Biotechnology Co., Ltd.; GV3101 Agrobacterium competent cells, purchased from Shanghai Weidi Biotechnology Co., Ltd.; primer synthesis and DNA sequencing were provided by Beijing Liuhe BGI Technology Co., Ltd. Ltd. offers completion.

Experimental reagents: RNA extraction kit (RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit), genome extraction kit (polysaccharide and polyphenol plant genomic DNA extraction kit) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; fluorescence quantification Reagent kit, reverse transcription kit (Transcriptor First Strand cDNA Synthesis Kit) was purchased from Roche Company of Switzerland; DNA amplification enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction endonuclease BsaI, plasmid extraction kit and DNA gel recovery kit was purchased from Bao Biotechnology Co., Ltd.

Experimental equipment: PCR instrument Tprofessional Thermocycler, Biometra Company; quantitative PCR instrument LightCycler96, Roche Company.

Example 1 Tobacco chloride channel protein encoding gene NtCLC13 gene

The nucleotide sequence of the tobacco chloride channel protein encoding gene NtCLC13 gene in this example is:

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

(2) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO. 1 is substituted and/or deleted and/or one or more nucleotides are added and expresses the same functional protein.

Example 2 Tobacco chloride channel protein NtCLC13

The amino acid sequence of tobacco chloride channel protein NtCLC13 in this example is:

(1) The amino acid sequence shown in SEQ ID NO.2;

(2) A derivative protein in which the amino acid sequence shown in SEQ ID NO. 2 is substituted and/or deleted and/or one or more amino acid residues are added and has the same function.

Example 3 Application of tobacco chloride channel protein encoding gene NtCLC13 gene in obtaining low-chlorine tobacco varieties

In this example, the NtCLC13 gene was successfully cloned through PCR. Subcellular localization found that the tobacco NtCLC13 protein was located in the cytoplasm. In order to further explore its function, a gene editing vector for the NtCLC13 gene was constructed, and the gene editing vector was transformed into tobacco plants using Agrobacterium transformation. , successfully constructed NtCLC13 gene knockout tobacco plants. The salt stress experiment found that the NtCLC13 gene is involved in the response of tobacco to salt stress. The _T1 generation of the gene knockout strain was cultured, and the test found that the chloride ion content in the leaves of the NtCLC13 knockout strain was significantly reduced. The specific implementation operations are as follows:

1. Cloning and subcellular localization of NtCLC13 gene encoding chloride channel protein

1) Cloning of NtCLC13 gene

The cloning and acquisition process of NtCLC13 gene is as follows:

(1) Extract RNA and reverse-transcribe cDNA

Sterilize K326 seeds and then inoculate them on MS medium for germination. Two weeks after germination, the seedlings are transplanted into pots and cultured in a plant culture room with a culture temperature of 23-26°C. When the tobacco seedlings grow to six leaves and one heart Transfer to 1/2MS liquid medium to continue culturing. Two weeks later, the roots were selected for sampling, frozen in liquid nitrogen for later use, and a plant RNA extraction kit was used to extract total RNA from tobacco roots. When extracting RNA, just refer to the instructions of the kit, and then reverse-transcribe it into cDNA for later use.

(2) Design primers and perform PCR amplification

The PCR amplification primer sequences are as follows:

NtCLC13-F: 5’-ATGACGGGAGGCGAGTAC-3’ (shown in SEQ ID NO.3);

NtCLC13-R: 5'-CTACATTGAAAGATGC-3' (shown in SEQ ID NO. 4).

Use the reverse-transcribed cDNA in step (1) as a template and use the above primers to perform PCR amplification.

The PCR amplification system is: 10 μL of 5×GCL buffer, 2 μL of cDNA, 1 μL of upstream and downstream primers, 6 μL of dNTP, 1 μL of GXL DNA Polymerase, and add sterile water to 50 μL.

PCR amplification conditions were: 98°C, 10sec; 55°C, 15sec, 68°C, 2min, 30 cycles. The PCR products were detected and analyzed by agarose electrophoresis, and the PCR amplified products were purified and recovered using the instructions of the DNA gel recovery kit.

The purified product was then connected to the pFF19 vector. The connection system was as follows: DNA amplification product, 6 μL; pFF19 vector, 1 μL; after mixing, ligation was carried out at 25°C for 25 minutes.

Transform the ligation product into E. coli DH5α competent cells. The specific transformation process is as follows:

Take out the competent cells from the -80°C refrigerator, place them on ice to dissolve, add the ligation product to 100 μL DH5α competent cells, mix gently, and keep in ice bath for 20 minutes; heat shock in 42°C water bath for 90 seconds, and place immediately Place on ice for 2 minutes; add 900 μL of LB (antibiotic-free) liquid culture medium that has been equilibrated to room temperature, shake and culture at 37°C for 1 hour; centrifuge at 4000 rpm for 3 minutes, remove part of the supernatant, leave 100 μL of the precipitate, mix evenly, and spread evenly on the LB solid plate (containing 50 μg/μL kanamycin), invert the culture dish, and culture at 37°C overnight.

The next day, single clones were selected and sent for sequencing.

Sequencing results and analysis results show that the length of the NtCLC13 gene coding region is 2238 bp nucleotides; analysis of the gene shows that the amino acid sequence of the NtCLC13 protein encoded by it is shown in SEQ ID NO. 2.

2) Subcellular localization of tobacco NtCLC13 protein

Design primers pCS1300S-NtCLC13F and pCS1300S-NtCLC13R, and clone NtCLC13 into the pCS1300S vector containing GFP tag. The primer sequence is:

pCS1300S-NtCLC13F: 5'- GCTTTCGCGAGCTCGGTACC ATGACGGGAGGCGAGTAC-3' (shown in SEQ IDNO.5);

pCS1300S-NtCLC13R: 5'- CCCTTGCTCACCATGGATCC CATTGAAAAGATGC-3' (shown in SEQ ID NO. 6).

Note: The underlined part is the adapter sequence.

The PCR amplification system and reaction procedures are the same as those in Part 1) Cloning of NtCLC13 gene. The obtained PCR product was connected to the pCS1300 vector using In-Fusion enzyme. The connection system was as follows: DNA amplification product, 2 μL; pCS1300S vector 2 μL; In-Fusion enzyme 1 μL. After mixing, connect at 50°C for 15 minutes. The ligation product was transformed into E. coli DH5α competent cells, and single colonies were picked for sequencing.

The correctly sequenced plasmid was extracted with a high-quality plasmid using a plasmid maximization kit and transformed into Nicotiana benthamiana protoplasts. The mitochondrial marker was used as a control (red). The results are shown in Figure 1. NtCLC13 is located in the cytoplasm.

2. Construction of gene editing vectors

In order to further understand the function of NtCLC13 gene in tobacco chloride ion absorption and transport, a CRISPR/Cas9 expression vector for knocking out NtCLC13 gene was constructed. The experimental process is as follows:

First, the target site was designed based on the recognition characteristics of the CRISPR/Cas9 system, and a 20bp sgRNA target sequence was designed in the first exon region of the NtCLC13 gene, as shown in Figure 2. The sgRNA sequence is: TCTCGGCGATAGTGCTCCAC (SEQ ID NO.7 As shown), the design knockout primer sequences NtCLC13-T1_F and NtCLC13-T1_R are as follows:

NtCLC13-T1_F: GTGGAGCACTATCGCCGAGAtgcaccagccgggaat (shown in SEQ ID NO. 8);

NtCLC13-T1_R: ttctagctctaaaacGTGGAGCACTATCGCCGAGA (shown in SEQ ID NO. 9).

Design the reaction system to obtain the DNA double strand (annealing) of the target site. The 20 μL reaction system is designed as follows: Annealing Buffer for DNA OLigos (5×), 4 μL; upstream and downstream primers (NtCLC13-T1_F, NtCLC13-T1_R), 4 μL each (50μmoL/μL); replenish Nuclease-free water to 20μL.

The reaction program is: 95°C for 5 minutes, decreasing by 0.1°C every 8 seconds to 25°C; the reaction product is stored at 4°C for later use, or the subsequent reaction can be carried out directly.

The above annealing product was connected to the CRISPR/Cas9 vector digested by BsaⅠ, and the CRISPR/Cas9 expression vector for knocking out the NtCLC13 gene was screened. The 20 μL connection system was designed as follows: annealing product, 6 μL; enzyme digestion product (BsaⅠ enzyme Cut CRISPR/Cas9 vector), 3 μL; 10×T4 DNA Ligase Buffer, 2 μL; T4 DNA Ligase, 1 μL; add sterile water to 20 μL, and connect at 37°C for 3 hours.

The ligation product is transformed into E. coli competent cells, and positive clones are picked, expanded and cultured. After the plasmid is extracted, the vector is confirmed to be successfully constructed by PCR and then stored at low temperature for Agrobacterium transformation.

3. Obtaining transgenic plants

The CRISPR/Cas9 expression vector constructed in the above steps was transformed into Agrobacterium and then into tobacco plants to construct transgenic plants with NtCLC13 gene knockout. The experimental process is as follows:

(1) Transformation of Agrobacterium

Take out Agrobacterium GV3101 competent cells from the -80°C refrigerator, freeze and thaw on ice, add 5 μL of the constructed gene editing vector when it is about to thaw, flick to mix; bath in ice for 30 minutes, freeze in liquid nitrogen for 5 minutes, and then Put it on ice immediately after 5 minutes in a 37°C water bath; add 900 μL of antibiotic-free LB liquid culture medium, shake and culture at 200 rpm for 4 hours; then centrifuge the bacterial solution at 4500 rpm for 3 minutes, discard half the volume of the supernatant, resuspend and spread evenly on the On the LB solid medium of Rif (100 μg/mL) and Kan (50 μg/mL), invert the culture at 28°C for about 2 to 3 days until a single colony is formed; pick a single colony, and conduct PCR identification of the bacterial solution after expansion. Identification of the correct positive clone strain is the transformation of the correct engineering bacteria.

(2) Transform tobacco plants

Take the leaves of tobacco (Zhongyan 100) sterile seedlings that have grown for about one month, use a hole punch to process the leaves into leaf discs with a diameter of 0.5cm, and pre-culture the treated leaf discs on MS solid medium for 3 days; The prepared transformed Agrobacterium engineered bacteria were cultured to about OD ₆₀₀ = 0.6, centrifuged at 4000 rpm for 5 minutes to collect the cells, and then suspended in 20 mL of MS liquid culture medium; then the above-mentioned pre-cultured leaf discs were placed in the bacterial liquid. Infect for 10 minutes; use sterile filter paper to absorb excess bacterial fluid around the leaf disc after infection, and cultivate in the dark for 3 days on a solid medium of MS+6-BA (2mg/L)+NAA (0.5mg/L); use Wash the leaf disc with sterile water containing Cef (400mg/L), absorb excess liquid with sterile filter paper, and transfer the leaf disc to a solution containing 6-BA (2mg/L), NAA (0.5mg/L), Cef ( 200mg/L) and Kan (50mg/L) on MS solid screening medium, culture at 28°C under light; when the adventitious buds grow to 0.5cm, transfer to Cef (200mg/L) and Kan (50mg/L) Roots were grown on MS solid medium.

(3) Identification of gene editing

After about a month of growth, take a small number of leaves, extract DNA according to the instructions of the plant genome extraction kit, and detect positive transgenic lines and mutant forms through PCR amplification, cloning, and sequencing. The specific identification methods are:

On the NtCLC13 genome, design a pair of detection primers located on both sides of the knockout target site, specifically:

NtCLC13-J-F: 5’-GGAGTGAGTACGGTGTGCCAGAAGGCGATTTAGAAAGC-3’ (shown in SEQ ID NO. 10);

NtCLC13-J-R: 5'-GAGTTGGATGCTGGATGGGCTGAAAACTAACCCCGC-3' (shown in SEQ ID NO. 11).

PCR amplification was performed using the DNA template of the T ₀ generation transgenic strain.

The PCR reaction system is: 2 μL of 10× buffer, 1 μL of cDNA, 0.5 μL of upstream and downstream primers, 3 μL of dNTP, 1 μL of EazyTaq enzyme, and add sterile water to 20 μL.

PCR amplification conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, a total of 25 cycles; and final extension at 72°C for 10 min. The PCR products were sent to Hi-tom sequencing.

The analysis and sequencing results are shown in Figure 3. In the T ₀ generation plants, it was detected that the NtCLC13 gene had a single-base deletion mutation at the knockout target site, while no mutation was detected in the NtCLC13 gene of the wild-type plant. This is This shows that the NtCLC13 gene has been successfully knocked out in the T ₀ generation plants, and the edited plant is named ntclc13 ^- .

4. Phenotypic observation of NtCLC13 gene-edited plants

(1)Seed disinfection

The seeds of Zhongyan 100 (control normal tobacco plants) and ntclc13 ^- edited materials were sterilized with 75% alcohol for 1-2 minutes, then sterilized with 10% NaClO for 10 minutes, and rinsed repeatedly with sterilized water 3-5 times.

(2) Observation of salt stress phenotypes

Sow the sterilized seeds on 1/2MS solid medium with 200mM NaCl added, and use 1/2MS solid medium without salt as a control. Observe the phenotype of the plants on the plate after 2-3 weeks.

The results are shown in Figure 4. Compared with the control, the growth of ntclc13 ^- plants in normal medium was not much different. After adding salt stress, the growth of ntclc13 ^- plants was significantly inhibited compared with the control, and the root length became shorter, indicating that the NtCLC13 gene is involved. Tobacco salt stress response.

5. Detection of chloride ion content in leaves of NtCLC13 gene-edited plants

(1)Hydroponics test

The T ₁ generation of NtCLC13 gene-edited plants and the control Zhongyan 100 plants were planted in the plant culture room of the National Tobacco Gene Research Center. The culture conditions were: temperature (23±1)℃, relative humidity 60%±2%, 16h light culture, 8h dark nourish. When the tobacco seedlings reach the six true leaf stage, they are moved to Hoagland's nutrient solution to continue culturing. The nutrient solution is replaced after 1 week. After another 3 days of cultivation, the leaves are taken for subsequent determination of chloride ion content. The leaves were quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator. Each strain was set up with 3 independent replications, and at least 3 tobacco seedlings with consistent growth were selected for each replication.

(2) Determination of chloride ion content

After freeze-drying the preserved leaf samples, use a mixed oscillating grinder to grind them into powder. Weigh about 0.0500g (accurate to 0.1mg) of the powder, place it in 10mL 5% (volume fraction) acetic acid, and shake at 30°C. Extract for 30 minutes, then filter with qualitative filter paper. After the filtrate is diluted, the chloride ion content is measured with an American Alalis MP6500 desktop pH meter.

The results are shown in Figure 5. After NtCLC13 gene editing, the chloride ion content in the leaves significantly decreased (** represents p<0.01).

In summary, the present invention found that the NtCLC13 protein is located in the cytoplasm through research on the tobacco NtCLC13 gene; the NtCLC13 gene knockout strain was obtained through gene editing technology. After salt stress, the growth of ntclc13 ^-edited plants was inhibited, and the root length was related to the The tobacco plants were significantly shorter than the original ones; the chloride ion content in the leaves of the edited plants was significantly reduced, and tobacco plants with reduced chloride ion content in the leaves were obtained.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention. .

Claims (9)

1. A tobacco chloride channel protein coding gene NtCLC13 gene, characterized in that: the nucleotide sequence is as follows:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.

2. A tobacco chloride channel protein NtCLC13, characterized in that: the amino acid sequence is as follows:

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.

3. Use of the tobacco chloride channel protein coding gene NtCLC13 gene of claim 1 for regulating and controlling tobacco chloride transport.

4. The use of the tobacco chloride channel protein coding gene NtCLC13 gene according to claim 3 for regulating and controlling tobacco chloride transport, characterized in that: inhibiting the expression of the NtCLC13 gene, and remarkably reducing the content of chloride ions in tobacco leaves.

5. Use of the tobacco chloride channel protein coding gene NtCLC13 gene according to claim 1 for obtaining low-chlorine tobacco varieties, characterized in that: the low-chlorine tobacco variety is obtained by inhibiting the expression of the NtCLC13 gene and reducing chloride ions in tobacco leaves.

6. The use of the tobacco chloride channel protein coding gene NtCLC13 gene according to claim 5 for obtaining low-chlorine tobacco varieties, characterized in that: the expression of the NtCLC13 gene is inhibited by constructing a gene editing vector and knocking out the tobacco chloride channel protein coding gene NtCLC13 gene.

7. The use of the tobacco chloride channel protein coding gene NtCLC13 gene according to claim 6 for obtaining low-chlorine tobacco varieties, characterized in that: the gene editing vector is obtained by the following method: and (3) connecting the double-stranded DNA of the target site to a gene editing empty vector, and sequencing and identifying.

8. The use of the tobacco chloride channel protein coding gene NtCLC13 gene according to claim 7 for obtaining low-chlorine tobacco varieties, characterized in that: the sgRNA sequence corresponding to the double-stranded DNA of the target site is as follows: TCTCGGCGATAGTGCTCCAC.

9. The use of the tobacco chloride channel protein coding gene NtCLC13 gene according to any one of claims 5-8 for obtaining low-chlorine tobacco varieties, characterized in that: the low-chlorine tobacco variety is obtained by the following method: and (3) transforming agrobacterium tumefaciens serving as an invader solution by using the gene editing vector, transforming tobacco, and screening and identifying to obtain a tobacco variety with obviously reduced leaf chloride ion content.