Tobacco NtTL17L Gene and Its Application

Abstract

This invention relates to the field of biotechnology, and more particularly to the tobacco NtTL17L gene and its applications. Experiments conducted in this invention have revealed that NtTL17L gene loss-of-function mutants exhibit increased levels of chlorophyll b, xanthophyll, and apocynin in their leaves, decreased chlorogenic acid content, and increased drought and salt tolerance. This has significant theoretical and applied implications for regulating tobacco photosynthesis, secondary metabolism, and improving tobacco leaf yield, providing new insights for tobacco breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the tobacco NtTL17L gene and its applications. Background Technology

[0002] Tobacco is an important economic crop in my country, and improving its yield and quality is a crucial direction for tobacco breeding. The content of tobacco pigments is closely related to the metabolic accumulation characteristics and processing methods during tobacco growth and development. Chlorophyll is the material basis for photosynthesis in green plants, and its content is an important indicator for measuring plant photosynthesis, nutritional status, and growth vigor. The content and properties of chlorophyll in tobacco leaves not only affect their appearance quality but also directly and indirectly influence their intrinsic quality.

[0003] Therefore, exploring and utilizing tobacco chlorophyll synthesis genes, studying the mechanisms of chlorophyll synthesis and decomposition metabolism, and creating tobacco materials with different chlorophyll contents can help us better understand the growth and development regulation process of tobacco. It can also provide theoretical basis and material resources for the genetic improvement of tobacco varieties and the increase of tobacco leaf production, and has important scientific research and practical value. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the tobacco NtTL17L gene and its application.

[0005] This invention provides the application of tobacco TL17L as a target in plant breeding and / or assisted plant breeding;

[0006] The amino acid sequence of the tobacco TL17L is shown in SEQ ID NO:2.

[0007] Furthermore, the plant breeding includes:

[0008] Regulating plant cytochrome content; and / or

[0009] Regulating plant secondary metabolites; and / or

[0010] Regulating plant salt tolerance; and / or

[0011] Regulate the drought tolerance of plants.

[0012] The cytochromes include: neoxanthin, azoxanthin, xanthin, chlorophyll a, chlorophyll b, and β-carotene;

[0013] The metabolites include chlorogenic acid;

[0014] Furthermore, the regulation includes increasing and / or decreasing.

[0015] This includes plants from the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, and / or Solanaceae families; further, the Solanaceae family includes tobacco.

[0016] This invention provides plant breeding or assisted breeding products, comprising at least one of the following: A) to C:

[0017] A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding the tobacco TL17L as a template;

[0018] B) Targeting the interfering fragments of the tobacco TL17L;

[0019] C) gRNA targeting the tobacco TL17L;

[0020] D) Expression cassettes containing interfering fragments as described in B), or gRNAs as described in C);

[0021] E) A recombinant vector containing the interfering fragment as described in B), or the gRNA as described in C), or the interfering fragment as described in D);

[0022] F) Transformation or transfection of host cells with the recombinant vector described in E);

[0023] G), a mixture obtained by culturing host cells as described in F).

[0024] In this invention, the nucleic acid includes DNA or RNA, which can be a nucleotide sequence as shown in SEQ ID NO:1; or it can be a transcription product of a nucleotide sequence as shown in SEQ ID NO:1—RNA. The selection of whether the template or target used in a specific experiment is DNA or RNA depends on the experimental purpose. In this invention, the DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acids can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be topologically linear or circular. Nucleic acids can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques.

[0025] This invention provides a gRNA fragment, which is a fragment located approximately 20 bp before the recognition site (PAM site) of the CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a, and CRISPR / Cas14a) editing system targeting the target gene. The recognition site of the CRISPR / endonuclease system editing system varies depending on the endonuclease used; typically, the recognition site includes NGG (…). Cas9) and / or TTN (Cpf1), etc.; In a specific embodiment of the present invention, the CRISPR / Cas9 system is used, and its recognition site is NGG, where N represents any one of the base species A, T, C or G. The position of NGG can be any position at the 5' end, middle or 3' end of the nucleic acid encoding tobacco TL17L, and the present invention does not limit this; In the present invention, tobacco TL17L is used as the target, and gene editing is performed using the CRISPR / Cas9 system. Specifically, the nucleotide sequence of the target segment of the gRNA fragment is shown in SEQ ID NO:7;

[0026] Furthermore, in this invention, a tobacco TL17L mutant strain was obtained after gene editing using the CRISPR / Cas9 system. This mutant strain has an A deletion inserted in the 167-168 bp region of the nucleotide sequence shown in SEQ ID NO:1, resulting in a frameshift mutation and causing loss of function of the tobacco TL17L mutant. The tobacco TL17L loss-of-function mutant strain exhibits increased contents of neoxanthin, apoxanthin, xanthin, chlorophyll a, chlorophyll b, and β-carotene, decreased chlorogenic acid content, and increased drought and salt tolerance.

[0027] The recombinant vector described in this invention is a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression, replication, or completion of the desired operation of the operatively linked coding gene in a specific host organism. In this invention, the recombinant vector includes a cloning vector, an expression vector, or a vector that performs functional interference or loss of function. In specific embodiments of this invention, a cloning vector is included for cloning and gene sequence analysis of the TL17L gene. In other specific embodiments of this invention, a CRISPR / Cas9 vector that performs loss of function is included. Selecting a suitable vector according to different experimental purposes is a conventional approach adopted by those skilled in the art, and this invention will not elaborate on this.

[0028] In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0029] In this invention, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, viral transfection, or Agrobacterium-mediated transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc. In a specific embodiment of this invention, the Agrobacterium-mediated leaf disc method is used to prepare a TL17L gene loss-of-function mutant strain for the study of TL17L gene function.

[0030] This invention provides a kit comprising the product and excipients described herein.

[0031] Furthermore, the excipients include at least one of the following: DNA extraction reagent, dNTP, DNA reverse transcription reagent, culture medium, antibiotic and / or buffer.

[0032] This invention provides the application of the product or kit described herein in plant breeding or assisted plant breeding.

[0033] This invention provides a method for plant breeding or assisted plant breeding, which includes the product or kit described in this invention for plant breeding.

[0034] The experiment of this invention found that the content of chlorophyll b, xanthophyll, and amethyst in the leaves of the NtTL17L gene loss-of-function mutant increased, while the content of chlorogenic acid decreased, and drought resistance and salt tolerance increased. This has important theoretical and applied significance for the regulation of tobacco photosynthesis, secondary metabolism and the improvement of tobacco leaf yield, and provides new ideas for tobacco breeding. Attached Figure Description

[0035] Figure 1 The image shows a PCR electrophoresis diagram of the NtTL17L gene.

[0036] Figure 2 This shows the expression characteristics of the NtTL17L gene in different tissues;

[0037] Figure 3 Map of pORE-CRISPR / Cas9 plasmid;

[0038] Figure 4 Analysis of mutation types in NtTL17L gene mutant plants;

[0039] Figure 5This indicates the pigment content in plants with the NtTL17L gene mutant;

[0040] Figure 6 Salt tolerance analysis of NtTL17L gene mutant plants;

[0041] Figure 7 Analysis of drought tolerance in plants with NtTL17L gene mutant. Detailed Implementation

[0042] This invention provides the tobacco NtTL17L gene and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0043] Biomaterials:

[0044] Tobacco variety: K326, a common cultivated tobacco variety. The seeds used in the examples were provided by Hunan Tobacco Industry Co., Ltd.

[0045] Vectors: pEASY-Blunt Zero cloning vector; pORE-CRISPR / Cas9 vector

[0046] Competent cells: Trans1-T1 competent cells; DH5α chemocompetent cells;

[0047] Strain: LBA4404 Agrobacterium strain;

[0048] Primer synthesis and DNA sequencing were provided by Beijing BGI Genomics Co., Ltd.

[0049] Experimental reagents:

[0050] RNA extraction kit, SuperPure Plant polyRNA Kit;

[0051] The real-time PCR enzyme (TB Green® Fast qPCR Mix) and reverse transcription kit were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0052] Restriction endonuclease BsaI and T4 ligase were purchased from NEB.

[0053] DNA amplification enzyme, purchased from Beijing TransGen Biotech Co., Ltd.

[0054] The plant genome extraction kit and DNA purification kit were purchased from QIAGEN;

[0055] The nucleotide sequence of tobacco NtTL17L gene: atggcgtccatatctattcctctggcgtataaatctcattccctcagtcgttcctcaagttatcgtcctcacttcactactccacaatttcacttacccatccaaatcaaatgttcagttaccaaggagggttcggataatgaggaaagctcgtgtcagttcaagcaacttaggaatgttgcttgtgggttccttgctgcttgggcagtggctaattctgtttctcccgtaattgctgcgggtcagagattgcctccattgtcaaccgacccagataggtgtgcacgggcctttgttggtaacacaataggtcaagctaatggagtttatgacaaaccacttgatctgcgcttttgtgattacacaaatgagaaaaataacctcaaggggaagtcacttgcagcagcacttatgtctggtgcaaaatttgatggtgcagacatgactgaagtgatcatgtctaaggcttatgccgttggagctagttttaaggggacagacttttcgaatgctgttctagatcgagtgaactttgagaaagccaacctccagggagcttcatttaagaacactgtactatcaggatctacctttaatgatgctcaacttgaagatgcagattttgaggacacaataattggctacattgatcttcagaagatatgtctgaataaaactattaatgaagaagggagagttaacttgggatgtagataa (SEQ ID NO: 1);

[0056] Tobacco NtTL17L gene amino acid sequence: MASISIPLAYKSHSLSRSSSYRPHFTTPQFHLPIQIKCSVTKEGSDNEESSCQFKQLRNVACGFLAAWAVANSVSPVIAAGQRLPPLSTDPDRCARAFVGNTIGQANGVYDK PLDLRFCDYTNEKNNLKGKSLAAALMSGAKFDGADMTEVIMSKAYAVGASFKGTDFSNAVLDRVNFEKANLQGASFKNTVLSGSTFNDAQLEDADFEDTIIGYIDLQKICLNKTINEEGRVNLGCR (SEQ ID NO:2);

[0057] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0058] Example 1: Cloning of the NtTL17L gene

[0059] (1) Preparation of cDNA as cloning template

[0060] Take 200mg of tobacco (K326) seedling roots as a sample, grind them thoroughly in liquid nitrogen, extract total RNA according to the RNA extraction kit instructions, and then reverse transcribe it into cDNA for later use;

[0061] (2) Design primers and perform PCR amplification.

[0062] The primer sequences designed for amplifying the NtTL17L gene are as follows:

[0063] NtTL17L-F: atggcgtccatatctattcc (SEQ ID NO:3);

[0064] NtTL17L-R: ttatctacatcccaagttaactctc (SEQ ID NO: 4);

[0065] Using the cDNA prepared in step (1) as a template, PCR amplification was performed using the above primers. The PCR amplification conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplified material was stored at 4℃ for later use, or directly subjected to electrophoresis detection and analysis.

[0066] Purify the PCR amplification product according to the instructions of the gel extraction kit. Then, ligate the purified product into the pEASY-BluntZero cloning vector. The ligation system is as follows:

[0067] DNA amplification product: 6 μL;

[0068] pEASY-Blunt Zero vector: 1 μL;

[0069] After mixing, connect at 25°C for 25 minutes.

[0070] The ligation product was transformed into competent E. coli cells. The specific transformation process is briefly described below:

[0071] Remove competent cells from the -80℃ freezer, place them on ice to thaw, add the ligation product to 50 μL of Trans1-T1 competent cells, gently tap to mix, and incubate on ice for 30 min.

[0072] Heat shock in a 42℃ water bath for 30 seconds, then immediately place on ice for 2 minutes; add 250 μL of LB (antibiotic-free) equilibrated to room temperature, and incubate at 37℃ with shaking for 1 hour.

[0073] Take 8 μL of the mixture and spread it evenly on an LB agar plate (containing 60 μg / μL ampicillin). Invert the culture dish and incubate overnight at 37°C.

[0074] After plaque amplification and culture, DNA from each plasmid was extracted. The recombinant plasmids were identified by plasmid PCR amplification, and the corresponding positive clones were sent for sequencing to obtain the NtTL17L gene sequence.

[0075] Sequencing analysis results show that the coding region of the NtTL17L gene is 717 bp in length, as shown in SEQ ID NO:1; analysis of this gene shows that the amino acid sequence of the NtTL17L protein it encodes is shown in SEQ ID NO:2.

[0076] Example 2: Expression of the NtTL17L gene in tobacco tissues and organs

[0077] In this embodiment, the inventors collected different tissues and organs and analyzed the expression pattern of the NtTL17L gene using quantitative real-time PCR. The relevant experiments are briefly described below.

[0078] Roots, stems, leaves, and flowers of tobacco plants in the budding stage were collected as samples, flash-frozen in liquid nitrogen, and then stored in a -80°C freezer for later use.

[0079] RNA was extracted from the preserved material, and cDNA was synthesized using a reverse transcription kit (follow the kit instructions). Using the tobacco NtL25 gene as an internal control, quantitative real-time PCR was performed. The primer sequences for detection were designed as follows:

[0080] The primers for quantitative real-time detection of the NtTL17L gene are as follows:

[0081] NtTL17L-qF: acatgactgaagtgatcatgtctaa (SEQ ID NO:5);

[0082] NtTL17L-qR: ggagcttcatttaagaacactgtac (SEQ ID NO: 6);

[0083] The conditions for quantitative real-time PCR are as follows: Step 1: pre-denaturation, 95℃ for 30 s; Step 2: PCR reaction, 95℃ for 3 s, 57℃ for 20 s, 40 cycles; Step 3: melting curve.

[0084] Each sample was biologically replicated three times, using 2 -△△CT Methods were used to analyze relative differences in gene expression. The results are as follows: Figure 2 As shown, the NtTL17L gene is expressed in leaves, flowers, and stems, with the highest expression level in leaves.

[0085] Example 3: Construction of the NtNL17L gene editing vector

[0086] To further understand the role of the NtTL17L gene, the inventors constructed an editing vector for the NtNL17L gene. The construction process is briefly described below.

[0087] First, based on the design principles of CRISPR / Cas9 target sites, cgtgtcagttcaagcaacttagg (gRNA target site, SEQ ID NO:7) was selected as the editing site for the NtTL17L gene, with AGG as the PAM region. Four bases of GATT were added to the 5' end of the forward primer, and four bases of CAAA were added to the 5' end of the reverse primer to synthesize the target site primers. The single-stranded Oligo DNA at the target site was annealed to form double-stranded DNA. 10 μL each of the forward and reverse primers were mixed and incubated at 95°C for 3 min in a PCR instrument. After incubation, the mixture was allowed to cool naturally to room temperature to obtain the double-stranded target site sequence.

[0088] The pORE-CRISPR / Cas9 editing vector was digested with BsaI. The reaction system consisted of 50 μL of editing vector, 10 μL of BsaI enzyme, 5 μL of CutSmart Buffer (10×) buffer, and 34 μL of ddH2O. The digestion was carried out at 37°C for 1 hour. The digested plasmid was recovered using a product recovery kit to obtain the digested editing vector.

[0089] The double-stranded target site sequence was ligated to the enzyme-digested editing vector using T4 ligase. The ligation system was 20 μL: 10×T4 DNA Ligase Buffer: 2 μL; double-stranded target site sequence: 5 μL; editing vector: 2 μL; T4 DNA Ligase: 1 μL; ddH2O was added to bring the total volume to 20 μL.

[0090] Ligation was performed at 25°C for 10 minutes. The ligation product was transformed into DH5α competent cells, and single clones were obtained. Positive clones were detected by colony PCR using detection primers (JC-F: ttaggtttacccgccaata, SEQ ID NO: 8) and target site reverse primers. Positive clones were expanded, plasmids (pORE-CRISPR / Cas9 editing vector plasmids) were extracted, and the cells were cryopreserved for Agrobacterium transformation.

[0091] Example 4: Construction of gene-edited plants

[0092] The gene-editing vector constructed in Example 3 was transformed into Agrobacterium and then into tobacco plants to construct NtNL17L gene-edited transgenic plants. The specific experimental process is briefly described below.

[0093] (1) Transformation of Agrobacterium

[0094] Remove Agrobacterium competent cells from a -80°C freezer and freeze-thaw them on ice. Just before thawing, add 10 μL of the pORE-CRISPR / Cas9 editing vector plasmid prepared in Example 3 and gently tap to mix. Incubate on ice for 10 min, then in liquid nitrogen for 5 min, then at 37°C for 5 min without shaking the surface, and then immediately freeze for 5 min. Add 600 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking at 200 rpm for 3 h. Spread 200 μL of bacterial cells onto YEB solid medium containing 50 mg / L rifampicin, 50 mg / L streptomycin, and 50 mg / L kanamycin. Incubate in the dark at 28°C for 2-3 days until single colonies form. Pick single colonies, expand them, and perform PCR identification. Correctly identified positive clones are the correctly transformed engineered bacteria.

[0095] (2) Identification of transformed tobacco plants and mutants

[0096] Take leaves from sterile tobacco seedlings that have grown for about one month, and use a punch to process the leaves into leaf discs with a diameter of 0.5 cm. Pre-culture the processed leaf discs on MS solid medium for 3 days.

[0097] The transformed Agrobacterium engineered bacteria prepared above were cultured to OD200. 600=0.6, centrifuge at 4000 rpm for 5 min to collect the bacterial cells, and then suspend the bacterial cells in 20 mL of MS liquid medium;

[0098] Then, the pre-cultured leaf discs were placed in the bacterial solution and incubated for 10 minutes.

[0099] Use sterile filter paper to blot away excess bacterial solution around the leaf disc after infection, and incubate in the dark for 3 days on MS medium containing 6-BA (2 mg / L) and NAA (0.5 mg / L).

[0100] Wash the leaf discs with sterile water containing Cef (400 mg / L) and absorb excess liquid with sterile filter paper. Transfer the leaf discs to MS solid selection medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L) and Kan (50 mg / L) and incubate at 28°C under light.

[0101] When the adventitious buds grow to 0.5 cm, they are transferred to MS solid medium containing Cef (200 mg / L) and Kan (50 mg / L) to root.

[0102] After about one month of growth, a small number of leaves were taken, and DNA was extracted according to the instructions of the plant genome extraction kit. Positive transgenic lines and mutation sites were detected using PCR amplification, cloning, and sequencing. The specific identification method is as follows:

[0103] A pair of detection primers were designed on the Cas9 sequence to detect transgenic lines, specifically:

[0104] Cas9-F:ctcaacacaacatatacaaaacaaa (SEQ ID NO:9);

[0105] Cas9-R:ctttggccatctcgtttga (SEQ ID NO:10);

[0106] Using T0 generation transgenic line DNA template, PCR amplification was performed. The PCR conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; and a final extension at 72℃ for 10 min. Transgenic positive plants were identified.

[0107] A primer was designed across the sgRNA target site of the NtNL17L gene to detect the mutation type at the target site, specifically:

[0108] NtTL17L-MF: cactactccacaatttcacttaccc (SEQ ID NO: 11);

[0109] NtTL17L-MR: aacaccaatcaatactacgaccatt (SEQ ID NO: 12);

[0110] Using DNA from positive transgenic lines as templates, PCR amplification was performed under the following conditions: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; followed by a final extension at 72℃ for 10 min. The mutation type of the NtTL17L gene target site in the transgenic positive plants was identified, and the sequencing results are as follows: Figure 4 As shown, there is a single-base "A" insertion in the target site region. This single-base insertion causes a change in the protein coding frame after the insertion site, leading to disordered protein translation and ultimately loss of function.

[0111] Example 5: Detection of pigment content and secondary metabolites in the NtTL17L gene-edited mutant.

[0112] The pigment content of the NtTL17L gene-edited mutant identified in Example 4 was detected. The specific experimental procedure is briefly described below:

[0113] Homozygous gene-edited lines with consistent seedling growth in a greenhouse and wild-type WT (K326) control plants were selected. A mixed sample of 1.0 mg was taken from every three plants. After freeze-drying the samples, approximately 0.05 g of the sample powder was weighed into a 2 mL centrifuge tube, 90% acetone (containing 0.1% BHT) was added, and the mixture was inverted and mixed thoroughly. The mixture was then sonicated for 20 min, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 1 mL filter and passed through a 0.22 μm organic membrane into a 1 mL brown sample bottle for injection. 10 μL of the sample was injected for HPLC detection.

[0114] HPLC results showed that, compared with the control, the NtNL17L gene-edited mutant had significantly increased levels of neoxanthin, xanthin, and chlorophyll b. Figure 5 The content of chlorogenic acid decreased.

[0115] Example 6: Salt tolerance analysis of the NtTL17L gene-edited mutant.

[0116] Salt tolerance analysis was performed on the NtTL17L gene-edited mutant identified in Example 5. The specific experimental procedure is briefly described below:

[0117] Seeds of the homozygous gene-edited line nttl17l and the wild-type control WT (K326) were selected. After surface sterilization, the seeds were inoculated onto MS solid medium and cultured vertically for germination. Once the nttl17l and wild-type WT (K326) seeds reached a certain length, they were transferred to a saline medium containing the appropriate concentration for salt stress treatment. The phenotypes of the salt-stressed nttl17l transgenic plants and the K326 control plants were observed to analyze their salt tolerance.

[0118] Under normal growth conditions (MS medium without NaCl), there was no significant difference in root length phenotype between nttl17l transgenic plants and wild-type tobacco seedlings, both around 8 cm. Under 100 mmol / L NaCl stress, root growth in wild-type tobacco seedlings was significantly inhibited, with root length decreasing to around 6 cm, while root length in nttl17l transgenic plants was almost uninhibited, remaining around 7.5 cm. The phenotypes of wild-type and mutant materials under 150 mmol / L NaCl stress were essentially consistent with those under 100 mmol / L NaCl stress. This indicates that nttl17l transgenic plants possess a certain degree of salt tolerance. Although salt stress inhibits root growth in tobacco seedlings, the inhibition effect on gene knockout lines is relatively mild, essentially not affecting root growth and development. Figure 6 ).

[0119] Example 7: Drought Resistance Analysis of the NtTL17L Gene-Edited Mutant

[0120] The NtTL17L gene-edited mutant identified in Example 5 was subjected to drought resistance analysis. The specific experimental procedure is briefly described below:

[0121] Nine wild-type tobacco plants (K326) and nine homozygous gene-edited mutants (nttl17l) with uniform growth were selected. These plants were subjected to drought and rehydration treatments, and phenotypic changes were observed. Figure 7 As shown, all tobacco plants lost water and wilted after drought treatment. After rehydration, 3 plants of the control K326 survived, while 6 plants of the homozygous gene-edited mutant nttl17l survived, a significantly higher survival rate than the control. This suggests that the deletion of the NtTL17L gene may enhance the tobacco's drought resistance.

[0122] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of tobacco TL17L as a target in increasing tobacco pigment content, reducing tobacco chlorogenic acid content, improving tobacco salt tolerance and / or improving tobacco drought resistance; The amino acid sequence of the tobacco TL17L is shown in SEQ ID NO:2; The pigments are neoxanthin, xanthin, and / or chlorophyll b; The improvement of tobacco pigment content, reduction of tobacco chlorogenic acid content, improvement of tobacco salt tolerance and / or improvement of tobacco drought tolerance are achieved by gRNA knockout. The nucleotide sequence of the target region of the gRNA is shown in SEQ ID NO:7.

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