Tobacco NtTL17L gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco NtTL17L gene and application thereof. Tests find that the contents of chlorophyll b, leaf yellow and violaxanthin in leaves of the NtTL17L gene function-deleted mutant strain are increased, the content of chlorogenic acid is reduced, and the drought resistance and the salt resistance are increased, so that the NtTL17L gene function-deleted mutant strain has important theoretical and application significance on regulation and control of tobacco photosynthesis, secondary metabolism and improvement of tobacco yield, and provides a new thought for tobacco breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a tobacco NtTL17L gene and an application thereof. Background Art

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

[0003] Therefore, exploring and utilizing tobacco chlorophyll synthesis genes, studying the mechanisms of chlorophyll synthesis and decomposition, 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 a theoretical basis and material resources for the genetic improvement of tobacco varieties and the increase in tobacco leaf production yields, which 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 a tobacco NtTL17L gene and application thereof.

[0005] The present invention provides the use 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 comprises:

[0008] Regulating plant cytochrome content; and / or

[0009] Regulation of plant secondary metabolites; and / or

[0010] Modulating plant salt tolerance; and / or

[0011] Regulates plant drought tolerance.

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

[0013] The metabolites include chlorogenic acid;

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

[0015] The invention comprises plants of the family Cruciferae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Leguminosae and / or Solanaceae; further, the plant of the family Solanaceae comprises tobacco.

[0016] The present invention provides a product for plant breeding or assisted breeding, which includes 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), an interference fragment targeting the tobacco TL17L;

[0019] C), gRNA targeting the tobacco TL17L;

[0020] D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C);

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

[0022] F), transforming or transfecting the host cell with the recombinant vector as described in E);

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

[0024] In the present invention, the nucleic acid includes DNA or RNA, which can be a nucleotide sequence as shown in SEQ ID NO: 1; it can also be a transcription product of the nucleotide sequence as shown in SEQ ID NO: 1 - RNA; it is selected whether the template or target used in a specific experiment is DNA or RNA according to different experimental purposes; in the present 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 linear or circular in topology. Nucleic acids can be obtained directly from natural sources, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques.

[0025] The present invention provides a gRNA fragment, which is a fragment targeting a target gene and located about 20 bp before a recognition site (PAM site) of a CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a and CRISPR / Cas14a) editing system; the recognition site of the CRISPR / endonuclease system editing system is different according to the different endonucleases, and generally, 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, wherein N represents any one of A, T, C or G base species, and the position of the NGG can be at any position of the 5' end, the middle or the 3' end of the nucleic acid encoding tobacco TL17L, and the present invention is not limited thereto; In the present invention, tobacco TL17L is used as a target, and the CRISPR / Cas9 system is used for gene editing, and specifically, the nucleotide sequence of the target segment of the gRNA fragment is shown in SEQ ID NO: 7;

[0026] Furthermore, in the present invention, a tobacco TL17L mutant was obtained after gene editing by the CRISPR / Cas9 system, wherein a base A deletion was inserted into the 167-168bp region of the nucleotide sequence shown in SEQ ID NO: 1, resulting in a frameshift mutation, which resulted in the loss of tobacco TL17L function. The tobacco TL17L function-loss mutant had increased content of neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b, and β-carotene, decreased content of chlorogenic acid, and increased drought resistance and salt tolerance.

[0027] The recombinant vector described in the present invention is a recombinant DNA molecule, which contains the desired coding sequence and the appropriate nucleic acid sequence or element that is essential for the expression, replication or completion of the target operation of the operably linked coding gene in a specific host organism; in the present invention, the recombinant vector includes a cloning vector, an expression vector or a vector that performs functional interference or functional loss; in a specific embodiment of the present invention, it includes a cloning vector, which is used for cloning and gene sequence analysis of the TL17L gene; in other specific embodiments of the present invention, it includes a CRISPR / Cas9 vector that causes functional loss; according to different experimental purposes, selecting a suitable vector is a conventional means adopted by the technical personnel in this field, and the present invention will not elaborate on this.

[0028] In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmid is the most commonly used vector form. However, the present invention is intended to include such other forms of expression vectors that play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genomic insertions.

[0029] In the present invention, the transformation method includes: chemical transformation and electrotransformation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, viral transfection or Agrobacterium-mediated transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc. In the specific implementation of the present invention, the Agrobacterium-mediated leaf disc method is used to prepare a TL17L gene function-deficient mutant for the study of TL17L gene function.

[0030] The present invention provides a kit, which comprises the product of the present invention and auxiliary materials.

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

[0032] The present invention provides application of the product or the kit in plant breeding or assisted plant breeding.

[0033] The present invention provides a method for plant breeding or assisted plant breeding, which comprises using the product of the present invention or the kit of the present invention to carry out plant breeding.

[0034] The experiment of the present invention found that the contents of chlorophyll b, lutein and violaxanthin in the leaves of the NtTL17L gene function-deficient mutant were increased, the content of chlorogenic acid was reduced, and the drought resistance and salt tolerance were increased. This has important theoretical and application significance for the regulation of tobacco photosynthesis, secondary metabolism and increasing tobacco leaf yield, and provides a new idea for tobacco breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shows the PCR electrophoresis of NtTL17L gene;

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

[0037] Figure 3 Shows the pORE-CRISPR / Cas9 plasmid map;

[0038] Figure 4 Shows the analysis of mutation types in NtTL17L gene mutant plants;

[0039] Figure 5Shows the pigment content in NtTL17L gene mutant plants;

[0040] Figure 6 Shows the salt tolerance analysis of NtTL17L gene mutant plants;

[0041] Figure 7 Shown is the drought tolerance analysis of NtTL17L gene mutant plants. DETAILED DESCRIPTION

[0042] The present invention provides tobacco NtTL17L gene and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] Biomaterials:

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

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

[0046] Competent cells: Trans1-T1 competent cells; DH5a chemical competent cells;

[0047] Strain: LBA4404 Agrobacterium strain;

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

[0049] Experimental reagents:

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

[0051] Fluorescence quantitative PCR enzyme (TB Green® Fast qPCR Mix) and reverse transcription kit were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

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

[0053] DNA amplification enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

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

[0055] 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 the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0058] Example 1 Cloning of NtTL17L gene

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

[0060] Take 200 mg of tobacco (K326) seedling roots as samples, grind them thoroughly in liquid nitrogen, extract total RNA according to the instructions of RNA extraction kit, and then reverse transcribe them 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] The cDNA prepared in step (1) was used as a template and PCR amplification was performed using the above primers. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s, for a total of 30 cycles; and final extension at 72°C for 10 min. The PCR amplification was stored at 4°C for later use, or directly subjected to electrophoresis detection and analysis.

[0066] Purify the PCR amplification product according to the instructions of the gel recovery kit, and then connect the purified product to the pEASY-BluntZero cloning vector. The connection 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 min.

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

[0071] Take out the competent cells from the -80℃ refrigerator, place them on ice to dissolve, add the ligation product to 50μL of Trans1-T1 competent cells, flick to mix, and place on ice for 30min;

[0072] Heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes; add 250 μL of LB (without antibiotics) equilibrated to room temperature, and culture at 37°C with shaking for 1 hour;

[0073] Take 8 μL of the mixture and spread it evenly on a LB solid plate (containing 60 μg / μL ampicillin), invert the culture dish, and culture at 37°C overnight.

[0074] After plaques were picked and cultured, the plasmid DNA was extracted, the recombinant plasmid was identified by plasmid PCR amplification, and the corresponding positive clones were sent for sequencing to obtain the NtTL17L gene sequence.

[0075] The results of sequencing analysis showed that the length of the coding region of the NtTL17L gene was 717 bp, as specifically shown in SEQ ID NO:1; after analyzing the gene, it was found that the amino acid sequence of the NtTL17L protein encoded by it was shown in SEQ ID NO:2.

[0076] Example 2 Expression of 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 fluorescent quantitative PCR. The relevant experiments are briefly introduced as follows.

[0078] The roots, stems, leaves and flowers of tobacco in the budding stage were collected as samples, quickly frozen with liquid nitrogen and placed in a -80°C refrigerator for future use.

[0079] Extract RNA from the stored materials, synthesize cDNA using a reverse transcription kit (refer to the kit instructions for operation), and perform fluorescent quantitative PCR detection using the tobacco NtL25 gene as an internal reference. During the detection, the primer sequences are designed as follows:

[0080] Fluorescence quantitative primers for detecting NtTL17L gene, the primer sequences are:

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

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

[0083] The conditions of fluorescence quantitative PCR were as follows: the first step was pre-denaturation at 95°C for 30 s; the second step was PCR reaction at 95°C for 3 s, 57°C for 20 s, and 40 cycles; the third step was melting curve.

[0084] Each sample was biologically replicated three times. -△△CT The relative gene expression differences were analyzed by the method. 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 editing vector of NtNL17L gene

[0086] In order 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, according to the design principle of CRISPR / Cas9 target site, cgtgtcagttcaagcaacttagg (gRNA target site, SEQ ID NO:7) was selected as the editing site of NtTL17L gene, the PAM region was AGG, and the four bases GATT were added to the 5' end of the forward primer, and the four bases CAAA were added to the 5' end of the reverse primer to synthesize the target site primer. The single-stranded Oligo DNA of the target site was annealed to form double-stranded DNA, and 10 μL of each upstream and downstream primers were mixed and placed in a PCR instrument for incubation at 95°C for 3 minutes. After incubation, it was naturally cooled to room temperature to obtain the double-stranded target site sequence.

[0088] Use BsaI to digest the pORE-CRISPR / Cas9 editing vector. The reaction system is 50 μL, 10 μL of editing vector, 1 μL of BsaI enzyme, 5 μL of CutSmart Buffer (10×), 34 μL of ddH2O, and digest at 37°C for 1 hour. Use the product recovery kit to recover the digested plasmid to obtain the digested editing vector.

[0089] The double-stranded target site sequence and the editing vector after restriction digestion were connected under the action of T4 ligase. The system was 20μL: 10×T4 DNA Ligase Buffer: 2μL; double-stranded target site sequence: 5μL; editing vector: 2μL; T4 DNA Ligase: 1μL; add ddH2O to 20μL.

[0090] Ligation was performed at 25°C for 10 minutes. The ligation product was transformed into DH5a competent cells and cultured to obtain a single clone. The detection primer (JC-F: ttaggtttacccgccaata, SEQ ID NO: 8) and the target site reverse primer were used as amplification primers, and the positive clones were detected by colony PCR. The positive clones were expanded and cultured, and the plasmids (pORE-CRISPR / Cas9 editing vector plasmids) were extracted and stored at low temperatures 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 as follows.

[0093] (1) Transformation of Agrobacterium

[0094] Take out the Agrobacterium competent cells from the -80℃ refrigerator, freeze and thaw on ice, and add 10μL of the pORE-CRISPR / Cas9 editing vector plasmid prepared in Example 3 when it is about to thaw, and flick to mix; ice bath for 10min, liquid nitrogen for 5min, 37℃ for 5min, do not shake the water surface, and then quickly put on ice for 5min. Add 600μL of antibiotic-free LB liquid culture medium, 28℃, 200r / min shaking culture for 3h. Apply 200μL of bacteria to YEB solid culture medium containing 50mg / L rifampicin, 50mg / L streptomycin and 50mg / L kanamycin, invert and culture in the dark at 28℃ for 2~3 days until a single colony is formed; pick a single colony, expand the culture, and perform PCR identification on the bacterial solution to identify the correct positive clone strain, which is the correct engineered bacteria for transformation.

[0095] (2) Transformation of tobacco plants and identification of mutants

[0096] Take the leaves of the sterile tobacco seedlings that have grown for about one month, use a hole puncher to process the leaves into leaf disks with a diameter of 0.5 cm, and pre-culture the processed leaf disks on MS solid culture medium for 3 days;

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

[0098] Then the pre-cultured leaf discs were placed in the bacterial solution and infected for 10 minutes;

[0099] Use sterile filter paper to absorb the excess bacterial liquid around the leaf disc after infection, and culture it in the dark on a solid medium of MS+6-BA (2 mg / L)+NAA (0.5 mg / L) for 3 days;

[0100] The leaf disc was washed with sterile water containing Cef (400 mg / L), and the excess liquid was removed with sterile filter paper. The leaf disc was transferred to MS solid screening medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L), and Kan (50 mg / L) and cultured at 28°C under light.

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

[0102] After about one month of growth, take a small amount of leaves and extract DNA according to the instructions of the plant genome extraction kit. Detect positive transgenic strains and mutation sites through PCR amplification, cloning, and sequencing. The specific identification method is:

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

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

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

[0106] PCR amplification was performed using the DNA template of the T0 transgenic strain. The PCR conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 25 cycles; and final extension at 72°C 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 of the target site, specifically:

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

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

[0110] The positive transgenic strain DNA was used as a template for PCR amplification. The PCR conditions were as follows: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 30 s, for a total of 30 cycles; and 72°C final extension for 10 min. The mutation type of the NtTL17L gene target site of the transgenic positive plant was identified. The sequencing results were as follows: Figure 4 As shown, there is a single base "A" inserted in the target site segment. The single base insertion causes a change in the protein coding frame after the insertion site, protein translation disorder, and thus loss of function.

[0111] Example 5 Detection of pigment content and secondary metabolites of NtTL17L gene-edited mutants

[0112] The NtTL17L gene editing mutant identified in Example 4 was tested for pigment content. The specific experimental process is briefly described as follows:

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

[0114] The HPLC test results showed that the contents of neoxanthin, violaxanthin, and chlorophyll b in the NtNL17L gene-edited mutant were significantly upregulated compared with the control ( Figure 5 ), the chlorogenic acid content decreased.

[0115] Example 6 Analysis of salt tolerance of NtTL17L gene-edited mutants

[0116] The NtTL17L gene-edited mutant identified in Example 5 was subjected to salt tolerance analysis. The specific experimental process is briefly described as follows:

[0117] The seeds of the homozygous gene-edited strain nttl17l and the control wild-type WT (K326) were selected, and the seeds were surface-sterilized and inoculated on MS solid medium for vertical culture and germination. After the nttl17l and control wild-type WT (K326) seeds grew to a certain length, they were moved to a medium containing salt at a corresponding concentration for salt stress treatment. The phenotypes of the salt-stressed nttl17l transgenic plants and the K326 control plants were observed, and the salt tolerance of the plants was analyzed.

[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 of which were about 8 cm. Under 100mmol / L NaCl stress, the growth of wild-type tobacco seedling roots was significantly inhibited, and the root length dropped to about 6 cm, while the root length of nttl17l transgenic plants was almost uninhibited, with a root length of about 7.5 cm. The phenotypes of wild-type and mutant materials under 150mmol / L NaCl conditions were basically consistent with those under 100mmol / L NaCl stress. This shows that nttl17l transgenic plants have a certain degree of salt tolerance. Although salt stress inhibits the growth of tobacco seedling roots, the inhibition on the gene knockout material strains is relatively mild and basically does not affect the growth and development of their root systems ( Figure 6 ).

[0119] Example 7 Drought resistance analysis of NtTL17L gene-edited mutants

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

[0121] Nine strains of wild-type tobacco K326 and homozygous gene-edited mutant nttl17l with consistent growth were selected and subjected to drought treatment and rehydration treatment to observe their phenotypic changes. Figure 7 As shown in the figure, all tobacco plants lost water after drought treatment and were in a wilting state. After rehydration, 3 control K326 plants survived. Compared with the control, 6 homozygous gene-edited mutant nttl17l plants survived, and the survival rate was significantly higher than that of the control. This suggests that the deletion gene NtTL17L may improve tobacco's resistance to drought.

[0122] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of tobacco TL17L as a target in plant breeding and / or assisted plant breeding; The amino acid sequence of the tobacco TL17L is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The plant breeding comprises: Regulating plant cytochrome content; and / or Modulation of plant secondary metabolites; and / or Modulating plant salt tolerance; and / or Regulates plant drought tolerance.

3. The use according to claim 1, characterized in that: The regulation includes increasing and / or decreasing.

4. The use according to any one of claims 1 to 3, characterized in that: The plants include tobacco.

5. A plant breeding or plant breeding assisted product, characterized in that: Including at least one of the following A) to C: A), amplification primers, detection primers and / or targeting primers using the nucleic acid encoding the tobacco TL17L as a template; B), an interference fragment targeting the tobacco TL17L; C), gRNA targeting the tobacco TL17L; D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C); E), a recombinant vector containing the interference fragment as described in B), or the gRNA as described in C), or the interference fragment as described in D); F), transforming or transfecting the host cell with the recombinant vector as described in E); G), culturing the mixture obtained by the host cells described in F).

6. The product according to claim 5, characterized in that The nucleotide sequence of the target region of the gRNA is shown in SEQ ID NO:

7.

7. A kit, characterized in that Including the product and auxiliary materials described in claim 5 or 6.

8. The kit according to claim 7, characterized in that The auxiliary materials include: at least one of a DNA extraction reagent, dNTP, a DNA reverse transcription reagent, a culture medium, an antibiotic and / or a buffer.

9. Use of the product according to claim 5 or 6 or the kit according to claim 7 or 8 in plant breeding or assisted plant breeding.

10. A method for plant breeding or assisted plant breeding, characterized in that The method comprises using the product according to claim 5 or 6 or the kit according to claim 7 or 8 for plant breeding.

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