Tobacco nicotine accumulation related gene NtGSTU19 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco nicotine accumulation related gene NtGSTU19 and application thereof. According to the invention, the expression mode of glutathione S-transferase NtGSTU19 in different tissues of tobacco is systematically analyzed, and the difference of the expression quantity of glutathione S-transferase NtGSTU19 in different parts of a tobacco plant is analyzed; by constructing a glutathione S-transferase NtGSTU19 gene knockout tobacco material, nicotine in leaves of a knockout strain is not changed, nicotine in a root system is obviously higher than that in a control group, and the synthesis capability of nicotine is enhanced after gene mutation is speculated; the improvement of the nicotine synthesis capability of the root system is beneficial to stabilizing the quality of the tobacco leaves, and plays an important role in tobacco molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the tobacco nicotine accumulation-related gene NtGSTU19 and its applications. Background Technology

[0002] Nicotine, a characteristic substance of tobacco, is mainly synthesized in the root system, primarily at the root tip, and transported upwards to the stems and leaves for accumulation. Therefore, the amount of nicotine synthesized is closely related to the size and growth quality of the root system. Studies report that leaves synthesize no more than 0.5% of their total nicotine content, while roots account for 99%. Increased root nicotine content helps optimize the source-sink relationship in tobacco plants: as the "source" of nicotine synthesis, increased root nicotine content promotes the translocation of nicotine to the leaves ("sink"), increasing the nicotine content of tobacco leaves and further stabilizing leaf quality. Currently identified genes regulating nicotine in tobacco include PMT and NtNAC-R1.

[0003] However, the effects of a single gene in improving tobacco quality may sometimes lead to other adverse factors in the tobacco plant. Therefore, discovering new nicotine-regulating genes can provide new ideas and methods for nicotine regulation in tobacco quality improvement and further enrich the genetic resources of tobacco. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the tobacco nicotine accumulation-related gene NtGSTU19 and its application.

[0005] This invention provides the application of tobacco glutathione S-transferase NtGSTU19 in plant quality improvement;

[0006] The amino acid sequence of the tobacco glutathione S-transferase NtGSTU19 is shown in SEQ ID NO:2.

[0007] Furthermore, the quality improvement includes increasing the nicotine content in the roots.

[0008] The plant mentioned includes tobacco.

[0009] This invention provides a product for improving plant quality, comprising at least one of the following: A) to C)

[0010] A) Amplification primers and / or quantitative detection primers using the nucleic acid encoding the tobacco glutathione S-transferase NtGSTU19 as a template;

[0011] B) gRNA targeting the tobacco glutathione S-transferase NtGSTU19;

[0012] C) A recombinant vector containing nucleic acid encoding tobacco glutathione S-transferase NtGSTU19, or gRNA as described in B).

[0013] Furthermore, the nucleotide sequence of the nucleic acid encoding the tobacco glutathione S-transferase NtGSTU19 is shown in SEQ ID NO:1.

[0014] The nucleotide sequence of the gRNA is shown in SEQ ID NO:7.

[0015] The amplification primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0016] The quantitative detection primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:6.

[0017] 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.

[0018] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of the operatively linked coding gene in a specific host organism or for performing the desired operation. In a specific embodiment of this invention, the sgRNA is integrated into a CRISPR / Cas9 gene editing vector to knock out the tobacco glutathione S-transferase NtGSTU19, achieving the purpose of gene silencing or loss of function.

[0019] 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.

[0020] The transformation methods include chemical transformation and electroconversion; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and Agrobacterium-mediated transfection. In a specific embodiment of the present invention, Agrobacterium-mediated transfection is used to obtain mutant plants for the study of the function of the tobacco glutathione S-transferase NtGSTU19 described in this invention.

[0021] This invention provides the application of the product in the improvement of plant traits.

[0022] The present invention provides a method for improving plant traits, characterized in that it includes using the product described in any one of claims 4 to 8.

[0023] This invention systematically analyzed the expression pattern of glutathione S-transferase NtGSTU19 in different tissues of tobacco and elucidated the differences in its expression levels in different parts of the tobacco plant. By constructing tobacco materials with the glutathione S-transferase NtGSTU19 gene knocked out, it was found that there was no change in nicotine in the leaves of the knockout strain, while the nicotine in the roots was significantly higher than that in the control, suggesting that the gene mutation enhanced the nicotine synthesis capacity. The improved nicotine synthesis capacity in the roots helps stabilize the quality of tobacco leaves and plays an important role in tobacco molecular breeding. Attached Figure Description

[0024] Figure 1 Electrophoresis diagram of NtGSTU19 gene PCR products;

[0025] Figure 2 Analysis of NtGSTU19 gene expression in different tissues;

[0026] Figure 3 This shows the expression characteristics of the NtGSTU19 gene under exogenous methyl jasmonate treatment;

[0027] Figure 4 This shows the gene mutation patterns in the NtGSTU19 transformed lines;

[0028] Figure 5 The nicotine content in the leaves and roots of the edited strains after jasmonic acid treatment was shown. Detailed Implementation

[0029] This invention provides the tobacco nicotine accumulation-related gene NtGSTU19 and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note 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.

[0030] Nucleotide sequence of GSTU19: (SEQ ID NO:1);

[0031] The amino acid sequence of GSTU19 (glutathione S-transferase NtGSTU19) is: MAEEVILLDCWCSMYGMRARIALAEKGVKYEYKEEDLNNKSPLLLEMNPIHKKVPVLIHNGKSICESLVIVQYIDEVWKGNGPLIPSDPYEKAQAWFWSDYMGNTVHEYAVKIWATKGEEQEQAVKDYLGGLKLIEGVLGDKPYFGGENFGFLDISLIGFYSWFLAYETFGKFSVEKECPKLILWVKRCLERDSVSKTLPDSEKVCEFAYEFGVE (SEQ ID NO:2);

[0032] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0033] Example 1: The nicotine accumulation-related gene NtGSTU19 and its application

[0034] I. Gene Cloning

[0035] Total RNA was extracted from root tissue of the K326 varietal variety using the SuperPure Plant polyRNA Kit. The extracted total RNA was then reverse transcribed into cDNA using the TransScript® II Reverse Transcriptase Kit. This cDNA was used as a template for PCR amplification.

[0036] Amplification primers:

[0037] F: atggcagaagaagtgattctgt, SEQ ID NO: 3;

[0038] R: tcattccactccaaactcatagg, SEQ ID NO: 4;

[0039] Referring to the 2×TransTaq High Fidelity (HiFi) PCR SuperMix instruction manual, using the prepared cDNA as a template, PCR amplification was performed using the designed amplification primers. The reaction system was added according to the instruction manual, and the PCR reaction was carried out under the following conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 40 s, 30 cycles; 72℃ extension for 10 min.

[0040] The PCR amplification products were detected by 1% agarose gel electrophoresis. The electrophoresis results are as follows: Figure 1As shown, there is a clear and single target band at around 650bp.

[0041] II. Gene Tissue Expression Analysis

[0042] Samples of different tissues, including leaves, stems, roots, and flowers, from different parts of the K326 variety during its full bloom period were collected, flash-frozen in liquid nitrogen, and then stored at -80°C for later use. Total RNA was extracted from the samples, and cDNA was synthesized using a reverse transcription kit. The expression pattern of the NtGSTU19 gene was detected using quantitative real-time PCR with the tobacco NtL25 gene as an internal control.

[0043] NtGSTU19 fluorescent quantitative primers:

[0044] QF: ggaaatcaatttgtgagtcacttgt, SEQ ID NO: 5;

[0045] QR: cttgctcttctccttttgttgc, SEQ ID NO: 6;

[0046] The specific primers used for detecting the tobacco NtL25 gene are:

[0047] NtL25-F: caaaagttacattccaccg, SEQ ID NO:7;

[0048] NtL25-F: tttcttcgtcccatcaggc, SEQ ID NO: 8;

[0049] The conditions for quantitative real-time PCR are as follows: Step 1: pre-denaturation, 95℃ for 30s; Step 2: PCR reaction, 95℃ for 3s, 60℃ for 20s, 40 cycles; Step 3: melting curve.

[0050] Each sample was biologically replicated three times, using 2 -△△CT Methods were used to analyze the differences in relative gene expression.

[0051] Analysis results as follows Figure 2 As shown, the NtGSTU19 gene is mainly expressed in the root system.

[0052] III. Jasmonic acid-induced gene expression analysis

[0053] Tobacco seeds were placed in petri dishes and cultured with Hoagland solution (light / dark = 18 / 6h, 23℃~28℃). After germination for 2 weeks, the seedlings were transplanted and immersed in a solution containing methyl jasmonate. The leaves were also sprayed with the corresponding concentration of methyl jasmonate for 5 hours. Samples were collected, flash-frozen in liquid nitrogen, and then stored at -80℃ for later use.

[0054] The specific treatment methods for the experimental group are as follows:

[0055] 50 μmol / L methyl jasmonate (MeJA);

[0056] A control group was set up, in which tobacco seedlings were treated with 1% (v / v) DMSO solution.

[0057] The treated tobacco seedlings were placed in 1.5 mL centrifuge tubes, flash-frozen in liquid nitrogen, and stored at -80 °C for later use.

[0058] RNA was extracted from the preserved material, and cDNA was synthesized using a reverse transcription kit (follow the kit instructions). The tobacco NtL25 gene was used as an internal control for quantitative real-time PCR detection.

[0059] Analysis results as follows Figure 3 As shown, compared with the control group, the relative expression level of the NtGSTU19 gene was downregulated by jasmonic acid.

[0060] IV. Construction of NtGSTU19 gene knockout vector

[0061] First, based on the design principles of CRISPR / Cas9 target sites, gtgcagtatgtatgggatgaggg (gRNA, SEQ ID NO:9) was selected as the editing site for the NtGSTU19 gene, with GGG 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, followed by natural cooling to room temperature. The pORE-CRISPR / Cas9 editing vector was digested with BsaI. The reaction mixture consisted of 50 μL of the editing vector (10 μL), 1 μL of BsaI enzyme, 5 μL of CutSmart Buffer (10X) buffer, and 34 μL of ddH2O. Digestion was carried out at 37°C for 1 hour, and the digested plasmid was recovered using a product recovery kit.

[0062] The annealed double-stranded target site sequence was ligated to the enzyme-digested editing vector using T4 ligase. The ligation system was 20 μL: 2 μL 10×T4 DNA Ligase Buffer, 5 μL double-stranded target site, 2 μL editing vector, 1 μL T4 DNA Ligase, and ddH2O was added to bring the total volume to 20 μL.

[0063] 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 using colony PCR with the detection primer (JC-F: ttaggtttacccgccaata, SEQ ID NO: 10) and the target site reverse primer. Positive clones were expanded, plasmids were extracted, and stored at low temperature for Agrobacterium transformation. The plasmid was named pORE-CRISPR / Cas9-NtGSTU19.

[0064] V. Obtaining Knockout Strains

[0065] 1. Transformation of Agrobacterium

[0066] Remove competent Agrobacterium cells and freeze-thaw them on ice. Just before thawing, add 10 μL of pORE-CRISPR / Cas9-NtGSTU19 and gently mix. Incubate on ice for 10 min, then in liquid nitrogen for 5 min, then at 37°C for 5 min, and then immediately freeze for 5 min. Add 600 μL of antibiotic-free LB broth and incubate at 28°C with shaking at 200 rpm for 3 h. Spread 200 μL of the 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 for PCR identification. Correctly identified positive clones are the successfully transformed engineered bacteria.

[0067] 2. Identification of transformed tobacco plants and mutants

[0068] 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.

[0069] The transformed Agrobacterium bacteria prepared above were cultured until OD600=0.6, the bacterial cells were collected, and then the bacterial cells were suspended in 20mL of MS liquid medium;

[0070] Then, the pre-cultured leaf discs were placed in the bacterial solution and incubated for 10 minutes.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] 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:

[0075] A pair of detection primers were designed on the Cas9 sequence to detect transgenic lines, specifically:

[0076] Cas9-F: ctcaacacaacatatacaaaacaaa, SEQ ID NO: 11;

[0077] Cas9-F: ctttggccatctcgtttga, SEQ ID NO: 12;

[0078] PCR amplification was performed using DNA template from the T0 generation transgenic line. 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.

[0079] A primer was designed across the NtFLS1 gene sgRNA target site to detect the mutation type at the target site, specifically:

[0080] NtGSTU19-MF: gcaaataagaaaaggtgaataatgc, SEQ ID NO: 13;

[0081] NtGSTU19-MR: cattgtgaattaagactgggacttt, SEQ ID NO: 14;

[0082] 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, 54℃ 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 NtGSTU19 gene target site in the transgenic positive plants was identified, and lines with one "A" base insertion and one "G" base deletion were obtained. Figure 4 Mutant strain 1 (KO-1) has an "A" base inserted after 49 bp of the nucleotide sequence shown in SEQ ID NO:1; mutant strain 2 (KO-1) has a "G" base inserted at positions 46-48 bp of the nucleotide sequence shown in SEQ ID NO:1.

[0083] VI. Nicotine Analysis of Wild-Type and Homozygous Mutant Lines

[0084] Wild-type and NtGSTU19 gene mutant lines (T1 generation) were cultured in a greenhouse. After two months of cultivation, they were sprayed three times with 50 μmol / L methyl jasmonate (MeJA). One week later, root and leaf tissues were collected, and the nicotine content was analyzed by ultra-high performance liquid chromatography.

[0085] The steps for analyzing nicotine content using ultra-high performance liquid chromatography are as follows:

[0086] After grinding, the sample was passed through a 40-mesh sieve. 150 mg of tobacco powder was accurately weighed into a 15 mL screw-top pressure-resistant test tube. 1.75 mL of 5% sodium hydroxide solution was added to moisten the sample. After standing for 15 min, 10 mL of 0.01% triethylamine / methyl tert-butyl ether solution was added. The tube was capped and sealed, and ultrasonic extraction was performed at room temperature for 15 min, followed by centrifugation at 6000 rpm for 5 min. 2 mL of the organic phase was collected, and the nicotine mass fraction was analyzed by GC-MS. The analysis was performed using two injections, with retention time and selected ion (SIM) mode for qualitative analysis and dual internal standard quantification. Analytical conditions:

[0087] Chromatographic column: DB‒35MS (30m×0.25mm id×0.25μm df); Temperature program: 100℃, 3 min; then ramp to 260℃ at 8℃ / min and hold for 10 min; Carrier gas: Helium; Column flow rate: 1.0 mL / min; Injector temperature: 250℃; Nicotine detection injection volume: 1 µL, split injection; Split ratio: 40:1; Other alkaloid detection injection volume: 2 µL, split injection; Split ratio: 10:1; Solvent delay: 8 min; Ionization voltage: 70 eV; Ion source temperature: 230℃; Transfer line temperature: 280℃; Scan mode: Selected ion mode (SIM).

[0088] The results showed that, after jasmonic acid treatment, there was no difference in nicotine levels in the leaves of the NtGSTU19 gene mutant line compared with the control, but the nicotine levels in the roots were significantly higher than the control, suggesting that the gene mutation enhanced the ability to synthesize nicotine. Figure 5 Increased nicotine content in the roots enhances the tobacco plant's natural defenses against pests and diseases, reducing their incidence and thus lowering pesticide usage, while improving the quality and safety of tobacco leaves. Furthermore, under adverse soil conditions such as drought and salinity, increased nicotine content in the roots helps the tobacco plant maintain normal physiological metabolism. As an important physiological regulator, nicotine can participate in regulating intracellular osmotic pressure, enhancing cellular resistance, and enabling the tobacco plant to grow normally under certain environmental stresses.

[0089] 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 glutathione S-transferase NtGSTU19 in plant quality improvement; The amino acid sequence of the tobacco glutathione S-transferase NtGSTU19 is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The quality improvement includes increasing the nicotine content in the roots.

3. The application according to claim 2, characterized in that, The plant mentioned includes tobacco.

4. A product for improving plant quality, characterized in that, Includes at least one of the following: A) to C) A) Amplification primers and / or quantitative detection primers using the nucleic acid encoding the tobacco glutathione S-transferase NtGSTU19 as a template; B) gRNA targeting the tobacco glutathione S-transferase NtGSTU19; C) A recombinant vector containing nucleic acid encoding tobacco glutathione S-transferase NtGSTU19, or gRNA as described in B).

5. The product according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid encoding the tobacco glutathione S-transferase NtGSTU19 is shown in SEQ ID NO:

1.

6. The product according to claim 4, characterized in that, The nucleotide sequence of the gRNA is shown in SEQ ID NO:

9.

7. The product according to claim 4, characterized in that, The amplification primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

4.

8. The product according to claim 4, characterized in that, The quantitative detection primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

6.

9. The use of the product according to any one of claims 4 to 8 in the improvement of plant traits.

10. A method for improving plant traits, characterized in that, This includes products using any one of claims 4 to 8.

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