Tobacco NtACS3 gene and its application

Abstract

This invention relates to the field of biotechnology, and more particularly to the tobacco NtACS3 gene and its applications. This invention provides the application of the tobacco NtACS3 gene in improving tobacco resistance to quincloracine. Knocking out the tobacco NtACS3 gene can confer resistance to quincloracine in tobacco, thereby eliminating or mitigating the losses caused by herbicide damage to tobacco production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the tobacco NtACS3 gene and its applications. Background Technology

[0002] Tobacco is an important economic crop, and tobacco leaves are the basic raw material for the tobacco leaf rolling industry. The quality of tobacco leaves determines its industrial usability.

[0003] Quinclorac is primarily used to control barnyard grass in rice paddies and is a major pre- and post-emergence herbicide in my country. However, due to its long-term and extensive use, quinclorac phytotoxicity has occurred in some parts of my country, causing severe economic losses. The accumulation of quinclorac causes tobacco leaves to curl and become deformed, leading to a significant decrease in tobacco yield or even crop failure. Simultaneously, it increases the nicotine content and decreases the reducing sugar content in tobacco leaves, affecting tobacco quality and further impacting the industrial usability and safety of tobacco.

[0004] It is evident that improving tobacco's resistance to quinclorac or reducing its use is urgently needed. However, quinclorac has already accumulated in large quantities in the soil, making tobacco breeding to enhance its resistance a primary means of addressing quinclorac-induced damage in tobacco. Therefore, identifying quinclorac resistance genes and applying them to tobacco breeding using genetic engineering techniques has significant practical implications. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the tobacco NtACS3 gene and its application.

[0006] This invention provides the application of ACS3 as a target in plant breeding and / or in the preparation of products that aid in plant breeding;

[0007] In the application described in this invention, the ACS3 includes at least one of the amino acid sequences shown in a) to c) below:

[0008] a) An amino acid sequence as shown in SEQ ID NO:2; or

[0009] b) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (a), and which has the same function as the amino acid sequence described in (a); or

[0010] c) An amino acid sequence having at least 90% sequence identity with the amino acid sequence described in (a) or (b).

[0011] Furthermore,

[0012] The plant breeding includes regulating plant resistance to dichloroquinoline acid;

[0013] The regulation includes increasing or decreasing; the decrease includes partial or complete loss of function; in a specific embodiment of the present invention, ACS3 function is completely lost, and the plant's resistance to dichloroquinoline acid is enhanced.

[0014] The plant in question is a member of the Solanaceae family; in a specific embodiment of the invention, it is tobacco.

[0015] This invention provides plant breeding or assisted breeding products, comprising at least one of the following: A) to C:

[0016] A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding ACS3 as a template;

[0017] B) Targeting the interference fragments of the ACS3;

[0018] C) gRNA targeting ACS3;

[0019] D) Expression cassettes containing interfering fragments as described in B), or gRNAs as described in C);

[0020] 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);

[0021] F) Transformation or transfection of host cells with the recombinant vector described in E);

[0022] G), a mixture obtained by culturing host cells as described in F).

[0023] Furthermore, in the product described in this invention,

[0024] The target nucleotide sequence of the gRNA is shown in SEQ ID NO:9. In this invention, the gRNA is recombined into a CRISPR / Cas9 vector, the target nucleotide sequence of which is shown in SEQ ID NO:9. Then, tobacco is transformed, and after gene editing by the CRISPR / Cas9 system, a tobacco ACS3 variant is obtained, which inserts a base A between 374 and 375 bp of the nucleotide sequence shown in SEQ ID NO:1, resulting in a frameshift mutation, leading to the loss and / or reduction of tobacco ACS3 function.

[0025] The nucleic acid encoding ACS3 includes at least one of the nucleic acids shown in i) to v) below:

[0026] i) Having a nucleotide sequence as shown in SEQ ID NO:1; or

[0027] ii) with at least one complementary nucleic acid molecule among the nucleic acid molecules shown in i); or

[0028] iii) Nucleic acid molecules that encode the same protein as those in i) or ii), but are different from those in i) or ii) due to the degeneracy of the genetic code; or

[0029] iv) Nucleic acid molecules obtained by substituting, deleting, or adding one or more bases to the nucleic acid molecules shown in i), ii), or iii), and having the same or similar function as the nucleic acid molecules shown in i), ii), or iii); or

[0030] A nucleic acid molecule that has at least 80% sequence identity with the nucleotide sequence described in v), i), ii), iii), or iv).

[0031] The nucleic acid described in this invention can be DNA, RNA, or cDNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded; this invention does not limit this.

[0032] The expression cassette described in this invention refers to a DNA sequence from the start of the promoter to the end of the terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, etc.

[0033] 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 ACS3 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.

[0034] 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.

[0035] In this invention, the transformation methods include chemical transformation and electrotransformation; the transfection methods include 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 a specific embodiment of this invention, the Agrobacterium-mediated leaf disc method is used to prepare ACS3 gene loss-of-function mutants for the study of ACS3 gene function.

[0036] This invention provides a kit comprising excipients and the product described herein.

[0037] The excipients are used to preserve the product, maintain the activity of the product, or assist the product in performing its function; in this invention, the excipients include at least one of the following: DNA extraction reagent, DNA reverse transcription reagent, buffer solution, transfection reagent, antibiotic and / or culture medium.

[0038] This invention provides the application of the product or kit described herein in plant breeding or assisted plant breeding.

[0039] This invention provides a method for plant breeding or assisted plant breeding, which includes plant breeding using the products or kits described in this invention.

[0040] This invention provides the application of the tobacco NtACS3 gene in improving tobacco resistance to quincloracine. Knocking out the tobacco NtACS3 gene can confer resistance to quincloracine in tobacco, thereby eliminating or reducing the losses caused by herbicide damage to tobacco production. Attached Figure Description

[0041] Figure 1 Electrophoresis diagram of NtACS3 gene CDS amplification products;

[0042] Figure 2 The diagram shows the induced expression analysis of the NtACS3 gene;

[0043] Figure 3 The image shows a CRISPR / Cas9 recombinant plasmid colony PCR detection result.

[0044] Figure 4 Diagram showing target site mutation types;

[0045] Figure 5 Leaf width analysis of mutants. Detailed Implementation

[0046] This invention provides the tobacco NtACS3 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.

[0047] Biological materials: Tobacco variety: K326, a common cultivated tobacco variety, the seeds of which were preserved by Hunan Tobacco Industry Co., Ltd. Tobacco was cultured in a growing room under the following conditions: temperature (27±1)℃, relative humidity (60±5)%, light / dark cycle 16 h / 8 h.

[0048]

[0049] Amino acid sequence of NtACS3: MGFENEKNSSILSKLATNEEHGENSPYFDGWKAYDNDPFHPLKNPNGVIQMGLAENQLCFDLIEEWIKRNPNASICTTEGIKSFRAIANFQDYHGLPEFRSAIAKFMEKTRGGR VTFDPERVVMAGGATGANETIIFCLADTGDAFLVPSPYYPAFNRDLRWRTGVQLIPIPCDSSNNFQITTKAVREAYENAQKSNIKVKGLILTNPSNPLGTTLDRDTLKNLLTFTNQHNIHLVCDEIY AATVFNTPQFVSIAEILDDETSHCNKDLVHIVYSLSKDMGLPGFRVGIVYSFNDAVVNCARKMSSFGLVSTQTQYLLAEMLSDERFVSNFLTESSKRLAKRHKHFTNGLEEVGIKCLRSNAGLFCWM DLRPLLKESTFDSEMSLWRVIINDVKLNVSPGSSFDCQEPGFFRVCFANMDDETVDIALARIRSFVGVKKSGDESTPILMEKKQQWKKNNLRLSFSKRMYDESVNLSPLSSPIPHSPLVRART(SEQ ID NO:2);

[0050] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0051] Example 1: Obtaining the tobacco auxin receptor NtACS3 gene

[0052] (1) PCR amplification primer design

[0053] Based on the existing tobacco genome sequence, primers for PCR amplification of the CDS region of the NtACS3 gene were designed, and the sequences are as follows:

[0054] Upstream primer ACS3-f: 5'-atgggatttgagaatgagaag-3' (SEQ ID NO:3);

[0055] Downstream primer ACS3-r: 5'-ttaagttctagctcgaacgagt-3' (SEQ ID NO:4);

[0056] (2) Preparation of PCR amplification template

[0057] Total RNA was extracted from K326 tobacco leaf material using the SuperPure Plant polyRNA Kit. TransScript was then used to analyze the RNA. ® The II Reverse Transcriptase kit reverse transcribes the extracted total RNA into cDNA. This cDNA is then used as a template for PCR amplification.

[0058] (3) PCR amplification

[0059] Referring to the instructions for the 2× TransTaq High Fidelity (HiFi) PCR SuperMix, the cDNA prepared in step (2) was used as a template, and PCR amplification was performed using the primers designed in step (1). The reaction system was added according to the instructions, and the PCR reaction was performed. The PCR reaction conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 100 s, 30 cycles; 72℃ extension for 5 min.

[0060] The PCR amplification products were detected by 1% agarose gel electrophoresis. The electrophoresis results are as follows: Figure 1 As shown, there is a clear and single target band at around 1476bp.

[0061] The target fragment was recovered via gel extraction and sent for sequencing. The gel extraction results are shown in SEQ ID NO:1.

[0062] Example 2: Analysis of the inducible expression of the NtACS3 gene

[0063] To clarify the expression pattern of the NtACS3 gene under dichloroquinoline acid induction, dichloroquinoline acid was used for treatment, and the relative expression level of the NtACS3 gene in tobacco leaves and roots was detected.

[0064] Using the common tobacco variety K326 at the 5-leaf stage as material, each tobacco plant was irrigated with 20 mL of a 0.2 mg / kg dichloroquinoline acid solution (75% wettable powder, Chengdu Kelilong Biochemical Co., Ltd.). Tobacco leaves and root tissues were collected at 2 h, 6 h, 1 d, 7 d, and 14 d after irrigation. The control (represented as 0 h) was irrigated with the same volume of water. Three plants were collected at each time point, with each plant representing one biological replicate.

[0065] RNA was extracted from the collected materials, 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:

[0066] The primers for quantitative real-time detection of the NtACS3 gene are as follows:

[0067] 20029f: 5'-cattagcgaggattcggagtt-3' (SEQ ID NO: 5);

[0068] 20029r: 5'-tggtgaatgagggataggaga-3' (SEQ ID NO: 6);

[0069] The specific primers used for detecting the tobacco NtL25 gene are:

[0070] NtL25-F: 5'-caaaagttacattccaccg-3' (SEQ ID NO:7);

[0071] NtL25-R: 5'-tttcttcgtcccatcaggc-3' (SEQ ID NO: 8);

[0072] The conditions for quantitative real-time PCR are as follows: Step 1: pre-denaturation, 95℃ for 10s; Step 2: PCR reaction, 95℃ for 5s, 60℃ for 30s, 39 cycles; Step 3: melting curve.

[0073] Each sample was biologically replicated three times, using 2 -△△CT Methods were used to analyze the differences in relative gene expression. The results of the relative expression level analysis are as follows: Figure 2 As shown in the figure, the expression level of the NtACS3 gene was upregulated by approximately 20-fold in leaves treated with quinclorac acid for 24 hours. This indicates that quinclorac acid can significantly induce the expression of the NtACS3 gene in leaves.

[0074] Example 3: Construction of gene editing vector

[0075] To obtain tobacco plants resistant to quinclorac, a gene-editing vector was constructed using CRISPR / Cas9 technology and transformed to obtain NtACS3 gene-knockout tobacco plants. This embodiment briefly describes the experimental details related to the gene-editing vector construction experiment.

[0076] (1) Design of primers for editing target sites

[0077] The following editing primer sequences were designed targeting the target region of the NtACS3 gene (specific target sequence: gagagagtagttatggctgg, SEQ ID NO:9):

[0078] ACS3f: 5'-gattgagagagtagttatggctgg-3' (SEQ ID NO: 10);

[0079] ACS3r: 5'-aaacccagccataactactctctc-3' (SEQ ID NO: 11);

[0080] (2) Primer annealing and vector ligation

[0081] Referring to the Annealing Buffer for DNA Oligos (5×) kit instructions, double-stranded DNA at the target site was obtained by primer annealing. The annealing reaction system was as follows: Annealing Buffer for DNA Oligos (5×), 4 μL; forward and reverse primers (ACS3f, ACS3r), 4 μL each (50 μmol / μL); Nuclease-free water was added to a final volume of 20 μL. The reaction program was: 95℃ for 5 min, decreasing by 0.1℃ every 8 s until reaching 25℃.

[0082] The annealing product was ligated with the BsaⅠ-digested CRISPR / Cas9 vector, and CRISPR / Cas9 expression vectors for knocking out the NtACS3 gene were obtained by screening. The 20 μL ligation system was designed as follows: annealing product, 6 μL; digestion product (BsaⅠ-digested CRISPR / Cas9 vector), 3 μL; 10×T4 DNA Ligase Buffer, 2 μL; T4 DNA Ligase, 1 μL; sterile water was added to 20 μL, and ligation was carried out at 37℃ for 3 h.

[0083] The ligation product was transformed into DH5α *E. coli* competent cells for replication, followed by screening and colony PCR identification. For identification, primer pairs U26-jiance-F (5'-ttaggtttacccgccaata-3', SEQ ID NO:12) and ACS3r were used to detect positive clones. Electrophoresis results are shown below. Figure 3 As shown, all clones exhibited a specific target band at approximately 500 bp, indicating they were all positive clones. Editing plasmids were extracted from the positive clones.

[0084] Example 4: Obtaining transgenic strains with NtACS3 gene knockout

[0085] The editing plasmid extracted in Example 3 was transformed into Agrobacterium and then into tobacco plants to construct transgenic plants with the NtACS3 gene knocked out. The specific experimental process is as follows.

[0086] (1) Transformation of Agrobacterium

[0087] Thaw competent Agrobacterium GV3101 cells on ice, add 6 μL of the editing vector prepared in Example 2, and mix gently. Place the mixture in a pre-chilled electroporation cuvette and incubate on ice for 5 min. Blot dry the cuvette with absorbent paper, then place it in the electroporation tank for electroporation transformation. Electroporation conditions: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω. After electroporation, quickly add 800 μL of YEB liquid medium preheated to 28°C, and incubate at 28°C for 3 h with shaking at 220 rpm. Spread the bacterial culture evenly onto YEB solid medium containing rifampicin (100 μg / mL), streptomycin (50 μg / mL), and kanamycin (50 μg / mL), and incubate at 28°C until single colonies form. Pick single colonies, expand the culture, and perform PCR identification using U26-jiance-F and TIRa1-r primers. The correctly identified strain is the positive engineered bacteria.

[0088] (2) Transformation of tobacco plants

[0089] The prepared positive-positive engineered bacteria 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. Take leaves from K326 sterile seedlings that have grown for about one month and cut them into 0.5-1 cm pieces. 2 Square leaf discs were prepared. The leaf discs were then placed in the bacterial solution and immersed for 10 minutes. Excess bacterial solution around the leaf discs was blotted dry with sterile filter paper, and the discs were incubated in the dark for 3 days on MS medium supplemented with 6-BA (2 mg / L) and NAA (0.5 mg / L). The MS medium formulation was: 4.4 g / L MS inorganic salts, 30 g / L sucrose, 2.5 g / L plant gel, pH 5.8–5.9.

[0090] 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.

[0091] 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.

[0092] After about a month of growth, the seedlings were transplanted into soil. A small number of leaves were taken, and genomic DNA was extracted. PCR was used to detect positive transgenic plants. The primers used were U26-jiance-F and ACS3r.

[0093] Based on the genome sequence, detection primers spanning the NtACS3 gene target site were designed to detect the mutation type at the target site. The specific primers are as follows:

[0094] 8890f: 5'-caagattatcacggcctac-3' (SEQ ID NO: 13);

[0095] 8890r: 5'-tgggtttacttgtctgtttg-3' (SEQ ID NO: 14);

[0096] The PCR amplification products are sent for sequencing, and the mutation type is determined based on the sequencing results. Target site mutation types include... Figure 4 As shown, in the T0 generation plant ACS3-11-13-34, a nucleotide insertion mutation was detected at the target site of the NtACS3 gene, inserting one base A (i.e., inserting one base A between 374 and 375 bp of the nucleotide sequence shown in SEQ ID NO:1), causing a frameshift mutation in the NtACS3 protein, thereby inactivating the NtACS3 protein. No mutation was detected at the target site in the NtACS3 gene of the wild-type plant.

[0097] Example 5: Dichloroquinoline acid resistance determination

[0098] To clarify the resistance level of NtACS3 gene knockout mutants to dichloroquinoline acid, the resistance of wild-type K326 and the NtACS3 gene mutant ACS3-11-13-34 to dichloroquinoline acid was measured using leaf width as an indicator.

[0099] A pot experiment was conducted to determine the resistance of the mutant in Example 5 to quinclorac acid by treating it with quinclorac acid. Specifically, mutant ACS3-11-13-34 and wild-type K326 were used as materials. At the 5-leaf stage, each tobacco plant was irrigated with 20 mL of quinclorac acid solution, with a final concentration of 0.1 mg / kg. Leaf width was measured 30 days after treatment. The top three leaves were measured. Untreated K326 (irrigated with the same volume of water) served as a control. Data were statistically analyzed, such as... Figure 5 As shown. Under conditions of dichloroquinoline phytotoxicity, compared to the control K326, the width of the upper three leaves of ACS3-11-13-34 increased by 40.9%, 44.4%, and 50.9%, respectively, demonstrating excellent resistance. Figure 5In the above, K326 served as the control; K326Q represented K326 treated with quinclorac; ACS3-11-13-34 represented ACS3-11-13-34 without quinclorac treatment; and ACS3-11-13-34Q represented ACS3-11-13-34 treated with quinclorac. Specifically, 47.5% and -0.50% represent the decrease in leaf width of the first leaf of K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively; 66.40% and 29.4% represent the decrease in leaf width of the second leaf of K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively; and 75.2% and 27.8% represent the decrease in leaf width of the third leaf of K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively.

[0100] 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 ACS3 protein as a target in improving tobacco resistance to dichloroquinolinic acid; The amino acid sequence of the ACS3 protein is shown in SEQ ID NO:2; The improvement of tobacco's resistance to dichloroquinoline acid is achieved by gRNA knockout; The target nucleotide sequence of the gRNA is shown in SEQ ID NO:9.

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