Tobacco NtACS3 gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco NtACS3 gene and application thereof. The invention provides application of a tobacco NtACS3 gene to improvement of quinclorac resistance of tobacco. The tobacco NtACS3 gene is knocked out, so that the tobacco can be endowed with quinclorac resistance, and the loss of tobacco production caused by phytotoxicity is eliminated or reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to tobacco NtACS3 gene and application thereof. Background Art

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

[0003] Quinclorac is mainly used to control barnyard grass in rice fields and is the main herbicide variety in rice fields before and after germination in my country. Due to long-term and large-scale use, quinclorac phytotoxicity has occurred in some parts of my country, causing serious economic losses. The accumulation of quinclorac causes tobacco leaves to curl and become deformed, resulting in a significant drop in tobacco yield or even total failure; at the same time, it causes an increase in the nicotine content of tobacco leaves and a decrease in the reducing sugar content, affecting the quality of tobacco and further affecting the industrial availability and safety of tobacco leaves.

[0004] It can be seen that it is urgent to improve tobacco's resistance to quinclorac or reduce the use of quinclorac; however, quinclorac has already accumulated in the soil in large quantities, so tobacco breeding and improving tobacco's resistance to quinclorac are the main means to solve the problem of quinclorac damage to tobacco. Therefore, it is of great practical significance to explore quinclorac resistance genes and apply them to tobacco breeding using genetic engineering methods. Summary of the invention

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

[0006] The present invention provides the use of ACS3 as a target in plant breeding and / or in the preparation of products assisting plant breeding;

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

[0008] a), the amino acid sequence 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 having 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 the resistance of the plant to quinclorac;

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

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

[0015] The present invention provides a product for plant breeding or assisted breeding, which includes at least one of the following A) to C:

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

[0017] B), an interfering fragment targeting the ACS3;

[0018] C), gRNA targeting the ACS3;

[0019] D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C);

[0020] 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);

[0021] F), transforming or transfecting the host cell with the recombinant vector as described in E);

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

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

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

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

[0026] i), having the nucleotide sequence shown in SEQ ID NO: 1; or

[0027] ii), a nucleic acid molecule complementary to at least one of the nucleic acid molecules as shown in i); or

[0028] iii), a nucleic acid molecule that encodes the same protein as the nucleic acid molecule of i) or ii), but is different from the nucleic acid molecule of i) or ii) due to the degeneracy of the genetic code; or

[0029] iv), a nucleic acid molecule obtained by replacing, deleting or adding one or more bases to the nucleic acid molecule shown in i), ii) or iii), and having the same or similar functions as the nucleic acid molecule shown in i), ii) or iii); or

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

[0031] The nucleic acid of the present invention may be DNA, RNA or cDNA. In an embodiment of the present 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 may be single-stranded or double-stranded; the present invention does not limit this.

[0032] The expression cassette of the present invention refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0033] The recombinant vector described in the present invention is a recombinant DNA molecule, which contains a desired coding sequence and an 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 ACS3 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 method adopted by technicians in this field, and the present invention will not elaborate on this.

[0034] 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.

[0035] 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 ACS3 gene function-deficient mutants for the study of ACS3 gene function.

[0036] The invention provides a kit, which comprises auxiliary materials and the product of the invention.

[0037] The auxiliary material is used to preserve the product, maintain the activity of the product, or assist the product to exert its function and complete the corresponding function; in the present invention, the auxiliary material includes: at least one of a DNA extraction reagent, a DNA reverse transcription reagent, a buffer, a transfection reagent, an antibiotic and / or a culture medium.

[0038] The present invention provides application of the product or the kit in plant breeding or assisted plant breeding.

[0039] 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.

[0040] The invention provides an application of tobacco NtACS3 gene in improving tobacco resistance to quinclorac. Knocking out tobacco NtACS3 gene can give tobacco resistance to quinclorac, thereby eliminating or reducing the loss caused by pesticide damage to tobacco production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shows the electrophoresis of the CDS amplification product of NtACS3 gene;

[0042] Figure 2 Shown is the analysis diagram of the induced expression of NtACS3 gene;

[0043] Figure 3 Shown is the PCR detection diagram of CRISPR / Cas9 recombinant plasmid colony;

[0044] Figure 4 A diagram showing the target site mutation types;

[0045] Figure 5 Leaf width analysis of mutants is shown. DETAILED DESCRIPTION

[0046] The present invention provides tobacco NtACS3 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.

[0047] Biological materials: Tobacco varieties: K326, a common cultivated tobacco variety, the seeds used were stored by Hunan China Tobacco Industry Co., Ltd. Tobacco was cultured in a growing room, and the culture conditions were: temperature (27±1)℃, relative humidity (60±5)%, light / dark 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 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:

[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 were designed for the NtACS3 gene. 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 leaves using the SuperPure Plant polyRNA Kit. ® II Reverse Transcriptase Kit was used to reverse transcribe the extracted total RNA into cDNA, and the cDNA was used as a template for PCR amplification.

[0058] (3) PCR amplification

[0059] Refer to the 2× TransTaq High Fidelity (HiFi) PCR SuperMix instruction manual, use the cDNA prepared in step (2) as a template, use the primers designed in step (1) to perform PCR amplification, add the reaction system according to the instruction manual, and perform the PCR reaction. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 100 s, 30 cycles; extension at 72°C for 5 min.

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

[0061] The target fragment was recovered from gel and sent for sequencing. The gel recovery result is shown in SEQ ID NO: 1.

[0062] Example 2 Analysis of Inducible Expression of NtACS3 Gene

[0063] In order to clarify the expression pattern of NtACS3 gene under quinclorac induction conditions, quinclorac was used to treat and the relative expression level of NtACS3 gene in tobacco leaves and roots was detected.

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

[0065] RNA was extracted from the collected materials, and cDNA was synthesized using a reverse transcription kit (refer to the kit instructions for operation). Fluorescence quantitative PCR was performed using the tobacco NtL25 gene as an internal reference. During the detection, the primer sequences were designed as follows:

[0066] Fluorescence quantitative primers for detecting NtACS3 gene, the primer sequences are:

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

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

[0069] When detecting tobacco NtL25 gene, the specific primers are:

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

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

[0072] The conditions of fluorescence quantitative PCR were as follows: the first step was pre-denaturation at 95°C for 10 s; the second step was PCR reaction at 95°C for 5 s, 60°C for 30 s, and 39 cycles; the third step was melting curve.

[0073] Each sample was biologically replicated three times. -△△CT The relative gene expression analysis results were as follows: Figure 2 The results showed that the expression of NtACS3 gene was upregulated by about 20 times in leaves after 24 hours of quinclorac treatment, indicating that quinclorac can significantly induce the expression of NtACS3 gene in leaves.

[0074] Example 3 Construction of gene editing vector

[0075] In order to obtain tobacco plants with quinclorac resistance, CRISPR / Cas9 technology was used to construct a gene editing vector and perform gene transformation to obtain tobacco plants with NtACS3 gene knockout. This example specifically introduces the experimental conditions related to the gene editing vector construction experiment as follows.

[0076] (1) Editing target primer design

[0077] For the target site region of the NtACS3 gene (specific sequence of the target site: gagagagtagttatggctgg, SEQID NO:9), the editing primer sequences were designed as follows:

[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] Refer to the Annealing Buffer for DNA Oligos (5×) kit instructions to obtain double-stranded DNA at the target site through primer annealing; the annealing reaction system is as follows: Annealing Buffer for DNA Oligos (5×), 4 μL; upstream and downstream primers (ACS3f, ACS3r), 4 μL each (50 μmoL / μL); Nuclease-free water is added to 20 μL. The reaction procedure is: 95℃ for 5min, and the temperature is reduced by 0.1℃ every 8s to 25℃.

[0082] The above annealing product was connected with the CRISPR / Cas9 vector after BsaⅠ digestion, and the CRISPR / Cas9 expression vector for knocking out the NtACS3 gene was screened. The 20μL connection system was designed as follows: annealing product, 6μL; digestion product (CRISPR / Cas9 vector after BsaⅠ digestion), 3μL; 10×T4 DNA Ligase Buffer, 2μL; T4 DNALigase, 1μL; sterile water was added to 20μL and connected at 37℃ for 3h.

[0083] The above ligation products were transformed into DH5α E. coli competent cells for replication, and then screened and identified by colony PCR. During identification, primer pair U26-jiance-F (5'-ttaggtttacccgccaata-3', SEQ ID NO: 12) and ACS3r were used for positive clone detection. The electrophoresis results were as follows: Figure 3 As shown in the figure, all clones have specific target bands at about 500 bp, and are positive clones. The editing plasmids of positive clones were extracted.

[0084] Example 4 Obtaining NtACS3 gene knockout transgenic strains

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

[0086] (1) Transformation of Agrobacterium

[0087] Thaw the competent cells of Agrobacterium GV3101 on ice, add 6 μL of the editing vector prepared in Example 2, and mix gently; place the mixture in a pre-cooled electroporation cup and place it on ice for 5 minutes. Use absorbent paper to absorb the water droplets on the outer wall of the electroporation cup, put the electroporation cup into the electroporation tank, and perform electroporation transformation. The electroporation conditions are: voltage 2.5kV, capacitance 25μF, resistance 200Ω. After the click is completed, quickly add 800μL of YEB liquid culture medium preheated to 28℃, and shake and recover at 28℃ for 3h in a shaker at 220rpm. The bacterial solution is evenly applied to the YEB solid culture medium containing rifampicin (100μg / mL), streptomycin (50μg / mL) and kanamycin (50μg / mL), and cultured at 28℃ until a single colony is formed. Pick a single colony, expand the culture, and perform PCR identification on the bacterial solution. The identification primers are U26-jiance-F and TIRa1-r. The correct strain is identified as the positive engineered bacteria.

[0088] (2) Transformation of tobacco plants

[0089] The positive engineered bacteria prepared above were cultured to OD 600 = 0.6, centrifuge at 4000rpm for 5min to collect the cells, and then suspend the cells in 20mL of MS liquid medium. Take the leaves of K326 sterile seedlings that have grown for about one month and cut them into 0.5~1cm 2 Square leaf disc. Then place the leaf disc in the bacterial solution and immerse for 10 minutes. Use sterile filter paper to absorb the excess bacterial solution around the leaf disc after immersion, and culture it in the dark on the solid medium of MS+6-BA (2mg / L)+NAA (0.5mg / L) for 3 days. MS solid medium formula: 4.4g / L MS inorganic salt, 30g / L sucrose, 2.5g / L plant gel, pH 5.8~5.9.

[0090] The leaf disc was washed with sterile water containing Cef (400 mg / L), and the excess liquid was absorbed 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.

[0091] 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.

[0092] After growing for about a month, transplant them into the soil. Take a small amount of leaves, extract genomic DNA, and use PCR method to detect positive transgenic strains. The detection primers are U26-jiance-F and ACS3r.

[0093] According to the genome sequence, detection primers spanning the target site of NtACS3 gene were designed to detect the mutation type of the target site. The specific primers are:

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

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

[0096] The PCR amplification product was sent for sequencing, and the mutation type was determined based on the sequencing results. Figure 4 As shown, in the T0 generation plant ACS3-11-13-34, a nucleotide base insertion mutation was detected in the target site of the NtACS3 gene, and one base A was inserted (i.e., one base A was inserted 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 in the target site of the NtACS3 gene of the wild-type plant.

[0097] Example 5 Quinclorac resistance assay

[0098] In order to clarify the resistance level of NtACS3 gene knockout mutants to quinclorac, the resistance of wild-type K326 and NtACS3 gene mutant plant ACS3-11-13-34 to quinclorac was determined using leaf width as an indicator.

[0099] A pot experiment was conducted, and dichloroquinoline was added for treatment to determine the resistance of the mutants in Example 5 to dichloroquinoline. Specifically, the mutant ACS3-11-13-34 and the wild type K326 were used as materials. Each tobacco plant was irrigated with 20 mL of dichloroquinoline solution at the 5-leaf stage, and the final concentration of dichloroquinoline was 0.1 mg / kg. The leaf width was measured after 30 days of treatment. The upper 3 leaves were measured. Untreated K326 (irrigated with the same volume of clean water) was used as the control. Data statistics were performed, as shown in Figure 2. Figure 5 As shown. Under the condition of quinclorac phytotoxicity, compared with 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, showing good resistance ( Figure 5In the data, K326 was the control; K326Q was K326 treated with quinclorac; ACS3-11-13-34 was ACS3-11-13-34 not treated with quinclorac; ACS3-11-13-34Q was ACS3-11-13-34 treated with quinclorac; among them, 47.5% and -0.50% represented the decrease in leaf width of the first leaf K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively; 66.40% and 29.4% represented the decrease in leaf width of the second leaf K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively; 75.2% and 27.8% represented the decrease in leaf width of the third leaf K326 and ACS3-11-13-34 before and after quinclorac treatment, respectively).

[0100] 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. Use of ACS3 as a target in plant breeding and / or in the preparation of products that assist plant breeding; The amino acid sequence of ACS3 is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The plant breeding comprises regulating the resistance of the plants to quinclorac.

3. The use according to claim 2, characterized in that: The regulation includes increasing or decreasing.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is a plant of the Solanaceae family.

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 ACS3 as a template; B), an interfering fragment targeting the ACS3; C), gRNA targeting the ACS3; 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 target nucleotide sequence of the gRNA is shown in SEQ ID NO:9; The nucleic acid encoding the tobacco ACS3 includes at least one of the nucleic acids shown in i) to v) below: i), having the nucleotide sequence shown in SEQ ID NO: 1; or ii), a nucleic acid molecule complementary to at least one of the nucleic acid molecules as shown in i); or iii), a nucleic acid molecule that encodes the same protein as the nucleic acid molecule of i) or ii), but is different from the nucleic acid molecule of i) or ii) due to the degeneracy of the genetic code; or iv), a nucleic acid molecule obtained by replacing, deleting or adding one or more bases to the nucleic acid molecule shown in i), ii) or iii), and having the same or similar functions as the nucleic acid molecule shown in i), ii) or iii); or v), a nucleic acid molecule having at least 80% sequence identity with the nucleotide sequence described in i), ii), iii) or iv).

7. A kit, characterized in that Comprising auxiliary materials and the product according to 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, a DNA reverse transcription reagent, a buffer, a transfection reagent, an antibiotic and / or a culture medium.

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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