Tobacco ntclpR4 gene and application

Abstract

The present application relates to the field of biotechnology, in particular to a tobacco NtClpR4 gene and application. The present application clones a nicotine transformation related gene NtClpR4 from tobacco for the first time, which encodes an ATP-dependent tyrosine protease Clp related subunit, the amino acid sequence is shown as SEQ ID NO: 2, and the nucleotide sequence is shown as SEQ ID NO: 1; the gene product affects the synthesis of nornicotine. After mutation of the NtClpR4 gene, the nicotine content of cured tobacco leaves decreases, and the nornicotine content increases, which provides a new technical means for nicotine transformation regulation and tobacco breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the tobacco NtClpR4 gene and its applications. Background Technology

[0002] Tobacco mainly contains four alkaloids: nicotine, nornicotine, neonicotine, and pseudoequisetine. Nicotine is the most abundant alkaloid in tobacco, accounting for 90%–95% of the total alkaloid content. When insects or herbivores infest tobacco leaves, nicotine synthesis mediated by jasmonic acid injury-induced signals immediately initiates in the roots. The root tip cortex and epidermal cells are the main sites of nicotine synthesis. The synthesized nicotine is transported to the tobacco leaves through vascular tissue and stored in cell vacuoles to defend against various attacks. Nicotine synthesis is regulated by multiple factors, such as insect bites, jasmonic acid, abscisic acid, ethylene, and auxins.

[0003] Nonoline is derived from nicotine and is highly unstable. During tobacco curing and aging, it readily undergoes biochemical reactions to produce nonoline derivatives, including oxidative formation of mesmine, acylated nonoline, and N-nitrosonicotine (NNN). Mesmine and acylated nonoline directly affect the chemical composition of tobacco smoke and are detrimental to the quality of tobacco leaves. NNN is an important tobacco-specific nitrosamine (TSNA) with carcinogenic effects. Therefore, the increase in nonoline content due to nicotine conversion has a significant impact on the quality and safety of tobacco leaves. Identifying genes that regulate nicotine conversion and reducing demethylated nicotine content has always been a goal of tobacco breeding. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the tobacco NtClpR4 gene and its application.

[0005] The present invention provides tobacco NtClpR4, which has the amino acid sequence shown in SEQ ID NO:2.

[0006] This invention marks the first cloning of a nicotine conversion-related gene, NtClpR4, from tobacco. NtClpR4 encodes the Clp-related subunit of an ATP-dependent tyrosine-based protease, with its amino acid sequence shown in SEQ ID NO:2. The gene product affects the synthesis of demethylnicotine. Mutagenesis of the NtClpR4 gene resulted in a decrease in nicotine content and an increase in demethylnicotine content in cured tobacco leaves.

[0007] This invention provides a nucleic acid encoding tobacco NtClpR4.

[0008] Furthermore, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO:1.

[0009] This invention provides a biomaterial comprising at least one of the following: A) to G)

[0010] A) An expression unit containing the nucleic acid as described in this invention;

[0011] B) Interference fragments targeting the tobacco NtClpR4 described in this invention;

[0012] C) Knockout and / or mutation of the gRNA fragment of tobacco NtClpR4 as described in claim 1;

[0013] D) A recombinant vector containing the expression unit as described in A), the interfering fragment as described in B), and / or the gRNA fragment as described in C);

[0014] E) Transformation or transfection of host cells with the recombinant vector described in D);

[0015] F) A mixture obtained by culturing host cells as described in E);

[0016] G) Amplification primers and / or detection primers for the nucleic acid as described in claim 2 or 3.

[0017] Furthermore, in the biological material described in this invention, the gRNA fragment is as shown in SEQ ID NO:5.

[0018] In this invention, the nucleic acid can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of RNA or DNA. 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.

[0019] In this invention, the nucleic acid may be optimized or unoptimized, and this invention does not limit the optimization. The optimization includes, but is not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0020] The present invention also provides an expression unit, which refers to a DNA sequence from the start of a promoter to the end of a 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.

[0021] This invention provides a gRNA fragment, which is a fragment located approximately 20 bp before the recognition site (PAM site) of a CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a, and CRISPR / Cas14a) editing system targeting the target gene. The recognition site of the CRISPR / endonuclease system editing system varies depending on the endonuclease used; typically, the recognition site includes NGG. (Cas9) and / or TTN (Cpf1), etc.; In a specific embodiment of the present invention, the CRISPR / Cas9 system is used, and its recognition site is NGG, where N represents any one of the base species A, T, C or G. The position of NGG can be any position at the 5' end, middle or 3' end of the nucleic acid encoding tobacco NtClpR4, and the present invention does not limit it; In the present invention, tobacco NtClpR4 is used as the target, and gene editing is performed using the CRISPR / Cas9 system. Specifically, the nucleotide sequence of the gRNA fragment is shown in SEQ ID NO:5;

[0022] Furthermore, in this invention, a tobacco NtClpR4 variant was obtained after gene editing using the CRISPR / Cas9 system. This variant has an A base inserted at 892 bp in the nucleotide sequence shown in SEQ ID NO: 1, resulting in a frameshift mutation that leads to the loss and / or reduction of NtClpR4 function.

[0023] The present invention provides a recombinant vector comprising at least one of the nucleic acid, expression unit, interfering fragment and / or gRNA fragment described in the present invention, and a vector backbone.

[0024] Furthermore, the vector backbone described in this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit this. The viral vector includes: tobacco mosaic virus, adenovirus vector, adeno-associated virus (AAV) vector, retroviral vector, or lentiviral vector, etc.

[0025] In specific embodiments of the present invention, the recombinant vector is derived from bacteria and / or plants. The bacterial vector is used for cloning, expressing, preserving, or performing a certain function of the NtClpR4 gene. In specific embodiments of the present invention, the vector specifically includes the pEASY-Blunt Zero cloning vector and / or the pORE-CRISPR / Cas9 vector.

[0026] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression or function (e.g., interference or knockout) of the operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in eukaryotic cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends 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.

[0027] The transformation methods include chemical transformation and electroconversion; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and Agrobacterium-mediated transfection, etc.

[0028] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention is not limited to these sources. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells. These transformed host cells are capable of replicating protein-encoding vectors, expressing desired proteins, or performing specific functions, such as interfering with or knocking out specific target nucleic acids or proteins.

[0029] The present invention provides any one of the following I) to III) for the application in regulating the content of nicotine, demethylnicotine and / or demethylnicotine derivatives in plants;

[0030] I) The protein described in this invention;

[0031] II) The nucleic acid described in this invention;

[0032] III) The biomaterials described in this invention.

[0033] Furthermore,

[0034] The regulation includes increasing and / or decreasing;

[0035] The plants include plants from the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, and / or Solanaceae families; the Solanaceae plants include tobacco.

[0036] The demethylated nicotine derivatives include mesmin, acylated nornicotine and / or N-nitrosonornicotine (NNN), etc., and the present invention does not limit them.

[0037] This invention provides a product for regulating the content of nicotine, demethylnicotine and / or demethylnicotine derivatives in plants, comprising excipients and the biomaterials described in this invention.

[0038] Furthermore, the excipients include at least one or more of buffer solutions, antibiotics, antioxidants, stabilizers, helper plasmids, viruses, and / or culture media, etc., and the present invention does not limit them. The excipients are used to preserve the biological material and / or to assist the biological material in functioning.

[0039] This invention utilizes overexpression and CRISPR / Cas9 technology to construct NtClpR4 gene overexpression and loss-of-function mutants. Experimental results show that the NtClpR4 gene regulates the content of nicotine and nornicotine in plants. Overexpression of the NtClpR4 gene reduces the content of nornicotine in plants; reducing or knocking out the NtClpR4 gene reduces the content of nicotine, increases the content of nornicotine, and increases the content of mesmin.

[0040] This invention provides a method for regulating the content of nicotine, demethylnicotine and / or demethylnicotine derivatives in plants, which includes utilizing biomaterials as described in this invention and / or products as described in this invention.

[0041] This invention provides at least one of the following applications in plant breeding: a) to c)

[0042] a) The tobacco NtClpR4 described in this invention;

[0043] b) The nucleic acid described in this invention;

[0044] c) The biomaterials described in this invention;

[0045] d) The product described in this invention;

[0046] e) The method described in this invention.

[0047] Furthermore, the plants include plants from the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, and / or Solanaceae families; the Solanaceae plants include tobacco.

[0048] The breeding program includes regulating and improving the quality and / or safety of salt production.

[0049] This invention marks the first cloning of a nicotine conversion-related gene, NtClpR4, from tobacco. NtClpR4 encodes the Clp-related subunit of an ATP-dependent tyrosine-based protease, with its amino acid sequence shown in SEQ ID NO:2 and its nucleotide sequence shown in SEQ ID NO:1. The gene product affects the synthesis of demethylnicotine. Mutagenesis of the NtClpR4 gene resulted in a decrease in nicotine content and an increase in demethylnicotine content in cured tobacco leaves, providing a new technical approach for nicotine conversion regulation and tobacco breeding. Attached Figure Description

[0050] Figure 1 The pORE-CRISPR / Cas9 vector spectrum is shown.

[0051] Figure 2 The image shows a PCR electrophoresis diagram of the NtClpR4 gene.

[0052] Figure 3 This shows the expression characteristics of the NtClpR4 gene in different tissues;

[0053] Figure 4 Analysis of mutation types in NtClpR4 gene mutant plants;

[0054] Figure 5 The phenotype of tobacco leaves after curing is shown in plants with NtClpR4 gene mutations;

[0055] Figure 6 Detection of alkaloids in tobacco leaves after curing from NtClpR4 gene mutant plants. Detailed Implementation

[0056] This invention provides the tobacco NtClpR4 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.

[0057]

[0058] NtClpR4 amino acid sequence: MEAVTIASQFSPATGVRLSSPASCRSSAPKRTLSFSPSPKSSLSTSFISPFVGGSVLADFSGHKIRPDSLRPSSSSSRPKRGVVTMVIPFSRGSAWEQPPPDLASYLYKNRIVYLGMSLV PSVTELILAEFLYLQYEDEEKPIYLYVNSTGTTKGGEKLGYETEAFAIYDVMRYVKPPIFTLCVGNAWGEAALLLAAGAKGNRAALPSSTIMIKQPIARFQGQATDVELMRKEVKNVKAELVKLYSKHIGKSP EEIEADIRRPKYFSPSEAVEYGIIDKWIGPIHGKWNFAYQFKPIRVTVQEFGTVFALGGACGFGEYGRKIYDGKVSGVSRLYKNGTGCGACYQVRCKIAGHCTDEGTKIVVTDYGDGDHTDFILSVRAYSEMA SQGMANHLLAYGVVDVEYRRIPCRYYGYNLMIKVHENSRFSNYLAIVPIYQSGAFDVEAVEVWQADCKEWRGMRKAYGAVWDMPNPPKGSLTFRVQVSVSGEVKWVQLADVLPDEWKAGIAYDTNLLLD (SEQ ID NO: 2);

[0059] The technical solution adopted in this application is described in detail below.

[0060] The application of the nicotine conversion regulatory gene NtClpR4 in tobacco inhibits the conversion of nicotine to nornicotine. After mutation of the NtClpR4 gene, the nicotine content of the flue-cured tobacco leaves of the mutant plants decreased, while the contents of nornicotine, neonicotine, and styramine increased. The tobacco nicotine conversion regulatory gene NtClpR4 has important application significance for improving the quality and safety of tobacco leaves.

[0061] The mutant vector for the tobacco alkaloid transformation regulatory gene NtClpR4 was obtained by designing a CRISPR / Cas9 gene mutation target site sequence based on the cloned NtClpR4 gene coding sequence; specifically, the sgRNA (gRNA) target site sequence was annealed and ligated into a pORE-CRISPR / Cas9 editing vector digested with BsaI (vector map shown in Figure 1). Figure 1 As shown in the figure, through transformation, screening, and identification, a gene editing vector for the mutant tobacco nicotine transformation regulatory gene NtClpR4 was obtained.

[0062] The mutant vector for regulating the tobacco alkali conversion gene NtClpR4 is applied in tobacco: after transforming the vector into tobacco, NtClpR4 mutant plants are obtained by molecular detection, and base insertion occurs at the target site.

[0063] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0064] Example 1 Cloning of the NtClpR4 gene

[0065] I. Materials, Reagents and Equipment

[0066] 1. Biomaterials:

[0067] Tobacco variety: K326, a common cultivated tobacco variety. The seeds used in the examples were provided by Hunan Tobacco Industry Co., Ltd.

[0068] Vectors: pEASY-Blunt Zero cloning vector; pORE-CRISPR / Cas9 vector

[0069] Competent cells: Trans1-T1 competent cells; DH5α chemocompetent cells;

[0070] Strain: LBA4404 Agrobacterium strain;

[0071] Primer synthesis and DNA sequencing were provided by Beijing BGI Genomics Co., Ltd.

[0072] 2. Experimental reagents:

[0073] RNA extraction kit, SuperPure Plant polyRNA Kit;

[0074] The real-time PCR enzyme (TB Green® Fast qPCR Mix) and reverse transcription kit were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0075] Restriction endonuclease BsaI and T4 ligase were purchased from NEB.

[0076] DNA amplification enzyme, purchased from Beijing TransGen Biotech Co., Ltd.

[0077] Plant genome extraction kit and DNA purification kit were purchased from QIAGEN.

[0078] 3. Experimental equipment:

[0079] QuantStudio 3 Real-Time PCR Instrument, Thermo Fisher Scientific;

[0080] Gel Doc™ XR+ UV gel imaging system, Bio-Rad;

[0081] GC-MS (Gas Chromatography-Mass Spectrometry and Mass Spectrometry) system, Agilent

[0082] II. Cloning of the NtClpR4 gene

[0083] The cloning process of the NtClpR4 gene is briefly described below.

[0084] (1) Preparation of cDNA as cloning template

[0085] Take 200mg of tobacco (K326) seedling roots as a sample, grind them thoroughly in liquid nitrogen, extract total RNA according to the RNA extraction kit instructions, and then reverse transcribe it into cDNA for later use;

[0086] (2) Design primers and perform PCR amplification.

[0087] The primer sequences designed for amplifying the NtClpR4 gene are as follows:

[0088] NtClpR4-F: 5'-atggaagctgtgactatcgctt-3' (SEQ ID NO: 3);

[0089] NtClpR4-R: 5'-ttagtcgagcagaaggttggtg-3' (SEQ ID NO: 4);

[0090] Using the cDNA prepared in step (1) as a template, PCR amplification was performed using the above primers. The PCR amplification conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplified material was stored at 4℃ for later use, or directly subjected to electrophoresis detection and analysis.

[0091] Purify the PCR amplification product according to the instructions of the gel extraction kit. Then, ligate the purified product into the pEASY-Blunt Zero cloning vector. The ligation system is as follows: DNA amplification product, 6 μL; pEASY-Blunt Zero vector, 1 μL; mix well and ligate at 25°C for 25 min.

[0092] The ligation product was transformed into competent E. coli cells. The specific transformation process is briefly described below:

[0093] Remove competent cells from the -80℃ freezer, place them on ice to thaw, add the ligation product to 50 μL of Trans1-T1 competent cells, gently tumble to mix, and incubate on ice for 30 min.

[0094] Heat shock in a 42℃ water bath for 30 seconds, then immediately place on ice for 2 minutes; add 250 μL of LB (antibiotic-free) equilibrated to room temperature, and incubate at 37℃ with shaking for 1 hour.

[0095] Take 8 μL of the mixture and spread it evenly on an LB agar plate (containing 60 μg / μL ampicillin). Invert the culture dish and incubate overnight at 37°C.

[0096] After selecting plaques for amplification and culture, the DNA of each plasmid was extracted, and the recombinant plasmids were identified by plasmid PCR amplification. The corresponding positive clones were sent for sequencing to obtain the NtClpR4 gene sequence.

[0097] Sequencing analysis results show that the coding region of the NtClpR4 gene is 1548 bp in length (e.g., Figure 2 As shown in SEQ ID NO:1, where M is the marker; analysis of this gene reveals that the amino acid sequence of the NtClpR4 protein it encodes is shown in SEQ ID NO:2.

[0098] Example 2: Expression pattern of the NtClpR4 gene

[0099] The expression pattern of the NtClpR4 gene was analyzed by collecting different tissues and organs and using quantitative real-time PCR. The relevant experiments are briefly described below.

[0100] Roots, stems, leaves, and flowers of tobacco plants in the budding stage were collected as samples, flash-frozen in liquid nitrogen, and then stored in a -80°C freezer for later use.

[0101] RNA was extracted from the preserved material, and cDNA was synthesized using a reverse transcription kit (follow the kit instructions). Using the tobacco NtL25 gene as an internal control, quantitative real-time PCR was performed. The primer sequences for detection were designed as follows:

[0102] The primers for quantitative real-time detection of the NtClpR4 gene are as follows:

[0103] NtClpR4-qF:5'-acagacgagggtacaaaaatagtag-3' (SEQ ID NO:5);

[0104] NtClpR4-qR: 5'-gggacaatggccaaatagtta-3' (SEQ ID NO: 6);

[0105] The conditions for quantitative real-time PCR are as follows: Step 1: pre-denaturation, 95℃ for 30 s; Step 2: PCR reaction, 95℃ for 3 s, 57℃ for 20 s, 40 cycles; Step 3: melting curve.

[0106] Each sample was biologically replicated three times, using 2 -△△CT Methods were used to analyze relative differences in gene expression. The results are as follows: Figure 3 As shown, the NtClpR4 gene is expressed in roots, stems, leaves, and flowers, with the highest expression level in roots.

[0107] Example 3: Preparation of NtClpR4 mutant vector and expression vector plasmid

[0108] To further understand the role of the NtClpR4 gene in nicotine conversion, the inventors constructed an editing vector for the NtClpR4 gene. The construction process is briefly described below.

[0109] First, based on the design principles of CRISPR / Cas9 target sites, tcttggacagtgaccctaatggg (SEQ ID NO:7) was selected as the editing site for the NtClpR4 gene, with the PAM region being GGG. Four GATT bases were added to the 5' end of the forward primer, and four CAAA bases 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 system consisted of 50 μL of the editing vector (10 μL), BsaI enzyme (1 μL), CutSmart Buffer (10×) buffer (5 μL), and ddH2O (34 μL). Digestion was carried out at 37°C for 1 hour, and the digested plasmid was recovered using a product recovery kit.

[0110] The annealed double-stranded target site sequence was ligated to the enzyme-digested editing vector using T4 ligase. The ligation system was 20 μL: 10×T4 DNA Ligase Buffer, 2 μL; double-stranded target site, 5 μL; editing vector, 2 μL; T4 DNA Ligase, 1 μL; ddH2O up to 20 μL.

[0111] Ligation was performed at 25°C for 10 minutes, and the ligation product was transformed into DH5α competent cells to obtain single clones. Positive clones were detected using colony PCR with the detection primer (JC-F: 5'-ttaggtttacccgccaata-3', SEQ ID NO:8) and the target site reverse primer. Positive clones were then expanded, plasmids were extracted, and the cells were cryopreserved for Agrobacterium transformation.

[0112] Example 4: Construction of NtClpR4 gene mutant lines and overexpression transgenic lines

[0113] The gene-editing vector constructed in Example 3 was transformed into Agrobacterium and then into tobacco plants to construct NtClpR4 gene-edited transgenic plants. The specific experimental process is briefly described below.

[0114] (1) Transformation of Agrobacterium

[0115] Remove Agrobacterium competent cells from a -80°C freezer and freeze-thaw them on ice. Just before thawing, add 10 μL of the pORE-CRISPR / Cas9 editing vector plasmid prepared in Example 3 and gently tap to mix. Incubate on ice for 10 min, then in liquid nitrogen for 5 min, then at 37°C for 5 min without shaking the surface, and then immediately freeze for 5 min. Add 600 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking at 200 rpm for 3 h. Spread 200 μL of bacterial cells onto YEB solid medium containing 50 mg / L rifampicin, 50 mg / L streptomycin, and 50 mg / L kanamycin, and incubate in the dark at 28°C upside down for 2-3 days until single colonies form. Pick single colonies, expand them, and perform PCR identification. Correctly identified positive clones are the correctly transformed engineered bacteria.

[0116] (2) Identification of transformed tobacco plants and mutants

[0117] 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.

[0118] The transformed Agrobacterium engineered bacteria prepared above were cultured to OD200. 600 =0.6, centrifuge at 4000 rpm for 5 min to collect the bacterial cells, and then suspend the bacterial cells in 20 mL of MS liquid medium;

[0119] Then, the pre-cultured leaf discs were placed in the bacterial solution and incubated for 10 minutes.

[0120] 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).

[0121] 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.

[0122] 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.

[0123] 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:

[0124] A pair of detection primers were designed on the Cas9 sequence to detect transgenic lines, specifically:

[0125] Cas9-F: 5'-ctcaacacaacatatacaaaacaaa-3' (SEQ ID NO:9);

[0126] Cas9-F: 5'-ctttggccatctcgtttga-3' (SEQ ID NO: 10);

[0127] Using T0 generation transgenic line DNA template, PCR amplification was performed. The PCR conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; and a final extension at 72℃ for 10 min. Transgenic positive plants were identified.

[0128] A primer was designed across the sgRNA target site of the NtClpR4 gene to detect the mutation type at the target site, specifically:

[0129] NtClpR4-MF: caagtgaaagctttttaatgcaagt (SEQ ID NO: 11);

[0130] NtClpR4-MR: taattgctgaccttctccatttatc (SEQ ID NO: 12);

[0131] 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, 58℃ 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 at the NtClpR4 gene target site in the transgenic positive plants was identified, and the results are as follows: Figure 4 As shown, it inserts an A base at 892 bp in the nucleotide sequence shown in SEQ ID NO: 1, resulting in a frameshift mutation that leads to the loss of NtClpR4 function.

[0132] Example 5: Determination of changes in phenotype and alkaloid content of NtClpR4 mutant.

[0133] The homozygous mutant of the NtClpR4 gene obtained in Example 4 was planted, and the tobacco was topped and cured at the industrial maturity stage. The alkaloid content of the cured tobacco leaves was then tested. The specific experimental process is briefly described below.

[0134] (1) Planting of materials

[0135] Select representative plots with uniform soil fertility, flat ground, convenient irrigation and drainage, and medium to high fertility. Apply nitrogen at a rate of 9.5 kg / mu, with N:P2O5:K2O = 1:1:3 and base fertilizer:topdressing = 70:30. Plant K326 variety and NtClpR4 gene mutant, with 200 plants for each material. Cultivation measures should be implemented in accordance with the requirements of the flue-cured tobacco standardization technical system.

[0136] (2) Baking of materials

[0137] After topping, the industrially mature leaves are harvested and then cured using a three-stage curing process, specifically:

[0138] After ignition, the temperature increases by 1°C per hour until the dry bulb reaches 38°C and the wet bulb reaches 36°C, at which point the leaves turn yellow. When the tobacco leaves turn yellow with green veins and become wilted and soft, the temperature increases by 1°C every two to three hours until the dry bulb reaches 54°C and the wet bulb reaches 38°C to 40°C. When the leaves are completely yellow and dry, the temperature increases by 1°C per hour until the dry bulb reaches 65°C to 68°C and the wet bulb reaches 42°C to 43°C, at which point the leaves become dry with veins.

[0139] (3) Detection of alkaloids in flue-cured tobacco leaves

[0140] Accurately weigh 150 mg of tobacco powder into a 15 mL screw-top pressure-resistant test tube, add 1.75 mL of 5% sodium hydroxide solution to moisten the sample, let it stand for 15 min, add 10 mL of 0.01% triethylamine / methyl tert-butyl ether solution, seal the tube, and ultrasonically extract at room temperature for 15 min, then centrifuge at 6000 rpm for 5 min. Take 2 mL of the organic phase and analyze the nicotine mass fraction by GC-MS; accurately transfer 5 mL of the organic phase and concentrate it to 0.5 mL, then analyze other low-mass-fraction alkaloids by GC-MS. The detection of nicotine and other low-mass-fraction alkaloids was completed by two injections, using retention time and selected ion (SIM) mode for qualitative analysis and dual internal standard quantification. Analytical conditions:

[0141] Chromatographic column: DB‒35MS (30m×0.25mm id×0.25μm df); Temperature program: 100℃ (3 min), 8℃ / min 260℃ (10 min); Carrier gas: Helium; Column flow rate: 1.0 mL / min; Injector temperature: 250℃; Nicotine detection injection volume: 1µL, split injection; Split ratio: 40:1; Other alkaloid detection injection volume: 2µL, 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).

[0142] (4) Changes in mutant phenotype and alkaloids

[0143] Compared to the wild type, the NtClpR4 gene mutant produces tobacco leaves that are initially orange-red after curing. Figure 5 Alkaloid analysis revealed a decrease in nicotine content and an increase in nornicotine content in the mutant. Figure 6 ).

[0144] 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. Tobacco NtClpR4 protein, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

2. The nucleic acid encoding the tobacco NtClpR4 protein of claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

1.

4. The application of the tobacco NtClpR4 protein as described in claim 1 in reducing tobacco nicotine content and increasing tobacco demethylnicotine content; The reduction of nicotine content and the increase of demethylated nicotine content in tobacco are achieved by knocking out the nucleic acid in tobacco as described in claim 2 or 3 using gRNA. The nucleotide sequence of the gRNA is shown in SEQ ID NO:7.

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