Tobacco NtClpR4 gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco NtClpR4 gene and application. The nicotine conversion related gene NtClpR4 is cloned from tobacco for the first time, the nicotine conversion related gene NtClpR4 encodes ATP-dependent tyrosine protease Clp related subunits, the amino acid sequence of the nicotine conversion related gene NtClpR4 is shown as SEQ ID NO: 2, and the nucleotide sequence of the nicotine conversion related gene NtClpR4 is shown as SEQ ID NO: 1; the gene product affects synthesis of nornicotine. After the NtClpR4 gene is mutated, the nicotine content of flue-cured tobacco leaves is reduced, the demethylated nicotine content is increased, and a new technical means is provided for nicotine conversion regulation and tobacco breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a tobacco NtClpR4 gene and application thereof. Background Art

[0002] Tobacco contains four main alkaloids, namely nicotine, demethylnicotine (nornicotine), anabasine and anabasine. Nicotine is the most important alkaloid in tobacco, accounting for 90% to 95% of the total alkaloid content of cultivated tobacco. When insects or herbivores infringe tobacco leaves, nicotine synthesis and metabolism mediated by jasmonic acid injury-induced signals will immediately start in the roots, among which the root tip cortex and epidermal cells are the main sites for nicotine synthesis. The synthesized nicotine is transported to the tobacco leaves through vascular tissues and stored in cell vacuoles to defend against various invasions. Nicotine synthesis and metabolism are regulated by many factors, such as insect biting, jasmonic acid, abscisic acid, ethylene, and auxin.

[0003] Nornicotine is converted from nicotine. Nornicotine is relatively unstable. During the process of tobacco leaf curing and aging, biochemical reactions are prone to produce nornicotine derivatives, including oxidation to form mesamine, acylated nornicotine and N-nitroso nornicotine (NNN). Mesamine and acylated nornicotine can directly affect the chemical composition of smoke and are unfavorable factors for the quality of tobacco leaves. NNN is an important tobacco-specific nitrosamine (TSNA) with carcinogenic effects. Therefore, the increase in nornicotine content caused by nicotine conversion has an important impact on the quality and safety of tobacco leaves. It has always been the goal of tobacco breeding to explore genes related to regulating nicotine conversion and reduce the content of demethylnicotine. Summary of the invention

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

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

[0006] The present invention clones a nicotine conversion-related gene NtClpR4 from tobacco for the first time, which encodes an ATP-dependent tyrosine protease Clp-related subunit, and its amino acid sequence is shown in SEQ ID NO: 2; the gene product affects the synthesis of nornicotine. After the NtClpR4 gene is mutated, the nicotine content of the flue-cured tobacco leaves decreases, and the nornicotine content increases.

[0007] The present 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] The present invention provides a biomaterial comprising at least one of the following A) to G):

[0010] A), an expression unit containing the nucleic acid according to the present invention;

[0011] B), the interference fragment targeting the tobacco NtClpR4 of the present invention;

[0012] C), knockout and / or mutation of the tobacco NtClpR4 gRNA fragment of claim 1;

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

[0014] E), transforming or transfecting the host cell with the recombinant vector as described in D);

[0015] F), culturing the mixture obtained by the host cell described in E);

[0016] G), amplification primers and / or detection primers for the nucleic acid according to claim 2 or 3.

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

[0018] In the present invention, the nucleic acid may be DNA, RNA, cDNA or PNA. In an embodiment of the present 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 may be single-stranded or double-stranded. Nucleic acid may 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 acid may be linear or circular in topology. Nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques.

[0019] In the present invention, the nucleic acid may be optimized or unoptimized, and the present invention is not limited thereto. The optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites 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 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.

[0021] The present invention provides a gRNA fragment, which is a fragment targeting a target gene and located about 20 bp before the recognition site (PAM site) of the CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a and CRISPR / Cas14a) editing system; the recognition site of the CRISPR / endonuclease system editing system is different according to the different endonucleases. Generally, the recognition site includes NGG (Cas9) and / or TTN (Cpf1), etc.; In a specific embodiment of the present invention, the CRISPR / Cas9 system is used, and its recognition site is NGG, wherein N represents any one of A, T, C or G base species, and the position of the NGG can be at any position of the 5' end, the middle or the 3' end of the nucleic acid encoding tobacco NtClpR4, and the present invention is not limited to this; In the present invention, tobacco NtClpR4 is used as a target, and the CRISPR / Cas9 system is used for gene editing, and specifically, the nucleotide sequence of the gRNA fragment is shown in SEQ ID NO:5;

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

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

[0024] Furthermore, the vector backbone of the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, which are not limited in the present invention. The viral vector may include tobacco mosaic virus, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, or lentivirus vector.

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

[0026] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression or function (such as interference or knockout) of an operably connected coding gene in a specific host organism. The nucleic acid sequence or element necessary for expression in eukaryotic cells includes a promoter, a ribosome binding site and possible other sequences. It is known that eukaryotic cells 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" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which 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 inserts.

[0027] The transformation methods include chemical transformation and electrotransformation; 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, Agrobacterium-mediated transfection, etc.

[0028] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited in the present invention. The present invention uses vectors constructed by recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins or perform specific functions, such as interfering with or knocking out specific target nucleic acids or proteins.

[0029] The present invention provides the use of any one of the following I) to III) in regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives;

[0030] 1), the protein of the present invention;

[0031] II), the nucleic acid of the present invention;

[0032] III), the biomaterial described in the present invention.

[0033] Furthermore,

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

[0035] The plants include plants of the family Cruciferae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Leguminosae and / or Solanaceae; the Solanaceae plants include tobacco;

[0036] The demethylnicotine derivatives include mesamine, acylated nornicotine and / or N-nitrosonornicotine (NNN), etc., which are not limited in the present invention.

[0037] The present invention provides a product for regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives, which comprises auxiliary materials and the biological material of the present invention.

[0038] Furthermore, the auxiliary materials include at least one or more of buffer, antibiotics, antioxidants, stabilizers, auxiliary plasmids, viruses and / or culture media, etc., which are not limited in the present invention. The auxiliary materials are used to preserve the biological material and / or to assist the biological material in exerting its function.

[0039] The present invention utilizes overexpression and CRISPR / Cas9 technology to construct and obtain NtClpR4 gene overexpression and function loss mutants, and test results show that the NtClpR4 gene regulates the nicotine and nornicotine (nornicotine) contents in plants, and overexpression of the NtClpR4 gene reduces the nornicotine content in plants; reduction or knockout of the NtClpR4 gene reduces the nicotine content in plants, and increases the nornicotine (nornicotine) content and the mysamine content.

[0040] The present invention provides a method for regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives, which comprises using the biological material and / or the product of the present invention.

[0041] The present invention provides the use of at least one of the following a) to c) in plant breeding:

[0042] a), the tobacco NtClpR4 of the present invention;

[0043] b), the nucleic acid of the present invention;

[0044] c) the biomaterial of the present invention;

[0045] d) the product of the present invention;

[0046] e), the method described in the present invention.

[0047] Furthermore, the plants include plants of the family Cruciferae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Leguminosae and / or Solanaceae; and the Solanaceae plants include tobacco.

[0048] The breeding includes regulation to improve salt quality and / or safety.

[0049] The present invention clones a nicotine conversion-related gene NtClpR4 from tobacco for the first time, which encodes an ATP-dependent tyrosine protease Clp-related subunit, and its amino acid sequence is shown in SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:1; the gene product affects the synthesis of nornicotine. After the NtClpR4 gene is mutated, the nicotine content of the flue-cured tobacco leaves decreases, and the nornicotine content increases, providing a new technical means for nicotine conversion regulation and tobacco breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shows the map of pORE-CRISPR / Cas9 vector;

[0051] Figure 2 Shows the PCR electrophoresis of NtClpR4 gene;

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

[0053] Figure 4 Shows the analysis of mutation types in NtClpR4 gene mutant plants;

[0054] Figure 5 The phenotype of tobacco leaves after flue-curing in NtClpR4 gene mutant plants is shown;

[0055] Figure 6 Shows the detection of alkaloids in tobacco leaves of NtClpR4 gene mutant plants after curing. DETAILED DESCRIPTION

[0056] The present invention provides tobacco NtClpR4 gene and application, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out 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.

[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 as follows.

[0060] The application of the nicotine conversion regulatory gene NtClpR4 in tobacco inhibits the conversion of nicotine to nornicotine; after the NtClpR4 gene is mutated, the nicotine content of tobacco leaves of the mutant plants after baking decreases, while the contents of nornicotine, anabasine and mesamine increase; the tobacco nicotine conversion regulatory gene NtClpR4 has important application significance for improving the quality and safety of tobacco leaves.

[0061] The mutation vector for the tobacco alkali conversion regulatory gene NtClpR4 is obtained by designing the CRISPR / Cas9 gene mutation target site sequence based on the cloned NtClpR4 gene coding sequence; specifically, the sgRNA (gRNA) target site sequence is annealed and connected to the pORE-CRISPR / Cas9 editing vector (vector map as shown in Figure 1 As shown in the figure, a gene editing vector for mutating the tobacco nicotine conversion regulatory gene NtClpR4 was obtained through transformation, screening and identification.

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

[0063] 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:

[0064] Example 1 Cloning of NtClpR4 gene

[0065] 1. 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 China Tobacco Industry Co., Ltd.

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

[0069] Competent cells: Trans1-T1 competent cells; DH5a chemical competent cells;

[0070] Strain: LBA4404 Agrobacterium strain;

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

[0072] 2. Experimental reagents:

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

[0074] Fluorescence quantitative PCR enzyme (TB Green® Fast qPCR Mix) and reverse transcription kit were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

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

[0076] DNA amplification enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

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

[0078] 3. Experimental equipment:

[0079] Fluorescence quantitative PCR instrument QuantStudio 3, Thermo Fisher;

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

[0081] GC-MS gas chromatography-mass spectrometry, Agilent.

[0082] 2. Cloning of NtClpR4 gene

[0083] The cloning process of the NtClpR4 gene is briefly introduced as follows.

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

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

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

[0091] Purify the PCR amplification product according to the instructions of the gel recovery kit, and then connect the purified product to the pEASY-BluntZero cloning vector. The connection system is as follows: DNA amplification product, 6 μL; pEASY-Blunt Zero vector, 1 μL; after mixing, connect at 25°C for 25 min.

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

[0093] Take out the competent cells from the -80℃ refrigerator, place them on ice to dissolve, add the ligation product to 50μL of Trans1-T1 competent cells, flick to mix, and place on ice for 30 min;

[0094] Heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes; add 250 μL of LB (without antibiotics) equilibrated to room temperature, and culture at 37°C with shaking for 1 hour;

[0095] Take 8 μL of the mixture and spread it evenly on a LB solid plate (containing 60 μg / μL ampicillin), invert the culture dish, and culture at 37°C overnight.

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

[0097] Sequencing analysis results showed that the length of the coding region of the NtClpR4 gene was 1548 bp nucleotides (e.g. Figure 2 As shown in SEQ ID NO: 1, wherein M is a marker; after analyzing the gene, it is known that the amino acid sequence of the NtClpR4 protein encoded by it is shown in SEQ ID NO: 2.

[0098] Example 2 Expression pattern of NtClpR4 gene

[0099] By collecting different tissues and organs, the expression pattern of NtClpR4 gene was analyzed by fluorescence quantitative PCR. The relevant experiments are briefly introduced as follows.

[0100] The roots, stems, leaves and flowers of tobacco in the budding stage were collected as samples, quickly frozen with liquid nitrogen and placed in a -80°C refrigerator for future use.

[0101] Extract RNA from the stored materials, synthesize cDNA using a reverse transcription kit (refer to the kit instructions for operation), and perform fluorescent quantitative PCR detection using the tobacco NtL25 gene as an internal reference. During the detection, the primer sequences are designed as follows:

[0102] Fluorescence quantitative primers for detecting NtClpR4 gene, the primer sequences are:

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

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

[0105] The conditions of fluorescence quantitative PCR were as follows: the first step was pre-denaturation at 95°C for 30 s; the second step was PCR reaction at 95°C for 3 s, 57°C for 20 s, and 40 cycles; the third step was melting curve.

[0106] Each sample was biologically replicated three times. -△△CT The relative gene expression differences were analyzed by the method. 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 mutation vector and expression vector plasmid

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

[0109] First, according to the design principle of CRISPR / Cas9 target site, tcttggacagtgaccctaatggg (SEQID NO:7) was selected as the editing site of NtClpR4 gene, the PAM region was GGG, 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 primer. The single-stranded Oligo DNA of the target site was annealed to form double-stranded DNA, 10 μL of upstream and downstream primers were mixed, placed in a PCR instrument and incubated at 95℃ for 3 minutes, and then naturally cooled to room temperature after incubation. The pORE-CRISPR / Cas9 editing vector was digested with BsaI, and the reaction system was 50 μL, 10 μL of editing vector, 1 μL of BsaI enzyme, 5 μL of CutSmartBuffer (10×) buffer, 34 μL of ddH2O, and digested at 37℃ for 1 hour. The digested plasmid was recovered by the product recovery kit.

[0110] The annealed double-stranded target site sequence and the edited vector after restriction digestion were connected under the action of T4 ligase. The system was 20μL: 10×T4 DNA Ligase Buffer, 2μL; double-stranded target site, 5μL; edited vector, 2μL; T4 DNA Ligase, 1μL; ddH2O up to 20μL;

[0111] The ligation was carried out at 25°C for 10 minutes, and the ligation product was transformed into DH5a competent cells and cultured to obtain a single clone. The detection primer (JC-F: 5'-ttaggtttacccgccaata-3', SEQ ID NO: 8) and the target site reverse primer were used as amplification primers, and the positive clones were detected by colony PCR. The positive clones were expanded and cultured, and the plasmids were extracted and stored at low temperature for Agrobacterium transformation.

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

[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 as follows.

[0114] (1) Transformation of Agrobacterium

[0115] Take out the Agrobacterium competent cells from the -80℃ refrigerator, freeze and thaw on ice, and add 10μL of the pORE-CRISPR / Cas9 editing vector plasmid prepared in Example 3 when it is about to thaw, and flick to mix; ice bath for 10min, liquid nitrogen for 5min, 37℃ for 5min, do not shake the water surface, and then quickly put on ice for 5min. Add 600μl of antibiotic-free LB liquid culture medium, 28℃, 200r / min shaking culture for 3h. Apply 200μL of bacteria to YEB solid culture medium containing 50mg / L rifampicin, 50mg / L streptomycin and 50mg / L kanamycin, invert and culture in the dark at 28℃ for 2~3 days until a single colony is formed; pick a single colony, expand the culture, and perform PCR identification on the bacterial solution to identify the correct positive clone strain, which is the correct engineered bacteria for transformation.

[0116] (2) Transformation of tobacco plants and identification of mutants

[0117] Take the leaves of the sterile tobacco seedlings that have grown for about one month, use a hole puncher to process the leaves into leaf disks with a diameter of 0.5 cm, and pre-culture the processed leaf disks on MS solid culture medium for 3 days;

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

[0119] Then the pre-cultured leaf discs were placed in the bacterial solution and infected for 10 minutes;

[0120] Use sterile filter paper to absorb the excess bacterial liquid around the leaf disc after infection, and culture it in the dark on a solid medium of MS+6-BA (2 mg / L)+NAA (0.5 mg / L) for 3 days;

[0121] The leaf disc was washed with sterile water containing Cef (400 mg / L), and the excess liquid was removed with sterile filter paper. The leaf disc was transferred to MS solid screening medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L), and Kan (50 mg / L) and cultured at 28°C under light.

[0122] 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.

[0123] After about one month of growth, take a small amount of leaves and extract DNA according to the instructions of the plant genome extraction kit. Detect positive transgenic strains and mutation sites through PCR amplification, cloning, and sequencing. The specific identification method is:

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

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

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

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

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

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

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

[0131] The positive transgenic strain DNA was used as a template for PCR amplification. The PCR conditions were as follows: 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; and 72℃ final extension for 10 min. The mutation type of the NtClpR4 gene target site in the transgenic positive plants was identified. The results are shown in Figure 2. Figure 4 As shown, a base A is inserted at 892 bp of the nucleotide sequence shown in SEQ ID NO: 1, resulting in a frameshift mutation, leading to the loss of function of NtClpR4.

[0132] Example 5 Determination of the phenotype and alkaloid content changes of NtClpR4 mutants

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

[0134] (1) Planting of materials

[0135] Representative plots with uniform soil fertility, flat ground, convenient irrigation and drainage, and medium to high fertility were selected. The nitrogen application rate was 9.5 kg / mu, N: P2O5: K2O = 1:1:3, base fertilizer: topdressing = 70:30, and K326 varieties and NtClpR4 gene mutants were planted. 200 plants of each material were planted, and the cultivation measures were carried out in accordance with the requirements of the standardized technical system for flue-cured tobacco.

[0136] (2) Baking of materials

[0137] After topping, the leaves at industrial maturity are harvested and then cured using a three-stage flue-cured tobacco curing process, specifically:

[0138] After ignition, the temperature rises by 1℃ per hour to 38℃ dry bulb, 36℃ wet bulb and turns yellow. When the tobacco leaves turn yellow, wither and become soft, the temperature rises by 1℃ every two or three hours to 54℃ dry bulb, 38℃~40℃ wet bulb and fixed color. When the leaves are completely yellow and dry, the temperature rises by 1℃ per hour to 65℃~68℃ dry bulb, 42℃~43℃ wet bulb and dry texture.

[0139] (3) Alkaloid detection in flue-cured tobacco leaves

[0140] Accurately weigh 150 mg of tobacco leaf powder in a 15 mL screw-capped pressure test tube, add 1.75 mL of 5% sodium hydroxide solution, wet the sample, let it stand for 15 minutes, add 10 mL of 0.01% triethylamine / methyl tert-butyl ether solution, seal it with a cap, and perform ultrasonic extraction at room temperature for 15 minutes, then centrifuge it at 6000 rpm for 5 minutes. Take 2 mL of organic phase and analyze the nicotine mass fraction by GC-MS; accurately transfer 5 mL of organic phase and concentrate it to 0.5 mL, and analyze other low-mass fraction alkaloids by GC-MS. The detection of nicotine and other low-mass fraction alkaloids was completed in two injections, using retention time and selected ion (SIM) mode for qualitative analysis and double internal standard for quantitative analysis. Analysis conditions:

[0141] Chromatographic column: DB‒35MS (30m×0.25mm id×0.25μm df); Program temperature: 100℃ (3 min), 8℃ / min 260℃ (10min); Carrier gas: Helium; Column flow rate: 1.0mL / min; Injection port 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: 8min; Ionization voltage: 70eV; Ion source temperature: 230℃; Transfer line temperature: 280℃; Scan mode: Selected ion mode (SIM).

[0142] (4) Mutant phenotype and alkaloid changes

[0143] Compared with the wild type, the NtClpR4 mutant tobacco leaves were orange-red after curing ( Figure 5 ), alkaloid detection found that the nicotine content in the mutant decreased, and the content of nornicotine (nornicotine) increased ( Figure 6 ).

[0144] 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. Tobacco NtClpR4, characterized in that It has an amino acid sequence as shown in SEQ ID NO:

2.

2. Nucleic acid encoding tobacco NtClpR4.

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

1.

4. Biomaterial, characterized in that Including at least one of the following A) to G): A), an expression unit containing the nucleic acid according to claim 2 or 3; B), an interference fragment targeting the tobacco NtClpR4 of claim 1; C), knockout and / or mutation of the tobacco NtClpR4 gRNA fragment of claim 1; D), a recombinant vector containing the expression unit as described in A), the interference fragment as described in B), and / or the gRNA fragment as described in C); E), transforming or transfecting the host cell with the recombinant vector as described in D); F), culturing the mixture obtained by the host cell described in E); G), amplification primers and / or detection primers for the nucleic acid according to claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that The gRNA fragment is shown in SEQ ID NO:

5.

6. Use of any one of I) to III) below in regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives; I), the protein according to claim 1; II), the nucleic acid according to claim 2 or 3; III), the biomaterial according to claim 4 or 5.

7. The use according to claim 6, characterized in that: The regulation includes increasing and / or decreasing.

8. A product for regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives, characterized in that: The method comprises auxiliary materials and the biological material according to claim 4 or 5.

9. A method for regulating the content of plant nicotine, nornicotine and / or nornicotine derivatives, characterized in that: Comprising the use of the biomaterial according to claim 4 or 5 and / or the product according to claim 7.

10. Application of at least one of the following a) to e) in plant breeding: a), the tobacco NtClpR4 according to claim 1; b), the nucleic acid according to claim 2 or 3; c) The biological material according to claim 4 or 5; d) The product according to claim 7; e) The method according to claim 8.

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