Genes associated with tobacco quality, biological materials, and uses thereof

Abstract

This invention discloses genes, biomaterials, and their applications related to tobacco quality, belonging to the field of biotechnology. The amino acid sequence of the protein encoded by the aforementioned gene is SEQ ID NO:1. This invention utilizes CRISPR / Cas9 technology to target and mutate the NtGDU1 gene to obtain a mutant without T-DNA insertion, which improves the quality of freshly cured tobacco leaves, especially increasing the content of the aroma precursor compound Amadori, without affecting tobacco plant development.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to genes, biomaterials and their applications related to tobacco quality. Background Technology

[0002] With the rapid development of the slim cigarette market and the increasing diversification of consumer demands, higher requirements have been placed on the sensory quality of cigarette raw materials, especially aroma quality and aroma quantity. Therefore, improving the intrinsic quality of tobacco leaves has become a key focus and challenge in current tobacco production and breeding. The Maillard reaction is a crucial pathway for the formation of aroma compounds in tobacco leaves during curing and aging. In this reaction, Amadori compounds, as important intermediates, can produce various aroma-producing components such as furans, pyrroles, aldehydes, and ketones through their cleavage. These components collectively endow flue-cured tobacco with rich aroma characteristics and significantly improve its flavor and taste. Therefore, the content of Amadori compounds in virgin flue-cured tobacco leaves is one of the key indicators for measuring the aroma potential of tobacco leaves. Increasing its content has a clear promoting effect on the overall improvement of tobacco leaf quality, especially aroma quality.

[0003] Currently, targeted regulation of metabolic pathways for key aroma precursors in tobacco leaves through genetic modification is an important breeding strategy for improving tobacco quality. However, in-depth exploration and application of key genes that directly regulate the formation of Amadori compounds are still lacking. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide novel genes related to tobacco quality. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] Based on this, in order to discover functional genes that can be used for quality improvement, our project team comprehensively analyzed tobacco transcriptome, miRNA expression profiles, and the content data of 36 nitrogenous metabolites in previous studies, and constructed a weighted gene co-expression network. Through in-depth analysis of this network, we screened out co-expression modules that were significantly related to glutamine content, and further identified a key candidate gene, named NtGDU1.

[0007] This invention aims to elucidate the role of the NtGDU1 gene in regulating tobacco growth and quality formation, specifically exploring its application potential in increasing the content of Amadori compounds in tobacco leaves and thus improving aroma quality. The implementation of this invention will provide new genetic resources and important theoretical basis for molecular breeding of tobacco.

[0008] The present invention provides the use of a protein or a substance that downregulates, inhibits, or reduces the expression of the gene encoding the protein, or a substance that downregulates, inhibits, or reduces the activity and / or content of the protein, in any of the following: M1) Improves tobacco quality; M2) is used to prepare products that improve tobacco quality; M3) cultivates tobacco with improved quality; M4) is used to prepare tobacco products with improved quality. M5) Tobacco breeding; The protein is any one of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0009] The purpose of the breeding is to cultivate tobacco with improved quality.

[0010] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the protein isolated in the present invention, as long as they encode and possess the aforementioned protein function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0011] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting Gapexistencecost, Perresiduegapcost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0012] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0013] In the above applications, improving tobacco quality includes increasing aroma quality and / or aroma quantity, and reducing off-flavors and / or irritation. The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity by increasing the content of Amadori compounds in the tobacco.

[0014] Reducing sugars in tobacco leaves directly react with amino acids and amines via the Maillard reaction, producing various Maillard reaction intermediates and aroma compounds. These aroma compounds have relatively low thresholds, low irritation, and contribute significantly to the aroma of tobacco leaves. They can remove nitrogenous compounds from tobacco while imparting a unique aroma profile. Amadori compounds are relatively stable intermediates produced during the Maillard reaction and can further react to generate various aroma compounds, making them important aroma precursors.

[0015] In the above applications, the Amadori compounds include Fru-Tyr (fructose-tyrosine), Fru-Phe (fructose-phenylalanine), Fru-Asp (fructose-aspartic acid), Fru-Glu (fructose-glutamic acid), Fru-Asn (fructose-asparagine), Fru-Val (fructose-valine), Fru-Ala (fructose-alanine), Fru-Ile (fructose-isoleucine), Fru-Leu (fructose-leucine), and Fru-Thr (fructose-threonine).

[0016] In the above applications, the substance includes biological materials, which are any of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0017] The expression cassette described herein refers to a DNA molecule capable of expressing the proteins described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0018] In this article, the vector may be a plasmid, granule, bacteriophage, or viral vector.

[0019] In this article, the microorganisms mentioned may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0020] In this article, the transgenic plant cell lines mentioned do not include propagation material.

[0021] In the above application, the RNA molecule described in B1) targets the gene encoding the protein.

[0022] In the above application, the gene encoding the protein is SEQ ID NO:2, B1), and the RNA molecule targets nucleotides 226-248 of SEQ ID NO:2.

[0023] The present invention also provides a method for improving tobacco quality, the method being M1 or M2; wherein M1 is to knock out the coding gene of the aforementioned protein in the recipient tobacco to improve the tobacco quality of the target tobacco, and the recipient tobacco contains the coding gene of the aforementioned protein. M2 is used to inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein in the target tobacco, or to inhibit, reduce, or downregulate the activity and / or content of the protein, thereby improving the tobacco quality of the target tobacco, which contains the gene encoding the aforementioned protein.

[0024] The present invention also provides a method for preparing tobacco with improved quality, the method being M1 or M2; wherein M1 involves knocking out the coding gene of the aforementioned protein in the starting plant to improve the quality of the starting plant and obtain the target tobacco, the quality of which is higher than that of the starting tobacco, and the starting tobacco contains the coding gene of the aforementioned protein. M2 is used to inhibit, reduce, or downregulate the expression of the coding gene of the aforementioned protein in the starting plant or to regulate the activity and / or content of the protein to obtain the target tobacco. The plant height of the target tobacco is lower than that of the starting tobacco, and the starting tobacco contains the coding gene of the aforementioned protein.

[0025] In the above method, the substance includes biological materials, and the biological materials are any of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0026] In the above method, B1) the RNA molecule targets the gene encoding the protein.

[0027] In the above method, the gene encoding the protein is SEQ ID NO:2, B1), and the RNA molecule targets nucleotides 226-248 of SEQ ID NO:2.

[0028] In the above method, improving tobacco quality includes increasing aroma quality and / or aroma quantity, and reducing off-flavors and / or irritation. The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity by increasing the content of Amadori compounds in the tobacco.

[0029] In the above method, the Amadori compounds include Fru-Tyr, Fru-Phe, Fru-Asp, Fru-Glu, Fru-Asn, Fru-Val, Fru-Ala, Fru-Ile, Fru-Leu, and Fru-Thr.

[0030] The present invention also provides a biomaterial, said biomaterial being any of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) Transgenic plant organs containing the gene described in B2), or transgenic plant organs containing the expression cassette described in B3), or transgenic plant organs containing the recombinant vector described in B4); B9) A transgenic plant containing the gene described in B2), or a transgenic plant containing the expression cassette described in B3), or a transgenic plant containing the recombinant vector described in B4).

[0031] In some specific embodiments, the plant is a genetically modified plant.

[0032] "Transgenic plant" refers to a plant whose genome has been altered by integrating or inserting recombinant DNA molecules, constructs, cassettes, or sequences for the expression of non-coding RNA molecules, mRNA, and / or proteins. Transgenic plants include R0 generation plants developed or regenerated from initially transformed plant cells and their offspring, or plants obtained by crossing with R0 generation transgenic plants containing recombinant DNA molecules, constructs, cassettes, or sequences. Plants with integrated or inserted recombinant DNA molecules, constructs, cassettes, or sequences are considered transgenic plants, even if the plant also has other mutations or edits that are not themselves considered transgenic.

[0033] "Plant cell" is the biological cell of a plant, which is taken from the plant or derived from a culture obtained by culturing cells taken from the plant.

[0034] "Transgenic plant cell" refers to any plant cell transformed with a stably integrated recombinant DNA molecule, construct, cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0035] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0036] In a specific embodiment of the present invention, the difference between the plant described in B9) and wild tobacco is that a T is inserted between the nucleotides at positions 242 and 243 of the tobacco SEQ ID NO:3.

[0037] The expression cassette described herein refers to DNA capable of expressing the aforementioned nucleotide molecules in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all regulatory sequences necessary for expressing any of the aforementioned nucleic acid molecules. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned nucleic acid molecules in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes.

[0038] This invention utilizes CRISPR / Cas9 technology to target and mutate the NtGDU1 gene to obtain a mutant without T-DNA insertion, which improves the quality of freshly cured tobacco leaves, especially increasing the content of aroma precursor Amadori compounds, without affecting tobacco plant development. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the NtGDU1 gene structure and sgRNA target sites of the present invention.

[0040] Figure 2 The results of PCR amplification of T0 generation plants in this invention (amplification target: HPT); Lane M: TAKARADL2000 marker; Lanes 1-12: T0 generation plants numbered 2#, 11#, 15#, 19#, 25#, 26#, 32#, 35#, 39#, 42#, 43#, and 48#, respectively.

[0041] Figure 3 The results of PCR amplification of the NtGDU1 target gene from T1-T2 generation plants, which were identified by this invention, show a size of 471 bp. Sequencing results indicate that the target gene was 471 bp.

[0042] Figure 4 The results of PCR detection of whether T-DNA was knocked out in T1 generation plants (amplification target: HPT) are shown in this invention; lanes 1-24 are 24 T1 generation plants obtained by self-pollination of T0 generation plant numbered 25#.

[0043] Figure 5 This is a sequencing peak diagram of the NtGDU1 target gene in the T1 generation gene-edited plant of this invention.

[0044] Figure 6 This is the gene editing result of the T2 generation T-DNA knockout plant of the present invention; wherein: A is the DNA sequence alignment of the T2-25-1 gene edited in the T2 generation T-DNA knockout plant (T represents base insertion); B is the amino acid sequence alignment obtained by translating the DNA sequence of the plant in A.

[0045] Figure 7 The results of phenotypic observation of T3-25-1-3 plants with T-DNA knockout in the T3 generation of this invention are shown; where: AE is the phenotype of seedling tobacco plants; F is the phenotype of tobacco plants in full bloom.

[0046] Figure 8 The present invention uses T3 generation T-DNA knockout plants T3-25-1-3 seedlings and mature plant botanical traits; wherein: A is the agronomic traits and biomass of seedling tobacco plants; F is the agronomic traits of tobacco plants in full bloom.

[0047] Figure 9 Analysis of Amadori compound content in the primary flue-cured tobacco leaves of the T3-25-1-3 generation T-DNA knockout plant of this invention.

[0048] Figure 10 Sensory quality evaluation of the T3-25-1-3 primary flue-cured tobacco leaves from the T3 generation T-DNA knockout plant of this invention.

[0049] Figure 11 Map of the pHSbdcas9i-tRNA(k5) vector. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0053] The experimental results of the following examples are expressed as mean ± standard deviation. An independent samples t-test was used, and P < 0.05 was observed. () indicates a significant difference, P < 0.01. () indicates a highly significant difference.

[0054] Example 1 1. Construction of CRISPR / Cas9-NtGDU1 gene editing vector An sgRNA target site was designed based on the NtGDU1 gene (CDS region sequence is SEQ ID NO:2, encoded protein sequence is SEQ ID NO:1). The nucleotide sequence of the sgRNA target site is 5'-GATGATACTGAGGCCGGCGC-3', which corresponds to nucleotides 507 to 526 of SEQ ID NO:3 (targeting nucleotides 226-245 of SEQ ID NO:2). The sgRNA was constructed into the pHSbdcas9i-tRNA(k5) vector (see map 11, also known as "zmpl-GDU1") to obtain the CRISPR / Cas9-NtGDU1 plasmid. The CRISPR / Cas9-NtGDU1 plasmid is a recombinant vector obtained by replacing the small fragment between the tRNA and gRNA scaffold of the pHSbdcas9i-tRNA(k5) vector with a DNA fragment whose nucleotide sequence is the gRNA expression unit, while keeping the other nucleotides of the pHSbdcas9i-tRNA(k5) vector unchanged.

[0055] The nucleotide sequence of the DNA fragment of the gRNA expression unit is as follows: 5'-GATCTTTGAACTATCGCCGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGC AAAGATCGCCATGGCATTCTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGATGATACTGAGGCCGGCGC-3'.

[0056] 2. Transformation of tobacco using CRISPR / Cas9-NtGDU1 gene editing vector (1) Cultivation of aseptic tobacco seedlings Tobacco K326 seeds (provided by the Seed Breeding Center of Guizhou Provincial Tobacco Science Research Institute) were disinfected with 75% (v / v) alcohol for 30 seconds, soaked in 10% (v / v) H2O2 for 10 minutes with constant shaking, rinsed 5 times with sterile water, dried and placed in 1 / 2 MS medium, and cultured under light at 26-28℃. After two months, sterile seedlings were taken for genetic transformation.

[0057] (2) Transformation The constructed CRISPR / Cas9-NtGDU1 plasmid was transformed into Agrobacterium competent cells GV3101 for Agrobacterium-mediated genetic transformation of tobacco plants.

[0058] Leaflets of sterile tobacco K326 seedlings were cut into cubes approximately 0.8 cm × 0.8 cm and inoculated into co-culture medium (MS medium + 1.5 mg / L 6-BA; pH 5.8-5.9) for 2 days. Agrobacterium tumefaciens in the optimal logarithmic growth phase (OD600 value approximately 0.6) was collected by centrifugation, diluted with sterile water, and the leaf discs were immersed in the bacterial solution for 10 minutes. The leaves were then removed, and the bacterial solution on the leaf surface was blotted dry with sterile filter paper. The leaf discs were then transferred to co-culture medium and incubated in the dark at 25°C for 2 days. The leaf discs were washed three times with sterile water, blotted dry, and transferred to selection medium (MS medium + 1.5 mg / L 6-BA + 10 mg / L hygromycin + 160 mg / L termethin; pH 5.8-5.9) and incubated at 25°C for 16 days. Under h / d light conditions, the seedlings were screened and cultured, and the culture medium was changed every 15-20 days. The resistant shoots that grew to more than 1 cm were cut off at the base and transferred to the rooting medium (1 / 2 MS medium + 0.1 mg / L NAA + 10 mg / L hygromycin + 160 mg / L termethin; pH 5.8-5.9). After the seedlings rooted, they were hardened off and transplanted. The seedlings were the T0 generation plants.

[0059] 3. Identification of T0 generation edited plants (1) PCR verification of T-DNA insertion in T0 generation plants DNA was extracted from T0 generation plants. Primers were designed based on the HPT gene (hygromycin phosphotransferase gene) sequence to amplify the T0 generation plant DNA by PCR. The primer sequences were: HPT-F: 5'-TTGACATTGGGGAGTTTAGCG-3'; HPT-R: 5'-CAGGACATTGTTGGAGCCG-3'. The 20 μL PCR amplification reaction system consisted of: DNA 2 μL, HPT-F 1 μL, HPT-R 1 μL, 2×Mix enzyme 10 μL, and ddH2O 6 μL. The PCR amplification program was: 95℃ for 4 min; 95℃ for 30 s, 63℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min. If a specific band of 383 bp was amplified, it indicates that the T-DNA has been inserted into the tobacco genomic DNA. Figure 2 As shown.

[0060] (2) Sequencing identification of gene editing in T0 plants Primers were designed at both ends of the sgRNA sequence to amplify the approximately 470 bp target sequence of NtGUU1 containing the sgRNA by PCR. The primer sequences were: NtGDU1-F: 5'-CTTCCATCACCTAAAACAGTACC-3'; NtGDU1-R: 5'-CCTCTTTAATTCTCTCTCTCTCATTCCC-3'. The 31 μL PCR amplification reaction system consisted of: 2 μL DNA, 12 μL ddH2O, 1 μL F primer, 1 μL R primer, and 15 μL q Mix enzyme (2×Magic Green Taq SuperMix). The PCR amplification program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 38 cycles; 72℃ for 2 min, 14℃ for 1 min. The PCR product, matching the size of the target sequence band, was sent to a biotechnology company for sequencing. The PCR size was 471 bp, and the target band was as follows. Figure 2 As shown. The editing pattern of the T0 generation plants was determined by sequencing peak diagrams.

[0061] 4. Identification of T1 generation edited plants (1) Verification of T-DNA knockout and genotyping of T1 generation plants T0 generation edited plants were self-pollinated and seeded. PCR was used to determine whether the HPT gene had been removed from the T1 generation plants. The HPT gene primers, PCR reaction system, and conditions were as described in "Steps (1) of the Identification of T0 Generation Edited Plants in Case Study 3". After the PCR reaction, if no specific 383bp band was amplified, it indicated that the T-DNA had been removed. Taking the T0 generation plant numbered 25# as an example, its offspring T1 generation plants showed segregation. Figure 4 The ratio of negative to positive plants was close to 1:3, indicating that the T-DNA insertion in the T0 generation was a single copy insertion.

[0062] Using the primers, PCR reaction system, and conditions described in "Implementation Case 3, Identification of T0 Generation Edited Plants (2)", the target sequence of approximately 470 bp containing sgRNA, NtGUU1, was amplified. After the PCR reaction, the PCR product was sequenced, and the sequencing results are as follows: Figure 5 As shown, this indicates that the plant is a homozygous biallelic mutant form of "+1(T)".

[0063] (2) T-DNA knockout retesting and screening of homozygous mutants in T2 generation plants T1 generation plants with T-DNA knockout were self-pollinated and harvested. To prevent false negatives in the PCR results of T1 generation plants, the knockout status of T-DNA in the T2 generation plant population was re-tested using HPT and Cas9 genes as targets. HPT amplification followed the primers, PCR reaction system, and conditions in step (1) of "Identification of T0 generation edited plants". The primer sequences for Cas9 gene amplification were Cas9-F: 5'-GCCTACCACGAGAAGTACCC-3'; Cas9-R: 5'-GGCTTGATGAACTTGTAGAACTC-3'. The 20μL PCR amplification reaction system consisted of: 2μL plant DNA, 1μL F primer, 1μL R primer, 10μL 2×Mix, and 6μL ddH2O. The PCR amplification reaction program was: 95℃ for 4min; 95℃ for 30s, 63℃ for 30s, 72℃ for 1min, 35 cycles; 72℃ for 1min. 5 min; PCR amplification product size was 755 bp.

[0064] Continue screening from the T2 generation until plants with homozygous biallelic mutations in the NtGDU1 gene and T-DNA deletion are selected (the HPT and Cas9 genes in the T2 generation plants cannot amplify specific bands of 383bp and 755bp, respectively, indicating that the HPT and Cas9 genes have been deleted, that is, the T-DNA fragment has been deleted from the plant).

[0065] Example 2 Single plants with T-DNA knockout and biallelic homozygous mutations were selected from the T2 generation plants of Example 1, such as... Figure 6 The mutation form is "+1(T)" and the number is T2-25-1.

[0066] Compared to the wild-type plant K326, the T2-25-1 plant exhibited the following change on both homologous chromosomes: an insertion of a T (corresponding to) between nucleotides 242 and 243 of SEQ ID NO:3. Figure 5 The "A" in the red box is marked as correct. Figure 5 The sequence described in the image is the reverse complementary sequence of SEQ ID NO:3, which causes a frameshift mutation in the encoded amino acid, resulting in the loss of function of the NtGDU1 gene.

[0067] 1. Phenotypic observation, agronomic traits and biomass measurement of T3 generation plants. The T3 generation (T3-25-1-3) of the T2-25-1 self-pollination progeny was used as experimental material, with the K326 variety as the control (WT). After sowing, the plants were cultured in an incubator (28℃, 65% humidity, 16 h light / 8 h dark, light intensity 20000 lux) and also grown in a greenhouse using pot experiments. Phenotypic development at seedling and maturity stages was observed. Figure 7 The agronomic traits of tobacco plants were measured in accordance with YCT 142-2010, the method for surveying and measuring tobacco agronomic traits. Figure 8 It was found that the NtGDU1 gene mutation had no significant effect on the growth of tobacco plants.

[0068] 2. Analysis of the content of multiple amino acid Maillard products Amadori compounds and sensory quality evaluation in the primary flue-cured tobacco leaves of the T3 generation strain.

[0069] The T3 generation plant population (T3-25-1-3) of the T2-25-1 self-pollination progeny was used as experimental material, and the K326 variety was used as the control (WT). The plants were planted under conventional field management conditions. After the tobacco leaves matured, they were cured using conventional intensive curing methods. Then, according to the national standard GB2635-92 "Flue-cured Tobacco", samples of C3F grade flue-cured tobacco leaves were selected, ground into powder, and passed through a 60-mesh sieve for the determination of Maillard product Amadori compounds and sensory quality evaluation.

[0070] Determination of Maillard product Amadori compounds: Standard products: Fru-Phe (purchased from TRC, USA, part number F792560), Fru-Ala (purchased from TRC, USA, part number F792520), Fru-Leu (purchased from TRC, USA, part number F792540), Fru-Pro (purchased from TRC, USA, part number F792565), Fru-Trp (purchased from TRC, USA, part number F792580), Fru-Val (purchased from TRC, USA, part number...). The following 10 Amadori compounds were used for quantification: Fru-Ile (purchased from TRC, USA, catalog number F792585), Fru-Arg (purchased from TRC, USA, catalog number F795655), Fru-Asn (purchased from TRC, USA, catalog number F792527), and Fru-Glu (purchased from TRC, USA, catalog number F795662). For compounds with available standards, external standard quantification was used; for compounds without available standards, internal standard quantification was used.

[0071] Sample pretreatment: Weigh 1g of tobacco powder, add 30mL of 30:70 (v / v) methanol / water solution, ultrasonically extract for 25min, centrifuge at 10000rpm for 5min, filter the supernatant through a 0.45μm organic filter membrane, take 50μL into a 10mL volumetric flask, dilute to volume with methanol, and take 1mL into a chromatographic bottle for LC-MS / MS analysis.

[0072] Chromatographic conditions: Waters Atlantis T3 column (4.6 × 250 mm, 5 μm); mobile phase: 0.2% formic acid aqueous solution (phase A) and acetonitrile (phase B); gradient elution program: 0–0.1 min, 89.5% A–78% A; 0.1–0 min, 78% A; flow rate: 600 μL / min; injection volume: 5 μL; equilibration with the initial mobile phase for 8 min between every two samples.

[0073] Mass spectrometry conditions: Electrospray ionization (ESI), multiple reaction monitoring positive ion mode (MRM); spray voltage: 5500V, ion source temperature: 550℃, curtain gas: 20psi, nebulizing gas: 60psi, drying gas: 75psi, collision gas: 9psi.

[0074] Analysis of Amadori compounds showed that the contents of various amino acid Maillard products, Amadori compounds, in the primary flue-cured tobacco leaves of the T3-25-1-3 strain were significantly increased compared with the control WT. Figure 9 (n=3), among which the Amadori compounds of aromatic amino acids (Fru-Tyr, Fru-Phe), Amadori compounds of acidic amino acids (Fru-Asp, Fru-Glu), Amadori compounds of amide amino acids (Fru-Asn), Amadori compounds of aliphatic amino acids (Fru-Val, Fru-Ala, Fru-Ile, Fru-Leu), and Amadori compounds of hydroxyl amino acids (Fru-Thr) were all significantly or substantially higher than the control WT, with increases ranging from 10.34% to 38.70%. This indicates that mutation of the NtGDU1 gene can increase the content of Amadori compounds, Maillard products of various amino acids.

[0075] Sensory quality of tobacco leaves refers to the smoking characteristics of the smoke produced by the combustion of tobacco leaves. It is the foundation and core indicator of the usability of tobacco leaves in the tobacco industry, accounting for up to 66% of the overall quality evaluation of tobacco leaves. Aroma quality and aroma quantity each account for up to 30% of the sensory quality evaluation, and aroma (aroma quality and aroma quantity) accounts for up to 60% of the total score. To further verify the impact of NtGDU1 gene mutation on the sensory quality of tobacco leaves, especially on aroma, seven professional smoke testers were organized to conduct sensory quality evaluation using a 9-point system according to YC / T 138-1998. Five quality evaluation indicators (aroma quality, aroma quantity, off-flavors, irritation, and aftertaste) were mainly evaluated, and the total score for the five quality indicators is the sum of the scores for the five quality evaluation indicators. Sensory quality evaluation results showed that the aroma quality, aroma quantity, off-odor score, and total score of the five quality items of the T3-25-1-3 strain of flue-cured tobacco leaves were significantly higher than those of the control K326, increasing by 7.22%, 5.42%, 8.30%, 5.00%, 7.22%, and 6.61%, respectively. Figure 10 The most prominent effect on aroma is a more delicate and mellow fragrance, a fuller and longer-lasting aroma, and a longer-lasting aftertaste. The evaluation results further verify that the NtGDU1 gene mutation significantly improves the sensory quality of tobacco leaves.

[0076] SEQ ID NO.1: MRTGSGFSTTSSPTIKATFSPPAIVQRSPWHSPVPYLFGGLAAMLGLIAFALLILACSYWKLSSHLREHDDNQSQDDTEAGAGEKPATGAVKAMPEFEEKIVVIMAGDLNPSFLATPMLMLSKVGTVLGDGSNLNKCEENLEKGSDEKEKLEQVVIEIREESNGDSQHSHGQNQ.

[0077] SEQ ID NO:2 Original NtGDU1 (>Ntab0546940.1) CDS sequence: 5'-ATGAGAACAGGAAGTGGTTTTTCTACAACTTCATCACCAACGATAAAAGCTACATTTTCACCGCCGGCGATAGTTCAAAGATCGCCATGGCATTCTCCGGTGCCGTATCTCTTCGGCGGATTAGCAGCCATGTTAGGATTAATAGCTTTTGCTCTATTAATATTAGCTTGTTCTTACTGGAAACTCTCTTCCCATTTAAGAGAACATGACGATAATCAGTCACAAGATGATACTGAGGCCGGCGCCGGTGAAAAACCGGCGACCGGAGCTGTGAAGGCGATGCCGGAGTTTGAGGAGAAGATTGTTGTTATAATGGCTGGAGATTTGAACCCAAGTTTTTTGGCTACGCCTATGTTAATGTTAAGTAAAGTTGGTACTGTTTTAGGTGATGGAAGTAATTTGAATAAATGTGAGGAAAATTTGGAAAAGGGAAGTGATGAGAAGGAGAAATTAGAACAAGTGGTGATTGAGATTAGAGAAGAGAGTAATGGAGATTCTCAACATAGCCATGGACAAAACCAGTGA-3'.

[0078] DNA sequence of original NtGDU1 (>Ntab0546940) of SEQ ID NO:3 (nucleotides 282-806 are identical to nucleotides 226-245 of SEQ ID NO:2):

[0079] The nucleotide sequence of the pHSbdcas9i-tRNA(k5) plasmid:

[0080] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance that downregulates, inhibits, or reduces the expression of the gene encoding the protein, or a substance that downregulates, inhibits, or reduces the activity and / or content of the protein, in any of the following: M1) Improves tobacco quality; M2) is used to prepare products that improve tobacco quality; M3) cultivates tobacco with improved quality; M4) is used to prepare tobacco products with improved quality. M5) Tobacco breeding; The protein is any one of the following: A1) The amino acid sequence of this protein is SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

2. The application according to claim 1, characterized in that, The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity, and reducing off-flavors and / or irritation. The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity by increasing the content of Amadori compounds in the tobacco.

3. The application according to claim 2, characterized in that, The Amadori compounds include Fru-Tyr, Fru-Phe, Fru-Asp, Fru-Glu, Fru-Asn, Fru-Val, Fru-Ala, Fru-Ile, Fru-Leu, and Fru-Thr.

4. The application according to any one of claims 1-3, wherein the substance comprises a biomaterial, and the biomaterial is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or RNA molecules that knock out the gene encoding the protein of claim 1. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

5. The application according to any one of claims 1-4, characterized in that, B1) The RNA molecule targets the gene encoding the protein.

6. The application according to any one of claims 1-5, characterized in that, The gene encoding the protein is SEQ ID NO:2, B1). The RNA molecule targets nucleotides 226-248 of SEQ ID NO:

2.

7. A method for improving tobacco quality, wherein the method is M1 or M2; wherein M1 is to knock out the gene encoding the protein of claim 1 in the recipient tobacco to improve the tobacco quality of the target tobacco, and the recipient tobacco contains the gene encoding the protein of claim 1; M2 is used to inhibit, reduce, or downregulate the expression of the gene encoding the protein described in claim 1 in the target tobacco, or to inhibit, reduce, or downregulate the activity and / or content of the protein, thereby improving the tobacco quality of the target tobacco, wherein the target tobacco contains the gene encoding the protein described in claim 1.

8. A method for preparing tobacco with improved quality, wherein the method is M1 or M2; wherein M1 is to knock out the gene encoding the protein of claim 1 in the starting plant to improve the quality of the starting plant and obtain the target tobacco, wherein the quality of the target tobacco is higher than that of the starting tobacco, and the starting tobacco contains the gene encoding the protein of claim 1. M2 is used to inhibit, reduce, or downregulate the expression of the gene encoding the protein described in claim 1 in the starting plant, or to regulate the activity and / or content of the protein, to obtain the target tobacco. The plant height of the target tobacco is lower than that of the starting tobacco, and the starting tobacco contains the gene encoding the protein described in claim 1.

9. The method according to claim 7 or 8, characterized in that, The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity, and reducing off-flavors and / or irritation. The improvement of tobacco quality includes increasing aroma quality and / or aroma quantity by increasing the content of Amadori compounds in the tobacco.

10. A biomaterial, characterized in that, The biomaterial is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or RNA molecules that knock out the gene encoding the protein of claim 1. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) Transgenic plant organs containing the gene described in B2), or transgenic plant organs containing the expression cassette described in B3), or transgenic plant organs containing the recombinant vector described in B4); B9) A transgenic plant containing the gene described in B2), or a transgenic plant containing the expression cassette described in B3), or a transgenic plant containing the recombinant vector described in B4).

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