Method for regulating content of tobacco aroma precursor and application thereof

Abstract

This invention discloses a method for regulating the content of aroma precursors in tobacco leaves and its application. The method includes overexpressing the NtMYB61 gene to reduce the content of aroma precursors in tobacco or mutating the NtMYB61 gene to increase the content of aroma precursors in tobacco. This invention is the first to discover that the NtMYB61 gene has the function of regulating the content of aroma precursors in tobacco. As an excellent gene resource for improving tobacco quality, the NtMYB61 gene can be used for the efficient cultivation of high-quality new tobacco materials, providing a new method for cultivating new tobacco materials with different aroma precursor contents and for plant genetic engineering research. It has significant value and importance for improving tobacco quality.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, and relates to a method for regulating the content of aroma precursors in tobacco leaves and its application. Background Technology

[0002] Flavonoids, as polyphenolic acid secondary metabolites widely found in plants, exhibit a wide variety of compound structures. Based on their chemical structures, flavonoids are further classified into different subclasses, including flavones, flavonols, chalcones, isoflavones, anthocyanins, and flavanones. Within plants, flavonoids play a crucial role in growth, development, and resistance to environmental stresses. For example, they participate in plant responses to ultraviolet (UV) stress, mitigating damage caused by UV radiation (UV-A and UV-B); act as important signaling molecules between plants and symbiotic microorganisms, promoting nitrogen-fixing symbiosis; inhibit the invasion and reproduction of pathogens, and enhance plant adaptability to biotic stresses. Natural flavonoids also play an important role in human health. Studies have found that flavonoids, as effective antioxidants, can help neutralize free radicals, reduce cellular oxidative stress, and protect the body from oxidative damage; some flavonoids possess anti-inflammatory properties, inhibiting inflammatory responses, reducing the release of inflammatory mediators, and regulating the immune system response.

[0003] Tobacco is one of my country's important economic crops. The composition, content, and distribution of aroma compounds in tobacco leaves are closely related to the quality and style of flue-cured tobacco. Among these, flavonoids synthesized via the phenylpropanoid metabolic pathway are important aroma precursors in flue-cured tobacco. For tobacco, flavonoids not only participate in tobacco growth and development and responses to biotic and abiotic stresses, but they are also important aroma precursors closely related to the quality of tobacco leaves. Studies have found that flavonoids can pyrolyze into some highly volatile substances during tobacco combustion, many of which directly affect the aroma of the smoke. During the curing, aging, and combustion of tobacco leaves, flavonoids can oxidize and decompose, imparting an elegant and refreshing aroma to tobacco and increasing the amount of aroma, thus playing a significant role in improving the quality of tobacco products. Furthermore, during tobacco curing, flavonoids undergo oxidation, and their oxidation products react with amino acids, minerals, and sugars through enzymatic browning reactions to generate a large number of complex pigments with varying molecular weights and colors. This causes the tobacco leaf color to change from yellow to orange-yellow or varying degrees of brown, playing a crucial role in the yellowing or browning of tobacco leaves. In summary, flavonoids have a significant impact on tobacco leaf quality, directly participating in the formation of aroma and the improvement of color. However, current research on the molecular-level regulation of tobacco flavonoid aroma precursor synthesis is relatively scarce. Therefore, there is an urgent need to provide a method that can effectively regulate tobacco flavonoid aroma precursors. Summary of the Invention

[0004] To address the shortcomings of existing technologies and practical needs, this invention provides a method for regulating the content of aroma precursors in tobacco leaves and its application. This invention is the first to discover that the NtMYB61 gene has the function of regulating the content of aroma precursors in tobacco. As an excellent gene resource for improving tobacco quality, the NtMYB61 gene can be used for the efficient cultivation of high-quality new tobacco materials. It provides a new method for cultivating new tobacco materials with different contents of aroma precursors and for plant genetic engineering research, which has important value and significance for improving tobacco quality.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for regulating the content of aroma precursors in tobacco leaves, the method comprising overexpressing the NtMYB61 gene to reduce the content of aroma precursors in tobacco leaves or mutating the NtMYB61 gene to increase the content of aroma precursors in tobacco leaves.

[0007] This invention is the first to discover that the NtMYB61 gene has the function of regulating the content of aroma precursors in tobacco, and can be effectively applied to the regulation of aroma precursor content in tobacco. As an excellent gene resource for improving tobacco quality, the NtMYB61 gene can be used for the efficient cultivation of high-quality new tobacco materials, providing new technical methods for cultivating new tobacco materials with varying aroma precursor content and for plant genetic engineering research, which has significant value and importance for improving tobacco quality.

[0008] Preferably, the tobacco aroma precursors include flavonoids.

[0009] Preferably, the nucleic acid sequence of the NtMYB61 gene includes the sequence shown in SEQ ID No. 1.

[0010] Preferably, the coding region sequence of the NtMYB61 gene includes the sequence shown in SEQ ID No. 2.

[0011] Preferably, the amino acid sequence of the protein encoded by the NtMYB61 gene includes the sequence shown in SEQ ID No. 3.

[0012] SEQ ID No. 1:

[0013]

[0014] SEQ ID No.2:

[0015] 。

[0016] SEQ ID No.3:

[0017] MGRPPCCDKIGIKKGPWTPEEDIVLVSYIQEHGPGNWRSVPTNTGLMRCSKSCRLRWTNYLRPGIKRGNFTPHEEGMIVHLQALLGNKWAAIASYLPQRTDNDIKNYWNTHLKKKLKKFQTGLDSHLSTPPADSSTCDHFSWNFMS DNNKQGSKLLLQQNHNSPNSSSSFYASSTENISRLLEGWMRSSPNPSRKVDEMILHENQNSQDQDLFKSRETSLVQDGGISNGIKTIPKEYMSVISDISACESSGVAEKTMNTNTPPPFTYLEKWLLEENAGQVEELMELPMIFT.

[0018] Secondly, the present invention provides the application of the method described in the first aspect in tobacco variety breeding.

[0019] Thirdly, the present invention provides a CRISPR / Cas9 gene editing system, wherein the CRISPR / Cas9 gene editing system includes the NtMYB61 gene described in the first aspect.

[0020] Fourthly, the present invention provides the application of the CRISPR / Cas9 gene editing system described in the third aspect in improving the synthesis and accumulation of aroma precursors in tobacco.

[0021] Fifthly, the present invention provides the application of the NtMYB61 gene and / or its gene editing reagent as described in the first aspect, the application including overexpressing the NtMYB61 gene to reduce the content of tobacco aroma precursors or mutating the NtMYB61 gene to increase the content of tobacco aroma precursors.

[0022] Preferably, the gene editing reagent includes an NtMYB61 gene overexpression reagent and / or an NtMYB61 gene mutation reagent.

[0023] It is understood that any reagents available in the art that can be used for overexpression or mutation of the NtMYB61 gene are applicable to this invention.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention clones the gene NtMYB61, which regulates the content of aroma precursors in tobacco, from tobacco. Overexpression of this gene using transgenic technology weakens the ability of tobacco to synthesize aroma precursors. However, mutants of this gene obtained through gene editing technology increase the synthesis content of aroma precursors in tobacco. Preferably, the aroma precursors include flavonoids. This research provides a theoretical basis and molecular targets for genetic research on improving tobacco quality (genetic regulation of tobacco aroma substances, application of genes related to the synthesis of tobacco aroma precursors in tobacco, and mining and analysis of genes related to the synthesis of tobacco aroma precursors), research on high-quality tobacco germplasm resources (collection and preservation of high-quality tobacco germplasm resources, utilization of high-aroma tobacco germplasm resources), and tobacco quality breeding research (genetic breeding strategies for the synthesis of tobacco aroma substances, application of molecular biotechnology in tobacco quality improvement, and breeding research on new tobacco varieties with high aroma substances). Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the NtMYB61 gene structure;

[0027] Figure 2 Figure 1 shows the agarose gel electrophoresis results of the NtMYB61 genome and CDS fragment;

[0028] Figure 3 A diagram illustrating the expression pattern of NtMYB61.

[0029] Figure 4 Figure showing the total flavonoid content at different leaf positions in wild-type K326;

[0030] Figure 5 This is a graph showing the expression levels of the NtMYB61 gene in wild-type K326 control and NtMYB61 overexpression materials.

[0031] Figure 6 Figure 1 shows the base changes and encoded amino acid results of the NtMYB61 gene in wild-type K326 control and NtMYB61 mutant materials;

[0032] Figure 7 Figure showing the total flavonoid content of NtMYB61 overexpression materials and mutant materials;

[0033] Figure 8 This figure shows the expression levels of key synthase genes in the flavonoid biosynthesis pathway in NtMYB61 overexpression materials and mutant materials. Detailed Implementation

[0034] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] Example 1

[0037] Tobacco seedling culture and NtMYB61 gene amplification.

[0038] Seedling culture: The wild-type tobacco seeds K326 were disinfected with 75% anhydrous ethanol for about 30 seconds, then soaked in 15% H2O2 for about 10 minutes, and finally rinsed three times with sterile water. After drying to semi-dryness, they were cultured on 1 / 2 MS solid medium and germinated and grew in a sterile tissue culture room with culture conditions of 25℃ and 16h / d light.

[0039] NtMYB61 gene amplification:

[0040] (1) When the tobacco plants have grown to three leaves and one bud or larger, take approximately 0.1g of sample with sterile scissors and obtain tobacco genomic DNA using the SLS DNA extraction method. Specific primers were designed upstream and downstream of the NtMYB611 gene; the primer sequences are shown in Table 1 below, and the amplified fragment length is 1805bp. Using the genomic DNA as a template, PCR amplification was performed using the sequences in Table 1 as upstream and downstream primers to obtain the NtMYB61 gene sequence.

[0041] Table 1

[0042] Primer 1 SEQ ID No. 4 TATATATCAACCAATTAAAGATCT Primer 2 SEQ ID No. 5 TTAAGTGAATATCATAGGAAGTTCC

[0043] Results of 1% agarose gel electrophoresis are as follows Figure 2 As shown, the results indicate that the target band size is consistent with the prediction. Further sequencing of the PCR product revealed the gene sequence as shown in SEQ ID No. 1; the full-length NtMYB61 gene is 1409 bp. A schematic diagram of the gene structure is shown below. Figure 1 As shown in the figure; further sequence analysis revealed that the NtMYB61 gene contains three exons and two introns. The three exons were spliced ​​together to obtain the full-length NtMYB61 CDS sequence of 876 bp, as shown in SEQ ID No. 2.

[0044] The detailed steps for DNA extraction are as follows:

[0045] a. Grind the sample with liquid nitrogen and place it into a 2.0 mL centrifuge tube. Add 800 μL of SLS extraction buffer (pH = 8.0).

[0046] b. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix thoroughly;

[0047] c. Centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.6 times the volume of pre-chilled isopropanol and mix well.

[0048] Centrifuge at 12000 rpm for 10 min, discard the supernatant, and rinse with 75% ethanol for a longer period of time.

[0049] e. Dissolve in ddH2O after drying.

[0050] (2) The above-mentioned tobacco seedlings were used for RNA extraction. The RNA extraction steps for tobacco leaves were performed according to the instructions of the RNA extraction kit: The sample was placed in liquid nitrogen and ground in a mortar and pestle, and then quickly transferred to a centrifuge tube with 1 mL Buffer RZ added. The sample was vortexed vigorously to mix. 200 μL of chloroform was added, and the sample was vortexed to mix. The sample was centrifuged at 13000 rpm for 1 min at 4 °C. 500 μL of supernatant was transferred to a new RNase-free centrifuge tube, and 0.5 times the volume of anhydrous ethanol was added. The sample was inverted and mixed, and then transferred to the adsorption column in the collection tube. The sample was centrifuged at 13000 rpm for 30 s and the waste liquid was discarded. 600 μL of Buffer RW2 was added to the adsorption column, and the sample was centrifuged at 13000 rpm for 30 s and the waste liquid was discarded. This step was repeated once. After 2 min of air centrifugation, the adsorption column was transferred to a new RNase-free centrifuge tube, and 100 μL of RNase-free H2O was added to the adsorption membrane. The sample was placed at room temperature for 2 min and centrifuged at 12000 rpm for 1 min to obtain the RNA solution.

[0051] According to the instructions of the RNA reverse transcription kit, using the RNA obtained in the above steps as a template, take about 1 μg of RNA and 4 μL of 4×gDNA wiper Mix, add RNase-free ddH2O to make up to 16 μL, mix well and incubate at 42℃ for 2 min; add 4 μL of 5x HiScriptⅢqRT SuperMix to the system and mix well, incubate at 37℃ for 15 min, then at 85℃ for 5 s to obtain the desired cDNA.

[0052] Table 2

[0053] Primer 3 SEQ ID No. 6 ATGGGAAGGCCTCCTTGTTGTG Primer 4 SEQ ID No.7 TTAAGTGAATATCATAGGAAGTTCC

[0054] Using the cDNA obtained in the above process as a template, and primers 3 and 4 in Table 2 as upstream and downstream primers for amplification, an 876bp PCR product of the NtMYB61 gene was obtained as its full-length CDS. The agarose gel electrophoresis results were then analyzed as follows: Figure 2 As shown, the results indicate that the PCR band size of the product is consistent with that of the target product, and the sequencing results are consistent with those shown in SEQ ID No. 2. The RNA extraction kit used in this process was purchased from Beijing Novell Biotechnology Co., Ltd.; the reverse transcription kit was purchased from Novizan Biotechnology Co., Ltd.; and all the pipette tips, centrifuge tubes, and other items used were RNase-free products purchased from Axygen Biotechnology Co., Ltd.

[0055] Example 2

[0056] Analysis of NtMYB61 tissue expression patterns.

[0057] Following the method described in Example 1, tobacco seedlings were transplanted into substrate nutrient soil. After one month of normal cultivation, samples were collected according to the method described in Example 1, and RNA was extracted from the corresponding samples. cDNA was then obtained through reverse transcription. The mixture was prepared according to the system in Table 3 below:

[0058] Table 3

[0059] Components Volume (μL) 2×SYBR GreenMasterMix 5 Primer 5 0.2 Primer 6 0.2 cDNA 1 <![CDATA[ddH2O]]> 3.6

[0060] Perform the qPCR reaction according to the procedure in Table 4 below:

[0061] Table 4

[0062]

[0063] Finally, the expression level was calculated using the ΔΔCt method, and the results are as follows: Figure 3 As shown, NtMYB61 is mainly expressed in leaves during the seedling stage, and the expression level of NtMYB61 gene gradually decreases as the leaf position of tobacco leaves increases. The expression level is relatively high in roots and low in stems.

[0064] The real-time quantitative PCR enzyme used in this process was purchased from Novizan Biotechnology Co., Ltd.; the primer sequences used are shown in Table 5 below:

[0065] Table 5

[0066] Primer 5 SEQ ID No. 8 GGTAACAAATGGGCAGCCA Primer 6 SEQ ID No. 9 ATCTGCAGGAGGAGTACTCAAA

[0067] Example 3

[0068] Detection of total flavonoid content in wild-type K326.

[0069] Following the method described in Example 1, tobacco seedlings were transplanted into substrate nutrient soil. After one month of normal cultivation, wild-type leaves were placed in kraft paper envelopes, labeled L1, L2, L3, and L4 from top to bottom, and quickly placed in liquid nitrogen. After all samples were collected, they were freeze-dried in a freeze dryer until constant weight. The method is as follows:

[0070] The sample was pulverized into powder, and the total flavonoid content of tobacco was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method.

[0071] Sample preparation: Take fresh tissue samples in aluminum foil or kraft paper envelopes, freeze dry to constant weight in a freeze dryer, crush the samples, weigh 0.05g of freeze-dried powder, add 1mL of 60% anhydrous ethanol, sonicate for 30min, and then place in a 4℃ refrigerator overnight for extraction; centrifuge at 12000rpm for 10min, and take the supernatant into a new centrifuge tube for testing.

[0072] Sample determination: Take 80 μL of supernatant into a new 5 mL centrifuge tube, add 60 μL of 5% NaNO2, mix well and let stand for 6 min; then add 60 μL of 10% Al(NO3)3, mix well and let stand for 6 min; finally add 800 μL of 4% NaOH, mix well and let stand for 15 min, and measure its absorbance at 510 nm.

[0073] Preparation of standard curve: Weigh 1 mg of rutin standard and add 60% anhydrous ethanol to prepare a 1 mg / mL stock solution standard; dilute the stock solution with 60% anhydrous ethanol to 0, 0.2, 0.4, 0.6, 0.8 and 1 mg / mL and prepare the standard curve according to the sample determination method.

[0074] Results processing: Based on the standard curve results, the absorbance values ​​measured for the samples are substituted into the values, and the concentration of flavonoids in the tobacco samples can be calculated after conversion.

[0075] The results are as follows Figure 4 As shown, during the seedling stage of wild-type K326 tobacco, the total flavonoid content in tobacco leaves continuously increased with the rising leaf position. Combined with the seedling tissue expression results of the NtMYB61 gene in Example 2, it is preliminarily inferred that NtMYB61 may regulate the synthesis of aroma precursors in tobacco, and that the total flavonoid content continuously decreased with increasing NtMYB61 expression, exhibiting a negative regulatory trend.

[0076] Example 4

[0077] Obtaining tobacco materials through NtMYB61 gene overexpression and knockout.

[0078] The product obtained by PCR using cDNA as a template in Example 1 was detected and purified by 1% agarose gel electrophoresis, ligated into the pEASY-Blunt Zero Cloning kit vector, and transformed into E. coli DH5α competent cells. Transformants were screened and cultured in LB solid medium containing kanamycin (50 mg / L) for 12 h. Single clones were picked and verified by bacterial PCR using universal primers for the vector. Positive clones were subjected to bidirectional sequencing to obtain the positive plasmid Blunt-NtMYB61 containing the NtMYB61 gene sequence.

[0079] Using the cloned Blunt-NtMYB61 plasmid as a template, PCR amplification was performed using Primer 7 & 8 (see Table 6). The fragment was then inserted into the plant binary expression vector pBWA(V)HS-ccdb-GLosgfp according to the standard vector construction method. The bacteria used for screening were resistant to kanamycin, resulting in the overexpression vector plasmid pBWA(V)HS-NtMYB61 of the NtMYB61 gene.

[0080] The NtMYB61 gene overexpression material was obtained using standard procedures in tobacco transgenic technology.

[0081] Table 6

[0082] Primer 7 SEQ ID No. 10 CAGTCGTCCACAACATGGGAAGGCCTCCTTGTTG Primer 8 SEQ ID No. 11 CAGTCGTCTCATACAAGTGAATATCATAGGAAGTT

[0083] RNA was extracted and reverse transcribed from NtMYB61 gene-overexpressing plants as described in Example 1, and the corresponding cDNA was obtained. The overexpression effect of NtMYB61 was detected using cDNA from wild-type tobacco K326 grown at the same time as a control. The expression effect of the NtMYB61 overexpression materials was calculated in the same manner as in Example 2. Figure 5 As shown, compared with the wild-type control K326, the expression level of the NtMYB61 gene in transgenic plants that overexpressed the NtMYB61 gene was significantly higher than that in the control.

[0084] Based on the sequence information of the NtMYB61 gene in the tobacco genome and the CDS sequence, a specific sgRNA target sequence for knocking out NtMYB61 was designed: GAGGCCAGGAATCAAAA. The sgRNA target sequence fragment was synthesized, and NtMYB61 mutant material was obtained using standard CRISPR / Cas9 technology and named NtMYB61.

[0085] Genomic DNA was extracted from K326 and NtMYB61 mutant tobacco using the SLS method. High-fidelity PCR amplification was performed using primers 9 and 10 (see Table 7), followed by 1% agarose gel electrophoresis and sequencing by a sequencing company. Homozygous mutant materials NtMYB61-2 and NtMYB61-4 were screened and identified. The mutation sites of the two obtained NtMYB61 homozygous mutants and their affected protein amino acids are as follows: Figure 6 As shown, the NtMYB61-2 line exhibits a homozygous deletion of 4 bp at +276 bp after the start codon of the NtMYB61 gene; the NtMYB61-4 line exhibits a homozygous insertion of base A at +281 bp after the start codon of the NtMYB61 gene. The amino acids encoded by the mutated NtMYB61 gene are completely different from those encoded by the normally encoded NtMYB61 gene. Figure 6 This result can lead to the loss or inhibition of activity of the protein encoded by the tobacco NtMYB61 gene.

[0086] Table 7

[0087] Primer 9 SEQ ID No. 12 TCCTTGTTGACCCCTACACC Primer 10 SEQ ID No. 13 TCCTTCGTTTATGCTCGTC

[0088] Example 5

[0089] Determination of total flavonoid content in NtMYB61 overexpression and mutants.

[0090] Unlike Example 3, the sample content in this example is the total flavonoid content of the whole tobacco plant. After one month of normal culture of NtMYB61 overexpression, mutant, and wild-type tobacco, leaves from the whole tobacco plant were collected, placed in kraft paper envelopes, and rapidly frozen in liquid nitrogen. The total flavonoid content was then determined according to the method described in Example 3. The results are as follows: Figure 7 During the same period, the total flavonoid content in the overexpression material was significantly lower than that in the wild-type K326; at the same time, the total flavonoid content in the wild-type K326 was significantly lower than that in the mutant tobacco. Combining the tissue expression pattern of NtMYB61 and the results of the total flavonoid content in tobacco leaves at different leaf positions in wild-type tobacco, it can be concluded that NtMYB61 negatively regulates the synthesis of flavonoid aroma precursors in tobacco leaves.

[0091] Example 6

[0092] NtMYB61 overexpression and expression of key synthase genes in the flavonoid aroma synthesis pathway in mutants.

[0093] In Example 5, before taking samples to determine the content of tobacco flavonoids, tweezers sterilized with alcohol were used to quickly cut approximately 2 cm of leaves from the same part of each tobacco plant. 2The RNA was placed in a 2 mL RNAase centrifuge tube containing zirconium beads and frozen in liquid nitrogen. After being pulverized using a tissue homogenizer, RNA extraction and reverse transcription were performed according to Example 1. Using primers listed in Table 8, the expression levels of key synthase genes in the tobacco flavonoid aroma synthesis pathway were determined according to the qPCR detection method described in Example 3.

[0094] Table 8

[0095]

[0096]

[0097] Expression levels were calculated using the ΔΔCt method, as shown below. Figure 8 The expression levels of key enzyme genes NtCHS, NtCHI, NtFLS, NtDFR, and NtANR in the tobacco flavonoid aroma synthesis pathway are shown. In the mutant, the expression levels of these key enzyme genes were significantly higher than those in the wild-type control K326, while the expression levels of these genes in the overexpression material were significantly lower than those in the wild-type control K326. This indicates that NtMYB61 can affect the content of flavonoid aroma precursors in tobacco by influencing the expression levels of these key enzyme genes.

[0098] In summary, the protein encoded by NtMYB61 plays an important role in the synthesis and accumulation of flavonoid aroma precursors in tobacco. Exploring the protein's ability to influence the synthesis of tobacco flavonoid aroma precursors will provide an important theoretical basis and molecular target for the screening and utilization of high aroma precursor germplasm resources in tobacco and for tobacco quality breeding research.

[0099] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for increasing the content of aroma precursors in tobacco leaves, characterized in that, The method includes mutating the NtMYB61 gene in tobacco leaves to reduce or inhibit the activity of its encoded protein, thereby increasing the content of aroma precursors in tobacco. The nucleic acid sequence of the NtMYB61 gene is as shown in SEQ ID No. 1; The aroma precursors in tobacco are flavonoids.

2. The method according to claim 1, characterized in that, The coding region sequence of the NtMYB61 gene is as shown in SEQ ID No.

2.

3. The method according to claim 1, characterized in that, The amino acid sequence encoded by the NtMYB61 gene is as shown in SEQ ID No.

3.

4. The application of the method according to any one of claims 1-3 in tobacco variety breeding.

5. The application of the NtMYB61 gene in enhancing aroma precursors in tobacco, characterized in that, The application includes mutating the NtMYB61 gene in tobacco leaves to reduce or inhibit the activity of its encoded protein, thereby increasing the content of aroma precursors in tobacco. The nucleic acid sequence of the NtMYB61 gene is as shown in SEQ ID No. 1; The aroma precursors in tobacco are flavonoids.

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