Method for regulating and controlling content of aroma precursor substances in tobacco leaves and application of method

Abstract

The invention discloses a method for regulating and controlling the content of aroma precursor substances in tobacco leaves and application of the method. The method comprises the following steps: overexpressing the NtMYB61 gene to reduce the content of the tobacco aroma precursor substance or mutating the NtMYB61 gene to improve the content of the tobacco aroma precursor substance. The function that the NtMYB61 gene has the function of regulating and controlling the content of the tobacco aroma precursors is found for the first time, the NtMYB61 gene serves as an excellent gene resource for improving the quality of tobacco leaves and can be used for efficient cultivation of high-quality new tobacco materials, a new method is provided for cultivation of the new tobacco materials with the different contents of the aroma precursors and plant genetic engineering research, and the application prospect is wide. The method has important value and significance for improving the quality of the tobacco leaves.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a method for regulating the content of tobacco aroma precursor substances and an application thereof. Background Art

[0002] Flavonoids are secondary metabolites of polyphenolic acids widely found in plants, and their compound structures are diverse. According to their different chemical structures, flavonoids are divided into different subclasses, including flavonoids, flavonols, chalcones, isoflavones, anthocyanins, flavanones, etc. In plants, flavonoids are of great significance for plant growth and development and for resisting external environmental stress. For example, they participate in the plant's response to external ultraviolet stress resistance and reduce the damage caused by ultraviolet rays (UV-A and UV-B) to plants; as important signal molecules between plants and symbiotic microorganisms, they promote the establishment of nitrogen-fixing symbiotic relationships; inhibit the invasion and reproduction of pathogens, and improve the adaptability of plants to biological stress, etc. In terms of human health, natural flavonoids also play an important role. Studies have found that flavonoids, as an effective antioxidant, can help neutralize free radicals, reduce cellular oxidative stress, and protect the body from oxidative damage; some flavonoids have anti-inflammatory properties, which can inhibit inflammatory reactions, reduce the release of inflammatory mediators, and regulate the response of the immune system.

[0003] Tobacco is one of the important economic crops in my country. The composition, content and distribution of aroma substances in tobacco leaves are closely related to the quality and style characteristics of flue-cured tobacco. Among them, flavonoids synthesized by the phenylpropanoid metabolic pathway are one of the important aroma precursors of flue-cured tobacco. For tobacco, in addition to participating in tobacco growth and development and the response to biotic and abiotic stresses, flavonoids are an important class of aroma precursors, which are closely related to the quality of tobacco leaves. Studies have found that flavonoids can be pyrolyzed into some highly volatile substances during the smoking of cigarettes, many of which have a direct effect on the aroma of smoke. During the process of tobacco leaf curing, aging and combustion, flavonoids can be oxidatively decomposed, giving tobacco an elegant and fresh aroma and increasing the amount of aroma, which has an important effect on the quality of tobacco products. In addition, during the tobacco leaf baking process, flavonoids undergo oxidation reactions, and their oxidation products react with amino acids, minerals, and sugars through enzymatic browning reactions to generate a large number of complex pigments with different molecular weights and colors, causing the color of tobacco leaves to change from yellow to orange or different degrees of brown, which plays an important role in the yellowing or browning of tobacco leaves. In summary, flavonoids have a significant impact on the quality of tobacco leaves, and they are directly involved in the formation of tobacco leaf aroma and the improvement of color. However, the research on the synthesis and regulation of tobacco flavonoid aroma precursors at the molecular level is relatively scarce. Therefore, it is urgent to provide a method that can effectively regulate tobacco flavonoid aroma precursors. Summary of the invention

[0004] In view of the deficiencies in the prior art and actual needs, the present invention provides a method for regulating the content of aroma precursors in tobacco leaves and an application thereof. The present invention discovers for the first time that the NtMYB61 gene has the function of regulating the content of aroma precursors in tobacco leaves. As an excellent gene resource for improving tobacco leaf quality, the NtMYB61 gene can be used for the efficient cultivation of high-quality new tobacco materials, and provides a new method for cultivating new tobacco materials with different contents of aroma precursors and for plant genetic engineering research, which is of great value and significance for improving tobacco leaf quality.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for regulating the content of tobacco aroma precursors, the method comprising 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.

[0007] The present invention discovered for the first time that the NtMYB61 gene has the function of regulating the content of tobacco aroma precursors, and can be effectively used to regulate the content of tobacco aroma precursors. As an excellent gene resource for improving tobacco leaf quality, the NtMYB61 gene can be used for the efficient cultivation of high-quality new tobacco materials, providing a new technical method for cultivating new tobacco materials with different aroma precursor content and plant genetic engineering research, which has important value and significance for improving tobacco leaf quality.

[0008] Preferably, the tobacco aroma precursor substances 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] MGRPPCCDKIGIKKGPWTPEEDIVLVSYIQEHPGGNWRSVPTNTGLMRCSKSCRLRWTNYLRPGIKRGNFTPHEEGMIVHLQALLGNKWAAIASYLPQRTDNDIKNYWNTHLKKKLKKFQTGLDSHLSTPPADSSTCDHFSWNFMS DNNKQGSKLLLQQNHNSPNSSSSFYASSTENISRLLEGWMRSSPNPSRKVDEMILHENQNSQDQDLFKSRETSLVQDGGISNGIKTIPKEYMSVISDISACESSGVAEKTMNTNTPPPFTYLEKWLLEENAGQVEELMELPMIFT.

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

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

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

[0021] In a fifth aspect, the present invention provides an application of the NtMYB61 gene and / or its gene editing reagent described in the first aspect, wherein the application includes 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 a NtMYB61 gene overexpression reagent and / or a NtMYB61 gene mutation reagent.

[0023] It is understood that any reagent that can be used for NtMYB61 gene overexpression or mutation in the art is suitable for the present invention.

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

[0025] The gene NtMYB61 regulating the content of tobacco aroma precursors is cloned from tobacco, and the gene is overexpressed by transgenic technology to weaken the ability of tobacco to synthesize aroma precursors, while the mutant of the gene obtained by gene editing technology increases the synthesis content of aroma precursors in tobacco. Preferably, the tobacco aroma precursors include flavonoids. This study provides theoretical basis and molecular targets for genetic research on improving tobacco quality (research on genetic regulation of tobacco aroma substances, application research of tobacco aroma precursor synthesis-related genes in tobacco, and mining and analysis of tobacco aroma precursor synthesis-related genes), research on high-quality tobacco germplasm resources (collection and preservation of high-quality tobacco germplasm resources, utilization of high-aroma tobacco germplasm resources), and research on tobacco quality breeding (genetic breeding strategies for tobacco aroma synthesis, application of molecular biotechnology in tobacco quality improvement, and research on breeding of new tobacco varieties with high aroma substances). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the NtMYB61 gene structure;

[0027] Figure 2 The results of agarose gel electrophoresis of NtMYB61 genome and CDS fragments;

[0028] Figure 3 This is the tissue expression pattern of NtMYB61;

[0029] Figure 4 This is the total flavonoid content in different leaf positions of wild-type K326;

[0030] Figure 5 It is a graph of the expression level of NtMYB61 gene in wild-type K326 control and NtMYB61 overexpression material;

[0031] Figure 6 The results of the base changes and encoded amino acids of the NtMYB61 gene in the wild-type K326 control and NtMYB61 mutant materials are shown;

[0032] Figure 7 This is the result diagram of total flavonoid content in NtMYB61 overexpression materials and mutant materials;

[0033] Figure 8 This is a graph showing the expression levels of key synthase genes in the flavonoid biosynthesis pathway in NtMYB61 overexpression materials and mutant materials. DETAILED DESCRIPTION

[0034] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] Example 1

[0037] Tobacco seedling culture and NtMYB61 gene amplification.

[0038] Seedling culture: Sterilize tobacco wild-type seeds K326 with 75% anhydrous ethanol for about 30 seconds, then soak in 15% H2O2 for about 10 minutes, and finally wash three times with sterile water. After drying to half dry, culture on 1 / 2MS solid culture medium, and germinate and grow in a sterile tissue culture room with culture conditions of 25°C and 16h / d of light.

[0039] NtMYB61 gene amplification:

[0040] (1) When tobacco leaves grow to three leaves and one heart or larger, use sterile scissors to sample about 0.1 g, and use the SLS DNA extraction method to obtain tobacco genomic DNA. Design specific primers upstream and downstream of the NtMYB611 gene. The primer sequences are shown in Table 1 below, and the length of the amplified fragment is 1805 bp. Use the genomic DNA as a template and the sequences in Table 1 below as upstream and downstream primers for PCR amplification 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] The results of 1% agarose gel electrophoresis were as follows Figure 2 As shown, the results show that the target band size is consistent with the prediction. The PCR product was further sequenced, and the sequencing results showed that the gene sequence is as shown in SEQ ID No.1, and the full length of the NtMYB61 gene is 1409bp. Figure 1 As shown; further sequence analysis found that the NtMYB61 gene contains three exons and two introns. The three exons were spliced ​​to obtain the NtMYB61 CDS full-length sequence of 876 bp as shown in SEQ ID No. 2.

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

[0045] a. The liquid nitrogen ground sample was placed in a 2.0 mL centrifuge tube and 800 μL of SLS extract (PH = 8.0) was added;

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

[0047] c. Centrifuge at 12000r / m for 10min, pipette the supernatant into a new 1.5mL centrifuge tube, add 0.6 times the volume of pre-cooled isopropanol and mix well.

[0048] d. Centrifuge at 12000r / m for 10min, discard the supernatant, and rinse with 75% ethanol. The rinsing time can be longer.

[0049] e. Dissolve in ddH2O after drying

[0050] (2) At the same time, the above tobacco seedlings were used for RNA extraction. The steps of RNA extraction from tobacco leaves were carried out according to the instructions of the RNA extraction kit: the sample was placed in liquid nitrogen and ground with a mortar, and then quickly transferred to a centrifuge tube with 1 mL of Buffer RZ added, and vigorously vortexed to mix; 200 μL of chloroform was added, vortexed to mix, and centrifuged at 13000 rpm for 1 min at 4°C; 500 μL of supernatant was transferred to a new RNase-free centrifuge tube, 0.5 times the volume of anhydrous ethanol of the supernatant was added, and the mixture was transferred to an adsorption column in a collection tube, 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 mixture was centrifuged at 13000 rpm for 30 s, and the waste liquid was discarded. This step was repeated once; the adsorption column was vacant for 2 min, and then transferred to a new RNase-free centrifuge tube, 100 μL of RNase-free H2O was added to the adsorption film, and the mixture was left at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min to obtain an RNA solution.

[0051] According to the instructions of the RNA reverse transcription kit, use the RNA obtained in the above step as a template, absorb about 1μg RNA, 4μL 4×gDNA wiper Mix, add RNase-free ddH2O to 16μL, mix well into an RNase-free centrifuge tube, and incubate at 42℃ for 2min; add 4μL 5x HiScriptⅢqRT SuperMix to the system and mix well, incubate at 37℃ for 15min, and 85℃ for 5s to obtain the required cDNA.

[0052] Table 2

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

[0054] The cDNA obtained in the above process was used as a template, and the sequences of Primer 3 and Primer 4 in Table 2 were used as upstream and downstream primers for amplification. The PCR product of 876 bp in size of the NtMYB61 gene was obtained, which was the full length of the CDS. According to the subsequent agarose gel electrophoresis results, Figure 2 As shown, the results show that the PCR band size of the product is consistent with the size of the target product, and the sequencing result is consistent with the result shown in SEQ ID No. 2. The RNA extraction kit used in the process was purchased from Beijing Nobel Biotechnology Co., Ltd.; the reverse transcription kit was purchased from Novazon Biotechnology Co., Ltd.; the gun tips, centrifuge tubes and other items used were all RNase-free products purchased from Axygen Biotechnology Co., Ltd.

[0055] Example 2

[0056] Analysis of tissue expression pattern of NtMYB61.

[0057] According to the method described in Example 1, the tobacco seedlings were transplanted into the matrix nutrient soil, and after normal cultivation for one month, samples were collected according to the method described in Example 1, the corresponding sample RNA was extracted, and reverse transcription was performed to obtain cDNA. The mixed solution was prepared according to the system in Table 3 below:

[0058] Table 3

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

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

[0061] Table 4

[0062]

[0063] Finally, the expression level was calculated according to the ΔΔCt method. The results are as follows: Figure 3 As shown in the figure, NtMYB61 was mainly expressed in leaves during the seedling stage, and the expression of NtMYB61 gene gradually decreased with the increase of leaf position of tobacco leaves. The expression level in roots was relatively high, while the expression in stems was low.

[0064] The real-time fluorescence quantitative PCR enzyme used in this process was purchased from Novozyme 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 flavonoids content in wild-type K326.

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

[0070] The samples were crushed into powder and the total flavonoids content in tobacco was determined by NaNO2-Al(NO3)3-NaOH colorimetric method.

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

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

[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, 1 mg / mL and prepare the standard curve according to the sample determination method.

[0074] Result processing: According to the standard curve results, the absorbance value of the sample is input and the flavonoid concentration in the tobacco sample can be calculated after conversion.

[0075] The results are as follows Figure 4 As shown, in the seedling stage of wild-type K326 tobacco, the total flavonoid content in tobacco leaves increases with the continuous increase of leaf position. Combined with the results of tissue expression of the NtMYB61 gene in the seedling stage in Example 2, it is preliminarily inferred that NtMYB61 may regulate the synthesis of aroma precursors in tobacco, and with the increase of NtMYB61 expression, the total flavonoid content continues to decrease, showing a negative regulation trend.

[0076] Example 4

[0077] Obtaining tobacco materials with 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, then connected to the pEASY-Blunt Zero Cloning kit vector and transformed into Escherichia coli DH5α competent cells. The transformants were screened and cultured for 12 hours using LB solid culture medium containing kanamycin (50 mg / L). Single clones were picked and verified by bacterial liquid PCR using universal primers for the vector. Positive clones were bidirectionally sequenced to obtain the positive plasmid Blunt-NtMYB61 containing the NtMYB61 gene sequence.

[0079] The cloned Blunt-NtMYB61 plasmid was used as a template and PCR was amplified using Primer 7&8 (see Table 6). The fragment was inserted into the plant binary expression vector pBWA(V)HS-ccdb-GLosgfp according to the common vector construction method. The bacteria used for screening resistance was kanamycin, and the NtMYB61 gene overexpression vector plasmid pBWA(V)HS-NtMYB61 was obtained.

[0080] The NtMYB61 gene overexpression material was obtained by conventional tobacco transgenic technology.

[0081] Table 6

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

[0083] RNA extraction and reverse transcription were performed according to Example 1 to obtain RNA and corresponding cDNA of NtMYB61 gene overexpression plants, and the cDNA of common wild-type tobacco K326 grown in the same period was used as a control to detect the NtMYB61 overexpression effect. The expression effect of NtMYB61 overexpression material 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 the transgenic plants overexpressing the NtMYB61 gene was significantly higher than that in the control.

[0084] According to the sequence information and CDS sequence of the NtMYB61 gene in the tobacco genome, the sgRNA target sequence for specific knockout of NtMYB61 was designed: GAGGCCAGGAATCAAAA. The sgRNA target sequence fragment was synthesized, and the NtMYB61 mutant material was obtained by conventional operation of CRISPR / Cas9 technology and named NtMYB61.

[0085] The K326 and NtMYB61 mutant tobacco genomic DNA was extracted by SLS method, and high-fidelity PCR amplification was performed with primers 9 and 10 (see Table 7). The results were detected by 1% agarose gel electrophoresis and sequenced by a sequencing company. The homozygous mutant materials NtMYB61-2 and NtMYB61-4 were screened and identified. The mutation sites of the two NtMYB61 homozygous mutants and the protein amino acids affected by them are shown in Figure 2. Figure 6 As shown. The NtMYB61-2 strain has a homozygous deletion of 4 bp at +276 bp after the start codon of the NtMYB61 gene; the NtMYB61-4 strain has a homozygous insertion of base A at +281 bp after the start codon of the NtMYB61 gene. The amino acids encoded by the NtMYB61 gene after the mutation are completely different from the amino acids normally encoded by NtMYB61 ( Figure 6 ), which can lead to the loss or inhibition of the 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] Different from Example 3, the sample content in this example is the total flavonoid content of the whole tobacco plant. After the NtMYB61 overexpression and mutant and wild-type tobacco plants were cultured normally for one month, the leaves of the whole tobacco plant were taken and placed in a kraft paper envelope, and quickly frozen with liquid nitrogen, and then the total flavonoid content was determined according to the total flavonoid content detection method in Example 3. The results are shown in FIG. 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. Combined with the tissue expression pattern of NtMYB61 and the total flavonoid content in tobacco leaves at different leaf positions in the wild type tobacco, it can be concluded that NtMYB61 negatively regulates the synthesis of flavonoid aroma precursors in tobacco leaves.

[0091] Example 6

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

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

[0094] Table 8

[0095]

[0096]

[0097] The expression level was calculated according to the ΔΔCt method and the results were as follows: Figure 8 The expression levels of the key synthase genes NtCHS, NtCHI, NtFLS, NtDFR, and NtANR in the synthesis pathway of flavonoid aroma substances in tobacco are shown. In the mutant, the expression level of the key synthase genes in the synthesis pathway of flavonoid aroma substances was significantly higher than that of the wild-type control K326, while the expression level of these genes in the overexpression material was significantly lower than that of the wild-type control K326, indicating that NtMYB61 can affect the content of flavonoid aroma precursor substances by affecting the expression level of the key synthase genes in the synthesis pathway of flavonoid aroma substances in tobacco.

[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 ability of this protein to affect the synthesis of tobacco flavonoid aroma precursors will provide an important theoretical basis and breeding molecular targets for the screening and utilization of tobacco high-aroma precursor germplasm resources and tobacco quality breeding research.

[0099] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various 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 regulating the content of aroma precursor substances in tobacco leaves, characterized in that: The method comprises overexpressing the NtMYB61 gene to reduce the content of tobacco aroma precursor substances or mutating the NtMYB61 gene to increase the content of tobacco aroma precursor substances.

2. The method according to claim 1, characterized in that The tobacco aroma precursor substances include flavonoid compounds.

3. The method according to claim 1 or 2, characterized in that: The nucleic acid sequence of the NtMYB61 gene includes the sequence shown in SEQ ID No.

1.

4. The method according to any one of claims 1 to 3, characterized in that The coding region sequence of the NtMYB61 gene includes the sequence shown in SEQ ID No.

2.

5. The method according to any one of claims 1 to 4, characterized in that The amino acid sequence of the protein encoded by the NtMYB61 gene includes the sequence shown in SEQ ID No.

3.

6. Use of the method according to any one of claims 1 to 5 in tobacco variety breeding.

7. A CRISPR / Cas9 gene editing system, characterized in that: The CRISPR / Cas9 gene editing system includes the NtMYB61 gene described in claim 3 or 4.

8. Use of the CRISPR / Cas9 gene editing system described in claim 7 in improving the synthesis and accumulation of tobacco aroma precursors.

9. The use of the NtMYB61 gene and / or its gene editing agent according to any one of claims 3 to 5, wherein the use comprises 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.

10. The use according to claim 9, characterized in that: The gene editing reagent includes a NtMYB61 gene overexpression reagent and / or a NtMYB61 gene mutation reagent.

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