Application of inhibiting or knocking out tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning

Abstract

The invention provides an application of inhibiting or knocking out a tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning. The important gene NtPPO12 related to tobacco browning is screened out through transcriptome sequencing and molecular biological experiments, the NtPPO12 promoter activity and the gene expression site are further verified, and the gene function is deeply studied through gene editing and overexpression. Molecular biology experiments prove that the NtPPO12 gene provided by the invention is a key gene related to browning in the tobacco maturing and baking process, and the browning degree of tobacco can be controlled by regulating the expression quantity of the NtPPO12 gene, so that the maturing resistance and baking resistance of tobacco are improved, the harvesting frequency is reduced, the baking characteristic is improved, and the yield of tobacco is improved. Therefore, the method has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tobacco genes, and specifically relates to the application of inhibiting or knocking out tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning and improving tobacco ripening and baking resistance during ripening and baking, and also relates to the application of inhibiting the gene protein, the application of the gene in screening low browning tobacco varieties, and the gene promoter and the application thereof. Background Art

[0002] The baking characteristics of tobacco leaves are important factors affecting the quality of tobacco leaves, including "easy baking" and "baking resistance". "Easy baking" mainly refers to the yellowing characteristics of tobacco leaves in the early stage of baking and the ease of color fixation after yellowing. Tobacco leaves that easily turn yellow and fix color are considered easy to bake. "Baking resistance" mainly refers to the tolerance of tobacco leaves to the baking environment in the color fixation stage in the later stage of baking. Tobacco leaves that are not easy to turn brown are considered to have good baking resistance.

[0003] During the baking process, the browning of tobacco leaves is mainly due to the fact that polyphenols in tobacco leaves are converted into dark substances such as quinones under the action of polyphenol oxidase. This complex process is called tobacco enzymatic browning reaction. The occurrence of tobacco enzymatic browning reaction seriously affects the appearance and intrinsic quality of tobacco leaves after baking. Whether tobacco leaves are prone to browning after entering the color-fixing period after baking is an important indicator for measuring the color-fixing characteristics of tobacco leaves. Tobacco enzymatic browning reaction can cause tobacco leaves to become ash-covered, steamed, black, etc., and can also cause tobacco leaves to become thinner, less elastic, and easy to break. At the same time, baking resistance and maturity resistance have a certain correlation. Improving the maturity resistance of tobacco leaves in the mature stage can reduce the number of harvests and promote the integration of agricultural machinery and agronomy.

[0004] The ease of baking and baking resistance of tobacco leaves are affected by genetic factors and are closely related to the activity of polyphenol oxidase PPO. Through bioinformatics analysis and expression spectrum analysis, 12 PPO family member genes in the tobacco genome were found. Although some genes were verified to have decreased PPO enzyme activity through gene knockout or RNA interference experiments, no evidence was found that these PPO genes regulate PPO enzyme activity and inhibit browning during the maturation and baking process. Tobacco leaf maturation and baking are two key periods in tobacco leaf production. Only when the maturity and baking quality of tobacco leaves meet the processability and availability required for cigarettes can they have the best commodity value, use value and economic value.

[0005] At present, there are almost no genes that significantly regulate the enzymatic browning reaction of tobacco during the tobacco ripening and baking period. There are no relevant reports on the use of genetic engineering methods to reduce the activity of polyphenol oxidase during this special period, thereby reducing the occurrence of enzymatic browning reactions. The NtPPO12 gene of the present invention plays a significant role in the two key periods of tobacco leaf production, ripening and baking, and has important application value for improving ripening resistance and baking resistance. Summary of the invention

[0006] In view of the above-mentioned status in the prior art, the primary purpose of the present invention is to provide an application of inhibiting or knocking out tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The primer sequences for amplifying the tobacco polyphenol oxidase gene NtPPO12 are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0008] The above application is specifically to improve the ripening resistance and baking resistance of tobacco by inhibiting the browning of tobacco.

[0009] More specifically, the invention is used in inhibiting tobacco browning during the tobacco ripening and baking stages.

[0010] Further,

[0011] The method for inhibiting the tobacco polyphenol oxidase gene NtPPO12 includes a reagent for inhibiting the expression of the gene; and the method for knocking out the tobacco polyphenol oxidase gene NtPPO12 includes targeted knockout using CRISPR / Cas9 technology.

[0012] Furthermore,

[0013] A targeted knockout site of the gene NtPPO12 was designed, and a mutant base was inserted into the gene NtPPO12 to inactivate the expression of the gene NtPPO12.

[0014] The knockout target sequences of gene NtPPO12 are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0015] The second aspect of the present invention aims to provide a protein for inhibiting the expression of tobacco polyphenol oxidase gene NtPPO12 for use in inhibiting tobacco browning; its amino acid sequence is shown in SEQ ID NO.2.

[0016] Specifically, it is used to inhibit tobacco browning, thereby improving tobacco's resistance to ripening and baking.

[0017] More specifically, the invention is used in inhibiting tobacco browning during the tobacco ripening and baking stages.

[0018] The third aspect of the present invention aims to provide the application of the tobacco polyphenol oxidase gene NtPPO12 in screening low browning tobacco varieties.

[0019] The fourth aspect of the present invention aims to provide a promoter for driving the expression of tobacco polyphenol oxidase gene NtPPO12, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0020] The primer sequences for amplifying the promoter are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0021] The fifth aspect of the present invention aims to provide the use of the promoter in promoting the expression of tobacco polyphenol oxidase gene NtPPO12.

[0022] Compared with the prior art, the advantages and technical effects of the present invention are as follows: the present invention first screened out the key gene NtPPO12 related to tobacco browning during the maturation and baking periods, further explored the activity and expression site of the NtPPO12 promoter by constructing a GUS staining vector, constructed a subcellular localization vector of NtPPO12 and transformed Nicotiana benthamiana to detect the expression of GFP protein, and conducted in-depth research on the function of the gene by gene editing and overexpression. The present invention proves through molecular biological experiments that the NtPPO12 gene provided by the present invention is an important gene related to browning during tobacco maturation and baking. By regulating the expression level of the NtPPO12 gene, the browning of tobacco can be effectively controlled, and its maturity resistance and baking resistance can be improved, which can be used to cultivate excellent varieties and improve the quality of tobacco leaves after baking, and has broad market prospects and economic benefits.

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the change of tobacco leaf appearance during dark treatment in dark box experiment;

[0025] Figure 2 It is the transcriptome expression result of the NtPPO12 tobacco PPO gene in the present invention;

[0026] Figure 3 is the expression level of NtPPO12 gene at 0 h, 24 h, 48 h, 72 h and 96 h of dark box treatment;

[0027] Figure 4 is the NtPPO12 promoter amplification map;

[0028] Figure 5 This is the GUS staining of leaf tissue of T0 generation of NtPPO12 promoter transgenic tobacco;

[0029] Figure 6 is the fluorescence signal diagram of NtPPO12 and GFP protein;

[0030] Figure 7 This is the expression result of NtPPO12 overexpression strain;

[0031] Figure 8This is a comparison of the appearance of the NtPPO12 overexpression strain (right) and the common tobacco K326 (left);

[0032] Fig. 9 The appearance changes of the middle leaves of the control (WT) and NtPPO12 overexpressing materials under dark box treatment;

[0033] Fig.10 The results of PPO activity in the middle leaves of the NtPPO12 overexpression line and the control (WT) line;

[0034] Fig.11 It is the appearance change of the middle leaves of the control (WT) WT and NtPPO12 gene-edited materials under dark box treatment;

[0035] Fig.12 These are the results of PPO activity in the middle leaves of the NtPPO12 gene-edited strain and the control (WT). DETAILED DESCRIPTION

[0036] In order to improve the understanding of the present invention, the present invention is further described below through preferred embodiments. Those skilled in the art should understand that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0037] Example 1: Transcriptome changes in tobacco leaves in response to dark stress

[0038] 1. Expression analysis of polyphenol oxidase gene in tobacco leaves under dark box simulated baking

[0039] According to the tobacco industry standard YC / T 311-2009, a dark box environment was constructed in a carton to simulate baking. The middle part of the mature K326 variety tobacco leaves were taken, and three replicates were placed in a carton. The tobacco leaves were taken out and photographed every 12 hours until the leaves turned 30% brown. Samples were taken every 24 hours and placed in a -80℃ low-temperature refrigerator for transcriptome sequencing.

[0040] The yellowing and browning phenotypes of tobacco leaves were observed under dark stress. Figure 1 As shown in the figure, tobacco leaves begin to brown at 60-72h. In order to identify the polyphenol oxidase gene that plays a role in the browning process of tobacco leaves, the present invention analyzed the expression of 12 PPO genes in tobacco, used HTSeq software to count the number of reads aligned to each gene, used FPKM value to standardize the expression, and drew a heat map. The results are shown in Figure 2 As shown in the figure, the expression level of NtPPO12 among the 12 NtPPO genes was significantly upregulated with the advancement of dark stress time. It can be judged that the NtPPO12 gene is involved in the browning reaction of tobacco leaves in the late stage of tobacco leaf maturity and during the baking process.

[0041] 2. qRT-PCR verification of NtPPO12 gene expression

[0042] To verify the differential expression of NtPPO12 gene in transcriptome sequencing analysis, qRT-PCR technology was used to verify its expression in samples treated with dark box for 0h, 24h, 48h, 72h and 96h. Figure 3 The experimental results showed that the expression level of NtPPO12 was significantly upregulated after 72 hours of dark box treatment, and qRT-PCR analysis further showed that the NtPPO12 gene was related to the browning of tobacco leaves during maturity and under dark stress.

[0043] Example 2: Analysis of the expression pattern of the NtPPO12 promoter

[0044] 1. Amplify the promoter of NtPPO12 gene

[0045] In order to study the expression characteristics of the NtPPO12 gene, the promoter of NtPPO12 was first cloned (eg Figure 4 ) and analyzed the cloned sequences.

[0046] Using the upstream sequence of NtPPO12 gene 3000bp as the reference sequence, design specific primers ProNtPPO12-F / R:

[0047] ProNtPPO12-F: AGAGGAACTTGTTAGCGTAT (SEQ ID NO.4);

[0048] ProNtPPO12-R: AGGAAGATGAAGAAGAGATGG (SEQ ID NO.5);

[0049] The fragment was cloned from the genome of common tobacco K326 using primers ProNtPPO12-F / R, and clone sequencing showed that the amplified promoter fragment ProNtPPO12 was 2074bp long (as shown in SEQ ID NO.3). The sequence was imported into the PlantCARE database, and the cis-acting elements in the promoter region of NtPPO12 were analyzed. It was found that in addition to a large number of TATA boxes and CAAT boxes that general promoters have, and a large number of light response elements (I-box, LTR, G-Box, GA-motif, GATA-motif, GT1-motif, etc.), the sequence also has many specific and stress response elements.

[0050] 2. Promoter function analysis and construction of plant expression vector

[0051] After the NtPPO12 promoter amplification product is purified, it is connected to the TA cloning vector, i.e., TA-PPO12-pro. Using this as a template, the primer GUS-NtPPO12-F / R with a linker is used for PCR amplification, and the product is cut and recovered by gel cutting. The PCAMBIA1301 vector is cut with BamHI and NcoI, the 35S promoter on the vector is removed, and gel recovery is performed. After that, the NtPPO12 promoter sequence is connected to the PCAMBIA1301 vector by seamless cloning, and the DH5α competent cells are transformed by heat shock method, identified by colony PCR, and the plasmid is extracted by expanded culture for sequencing verification. Finally, the correct expression vector ProNtPPO12-1301 is constructed for genetic transformation of tobacco.

[0052] GUS-NtPPO12-F:

[0053] AATTCGAGCTCGGTACCCGGGGATCCAGAGGAACTTGTTAGCGTAT;

[0054] (SEQ ID NO.12)

[0055] GUS-NtPPO12-R:

[0056] TTACCTCAGATCTACCATGGTACTATAATTTTTGCTTGGTTTGAG (SEQ ID NO. 13).

[0057] Figure 5 The results showed that GUS staining was positive in the leaves and flowers of the transformed plants, indicating that the promoter of NtPPO12 regulated the expression of the GUS gene in leaves and flowers, especially the darker blue in the vascular bundles and glandular hairs in the leaves.

[0058] Example 3: Subcellular localization

[0059] The localization of PPO12 protein in tobacco cells was detected by fusing the GFP fluorescent protein gene to the C-terminus of PPO12. First, the PCAMBIA35S-EGFP vector was cut with KpnI, and the target gene was connected to the vector by seamless cloning after recovery. Then, Escherichia coli DH5α was transformed. After positive colonies were obtained by colony PCR identification, the plasmid was expanded and extracted. The PPO12 subcellular localization vector PPO12-PCAMBia35S-EGFP was obtained by sequencing verification. It was transiently expressed in Nicotiana benthamiana through Agrobacterium-mediated transient transformation of tobacco, and the subcellular localization was verified by detecting the GFP fluorescence signal using a laser confocal microscope. The results are shown in Figure 6 shown.

[0060] Figure 6Shown in the figure are the GFP signal (GFP), the chlorophyll autofluorescence signal (Chlorophyll), the superposition signal of the GFP signal, the chlorophyll autofluorescence signal and the bright field (Merged). In the figure, the green fluorescence of the PPO12-PCAMBia35S-EGFP fusion protein is superimposed with the chloroplast autofluorescence (red fluorescence) and mixed into yellow fluorescence. Therefore, it can be determined that the PPO12-PCAMBia35S-EGFP fusion protein is mainly located in the chloroplast, the polyphenol oxidase is located on the thylakoid membrane in the chloroplast, and the enzyme product is located in the thylakoid cavity. Example 4: NtPPO12 gene overexpression experiment

[0061] 1. Cloning of target gene and construction of vector

[0062] The nucleotide sequence of the NtPPO12 gene provided by the present invention is shown in SEQ ID NO.1, and its corresponding amino acid sequence is shown in SEQ ID NO.2. The cDNA extracted from the cultivated tobacco K326 treated in a dark box experiment for 72 hours was used as a template to amplify the NtPPO12 gene coding sequence, and the upstream and downstream primer sequences for amplifying the NtPPO12 gene are shown in SEQ ID NO.6 and SEQID NO.7. The homologous recombination primers PO12-KpnI-F-2.0 and PO12-KpnI-R-2.0 were used to construct an overexpression vector NtPPO12-PCAMBIA35S-eGFP containing the target fragment NtPPO12 gene, and Escherichia coli DH5α was transformed. Single clones were picked, PCR amplified and electrophoresis identified. After the positive strains were expanded and cultured, the plasmids were extracted and transformed into Agrobacterium to infect tobacco leaves.

[0063] NtPPO12-F: AGTAATGGCTTCTCTTCCACT (SEQ ID NO. 6);

[0064] NtPPO12-R: AGGTGTCAATCAATCCTCAAGC (SEQ ID NO.7);

[0065] PO12-KpnI-F-2.0:TTACGAACGATAGCCATGGCTTCTCTTCCACTCC (SEQ ID NO.8);

[0066] PO12-KpnI-R-2.0: TGTTAACAAGGCCTGTCAATCCTCAAGCACAATCTTGACA (SEQ ID NO. 9).

[0067] 2. Screening of positive seedlings of T0 generation plants

[0068] The primers were designed as follows, and the genomic DNA of the regenerated seedlings was extracted and used as a template for PCR amplification verification.

[0069] 35s-F: AGCAAGTGGATTGATGTGA; (SEQ ID NO.14)

[0070] OE-NtPPO12-R:GATGATGTTCGCCGTTGTCG; (SEQ ID NO.15)

[0071] A total of 10 strains overexpressing NtPPO12 (NtPPO12-OE) were obtained by Agrobacterium-mediated genetic transformation of tobacco. Young leaves of T0 generation plants were selected as samples, DNA was extracted, and PCR reaction was performed using the extracted DNA as a template. Figure 7 As shown, Figure 7 1, 2, 3, 4, 7, 8, 9, 10, and 11 are samples, and WT is the wild type of common tobacco K326. The results showed that 7 of the 9 PPO12-OE strains could amplify bands, while the negative control wild type plants could not amplify bands, indicating that the 7 PPO12-OE transgenic seedlings were positive seedlings.

[0072] Total RNA was extracted from leaves of transgenic positive seedlings and reverse transcribed into cDNA for fluorescence quantitative PCR analysis. The quantitative primers were: RTPO12-F: ACTCTTGTTGGTGGTCATC (SEQ ID NO. 16); RTPO12-R: ATCATCGTCTGGAAGTTGTT (SEQ ID NO. 17).

[0073] The results showed that the expression levels of the seven NtPPO12 overexpression strains were significantly higher than those of wild-type tobacco, and the strain NtPPO12-OE-3 had the highest expression level ( Figure 7 ).

[0074] 3. Phenotypic analysis of overexpression strains under dark box treatment

[0075] There was no significant difference in plant type and appearance between the NtPPO12 overexpression strain and the common tobacco K326. Figure 8 At the bud stage, the middle leaves of the T0 generation NtPPO12-OE overexpression positive plants and the control K326 plants were selected for dark treatment to observe the leaf browning phenotype. The results are shown in Fig. 9 shown.

[0076] Referring to Gao Yunpeng et al., the 2023 method was used to quantitatively determine the proportion of green, yellow and brown areas of tobacco leaves (Gao Yunpeng, Fang Song, Wang Yihui et al., Effect of harvest maturity on color change of cigar tobacco leaves during air-drying [J]. Chinese Tobacco Science, 2023, 44(04): 87-93.). The results are shown in Table 1. During the dark box treatment for 0-72h, the green area of ​​the middle leaves gradually decreased, and the yellow area gradually increased. At 72h, the tobacco leaves turned completely yellow, and the NtPPO12-OE strain tobacco leaves turned yellow by 78.49%, which was higher than the control 71.95%. The browning of the middle tobacco leaves mainly occurred after yellowing (72h), and the degree of browning gradually stabilized from 168 to 192h. The degree of browning of tobacco leaves in the NtPPO12-OE overexpression strain was significantly higher than that of the wild-type control. At 192h, the browning area of ​​tobacco leaves in the NtPPO12-OE strain was 67.97%, while that in the control was 53.21%.

[0077] Table 1 The percentage of color change area of ​​the middle leaves of overexpression materials and wild-type tobacco under dark box treatment

[0078]

[0079]

[0080] 4. Determination of polyphenol oxidase activity

[0081] The PPO activity of the NtPPO12-OE overexpression strain and the wild-type strain treated in the dark was determined using a polyphenol oxidase (PPO) kit (Suzhou Grace Biotechnology Co., Ltd., catalog number G0113W).

[0082] Weigh 0.1 g of dried sample (the sample was inactivated at 105°C for 3 min, then dried at 60°C to constant weight, crushed, and passed through a 40-60 mesh sieve to obtain a dried sample), add 1 mL of the extract, homogenize in an ice bath, centrifuge at 4°C × 12000 rpm for 15 min, take the supernatant, and determine the polyphenol oxidase activity.

[0083] The results are as follows Fig.10 As shown, at each time point after dark box treatment, the PPO activity of the middle tobacco leaves of the NtPPO12-OE overexpression line was higher than that of the WT; and the PPO activity was highest at 72h.

[0084] Example 6: Gene Editing

[0085] 1. Gene knockout

[0086] According to the sequence of tobacco NtPPO12 gene (shown in SEQ ID NO.1), the knockout target was designed, and the design of NtPPO12 target sequence is shown in Table 2; the CRISPR / Cas expression vector backbone was linearized and connected with the annealing product, and introduced into the downstream of U6 promoter and upstream of gRNA. The vector also enhances the expression of Cas9 protein through two tandem strong 35S promoters.

[0087] Table 2 Gene editing vector target site sequences

[0088]

[0089] The constructed NtPPO12-CRISPR / Cas9 recombinant plasmid was transformed into tobacco by Agrobacterium-mediated transformation. DNA was extracted from T0 plants after sampling, and Sanger sequencing was performed after PCR amplification (Shenzhen BGI Co., Ltd.). The primers used are shown in Table 3;

[0090] Table 3 Gene editing detection primers

[0091]

[0092] The sequencing results showed that 5 NtPPO12 gene knockout plants were obtained, among which there was a homozygous plant NtPPO12-5-GE, and the mutation type was the insertion of a T base. As shown in Table 4:

[0093] Table 4 Types of homozygous mutations in gene editing

[0094]

[0095] 2. Phenotypic analysis of NtPPO12-GE knockout plants

[0096] At the bud stage, the middle leaves of the T0 generation NtPPO12 gene-edited strain and the wild-type control plants were treated with darkness to observe the browning phenotype. The results are as follows: Fig.11 As shown. Similarly, the proportion of green area, yellow area and brown area of ​​tobacco leaves was determined. The results showed that during the dark box treatment process, the green area of ​​leaves of both NtPPO12 knockout strains and WT gradually decreased and the yellow area increased over time, and the overall browning degree of tobacco leaves showed an upward trend. At 96-120h, the middle leaves of the NtPPO12 knockout strains turned completely yellow, and then the tobacco leaves showed obvious browning at 120h. At 192h, the browning degree of WT was 60.5%, and that of NtPPO12-GE was 39.06%. Overall, the browning degree of the middle leaves of the NtPPO12 knockout strains was significantly reduced compared with the control WT, indicating that the baking resistance and ripening resistance were significantly improved.

[0097]

[0098]

[0099] Table 5 The percentage of color change area of ​​the middle leaves of gene-edited materials and wild-type tobacco under dark box treatment

[0100] 4. Analysis of polyphenol oxidase activity in gene-edited materials

[0101] The PPO activity of the leaves treated in the dark box was further determined. Fig.12 As shown, it can be seen that during the entire dark treatment process, the PPO activity of the NtPPO12 gene-edited strain was lower than that of the control group WT; at 96h, the PPO activity of the NtPPO12-GE strain was significantly lower than that of the control.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

[0103] Sequence Listing

[0104] SEQ ID NO.1: NtPPO12 gene

[0105] ATGGCTTCTCTTCCACTCCCCACCACCAACGCCATCTCTTCTTCATCTTCCT

[0106] CTTCAACTACTTCAACTCTTTCCAATTTGCATTCTTCTACTTTCTTTACGAAG

[0107] ACATCAAAAGTTTCCACTTTAAGAAAGTACGGTAACCATAGTTTCCAAGTC

[0108] TCATGCAAGGGTACAGAAGATGACCAAACTATTAACACTTCCAAATCTTCT

[0109] GATTCTTCAAACAAATAAGATCATTGATAGAAGAAACATGCTACTTGGATTAG

[0110] GAGGCATTTATGGTGCTGCTACTCTTGTTGGTGGTCATCCCTTTGCCTTCGC

[0111] GGCTCCTGTGCCCGGACCTGACGTTTCCAAATGTGGCGCTGCAGATTTGCC

[0112] ACCAGGTGCAGCACCAGTCAACTGTTGTCCTCCGACAACGGCGAACATCA

[0113] TCGACTTCCAACTTCCACCACCGTCAGCCACCCTCCGTACACGGCCAGCAG

[0114] CTCATTCCGCCGATAGTGCCTATATAGAGAAATTCAAACAGAGCTATTCAGCT

[0115] CATGAAACAACTTCCAGACGATGATCCACGTAGCTTCAGGCAACAAGCAA

[0116] ATGTTCATTGTGCTTACTGTGACGGTGCCTATGACCAACTAGGTTTCCCAAA

[0117] CTCTGAACTCCAAGTTCATTTCTCTTGGCTTTTCCTCCCTTTTCATCGTTGTT

[0118] ATCTCTACTTCTTCGAAAAAATCTTGGGAAGTTTGATAAATGACCCTACTTT

[0119] CGTTATCCCATTTTGGAACTGGGATCATCCTGATGGCATGAGACTTCCTGCC

[0120] ATGTATGCGAACCGTAGTTCTTCTCTCTTTGATCCTCTCCGTGATCGGAGGC

[0121] ATCAGCCTCCGGTCATGGTCGATCTCGACTTCCAATGGAACGGATCCTAACAT

[0122] AAGTAACGCTCAACAAACTTCCCAAAATCTCACTATCATGTATAGGCAAAT

[0123] GGTTTCACTAGGAAGTACTCCAGCGACTTTCCTCGGAGACCCTTACCGTGC

[0124] CGGTGGCGAACCGGGTGGTGCTGGGTCCCTCGAGAACATTCCACATGGAC

[0125] CGGTCCATGTTTGGACCGGTGATAGAACCCAACCTAATTTTGAGAACATGG

[0126] GAGATTTTTATTCAGCTGCTAGAGACCCTATTTTCTATGCTCATCATTCTAAT

[0127] ATTGATAGATTGTGGAGTGTTTGGAAAACCCTAGGTGGAAGACGTCAAGAT

[0128] TTTACTGACCCTGATTTCTTAAATGCTTCGTTTTTGTTTTATGATGAGAAAGC

[0129] ACAAATGGTACGTATTAGGGTACGTGACTGTTTGGATACAACAAGACTTGG

[0130] ATACGTTTATCAAGGTGTAGCTAATCCGTGGATAAATTCTCGTCCAAGGGCT

[0131] AGGGTTTCAAGTGCTTTGAGTAGTGTAAGGAGGCTTGTTGAAGCAAGAGC

[0132] AGCTGATAATTTTCCAAGTGCAAAAGATGTTTTCCCAACGAAACTTGACCA

[0133] TGTGATAAGAGTTATGGTAAAGAGGCCAATTAAGAAGAGAAACAAGAAGG

[0134] AGAAAGATGCAAAAGAGGAGTTTTTAGTAGTTGAAGGGATAGAGCTGGAA

[0135] ACTGATGTTTTTGTCAAGTTTGATGTGTTGATTAATGATGAAGATGAGACTG

[0136] TAATTTCGCCGAATAATGCTGAGTTTGCAGGTAGTTTTGTTAACGTGCCACA

[0137] TCATAGTCATGGTAAGAGTGACAAGAAACGTAAAACTAAGTTGAAGTTGG

[0138] CTATAACTGAGCTGTTGGAAGATTTAGATGCTGAGGATGATGATCATGTGGT

[0139] GGTGACTTTTGTTCCAAAGAATGGTTCTGGTGCTGTAAAAATTGGAGGTGT

[0140] CAAGATTGTGCTTGAGGATTGA

[0141] SEQ ID NO.2:NtPPO12 protein

[0142] MASLPLPTTNAISSSSSSSTTSTLSNLHSSTFFTKTSKVSTLRKYGNHSFQVSCK

[0143] GTEDDQTINTSKSSDSSNNKIIDRRNMLLGLGGIYGAATLVGGHPFAFAAPVPG

[0144] PDVSKCGAADLPPGAAPVNCCPPTTANIIDFQLPPPSATLRTRPAAHSADSAYIE

[0145] KFNRAIQLMKQLPDDDPRSFRQQANVHCAYCDGAYDQLGFPNSELQVHFSW

[0146] LFLPFHRCYLYFFEKILGSLINDPTFVIPFWNWDHPDGMRLPAMYANRSSSLFD

[0147] PLRDRRHQPPVMVDLDFNGTDPNISNAQQTSQNLTIMYRQMVSLGSTPATFLG

[0148] DPYRAGGEPGGAGSLENIPHGPVHVWTGDRTQPNFENMGDFYSAARDPIFYA

[0149] HHSNIDRLWSVWKTLGGRRQDFTDPDFLNASFLFYDEKAQMVRIRVRDCLDT

[0150] TRLGYVYQGVANPWINSRPRARVSSALSSVRRLVEARAADNFPSAKDVFPTK

[0151] LDHVIRVMVKRPIKKRNKKEKDAKEEFLVVEGIELETDVFVKFDVLINDEDET

[0152] VISPNNAEFAGSFVNVPHHSHGKSDKKRKTKLKLAITELLEDLDAEDDDHVV

[0153] VTFVPKNGSGAVKIGGVKIVLED

[0154] SEQ ID NO.3: NtPPO12 promoter sequence

[0155] AGAGGAACTTGTTAGCGTATATAAATTAAATCCACGTGATATGAAAATTATA

[0156] ACGCTGTTGCTAGTCAATCTTTACCTAACTTTGTTCTGAGATTTTTCTGTCAC

[0157] TTATCCATTTACAATAGCGTCTTAATATTTAAAGCGATACAAGTTGGACGAA

[0158] GATGAACTTGATTAATACTTTAATTAACTAGTTTTAGACCTTGTTGTTGACTA

[0159] AACATGAAAGAACGTGCATTTTCATCTCTCCGTCAATTTTACATAAATGACT

[0160] AAAATTCATTATAAAAACTGGTCGCAAGTCTTCGCTAAGATTGATGTACGCA

[0161] ACTATCAGAAATGACAATTTGGGAGAAAGAAGAAAATACACCCCGTTCAA

[0162] AGTATTTGTGTTTTCCAGCTGTACACTAAGCAACCAAAGTACATTCAATACC

[0163] TTTACTTTGTTTAGGGGATTCAAATAAATGAGCTCCTCTCTAACTTTTTCACT

[0164] CATTTTCCCCTCTAATGGAACAAAAACAGTATAACGTATAAAAGAATACC

[0165] ACTTTGCAGGGGTAAGATATGATATAGTAGTAACGGCTTGAAGTTGGA

[0166] AAAGAAGAGAGAAATAGTGAATTACCTTATAAAGAATCATACAGTCAATGT

[0167] GCAGTGCATATATAAAATATTTAGCACACACCAACAAAACGACTTTATTAAA

[0168] GTACCAAAGACCACGTACGAAAGTATAGATTTCGTGAATTTAGCAATATATA

[0169] TTGATTTAAAGGTTTTTTTTGGAATCATCATTAATAGTTGTTTGGAAAATATTT

[0170] GGTTAAGTTGGAAAAAAAAAGTATTTGAAATTGAAGTTGAAAAAAAATACTT

[0171] AGAGCCCGTTTGGATTGACTTATTTTAATAGCCCGTTTGGACATAAGAAATT

[0172] TTTCCCCTTTTTAAAAAAATTCATTTTTTTTTAAATCAGCATTTGTTTATAA

[0173] AATTTTCCAATTTTTCAATTGAAGATTATTTTGGAATTAGCAAGATTATTAAT

[0174] TTATTACTTTTGTGCATTGGAGAAAGATAAGGAGAGTTTTATTAATTTCCATT

[0175] TATTCTACTCTTTCGACATTATCGCTGAATGGTCGGTTGATGTTATACAAGGC

[0176] TGCACGTCTTGATTCTTGCCTAACTGGATAAATAAAATAGCACGTACTTGT

[0177] AAACCTGATATACTTGTTAATTATGAGCAATATTTTCCCCCTGTTTATTTAAATT

[0178] TCCTGTTACAATATAATATTGGTTAACACCATTATCTTTCTACATGTTGAATAA

[0179] TTGTTGAAAAAATGAAGGCGTTGAAGCTTCATAACGGCTTCCTATTTGAG

[0180] ATTGCATCGAAACTTGCTGCTATATTGTAAAAAAAGAAGTCTAAACTAATA

[0181] TTAATTTTGACACGGCAGACATAATATTTGAAAAAATCACTAATTTCTTCT

[0182] AAACCAACCAGCTATATCTAGAACTTGAAGGACAATTCCATTTCTCTGTGCA

[0183] GTGATTATTCATAAAACAAATAAGGTTTTTGCTCATTCCGGTCCAAAATAA

[0184] GTGATTTTTTGGTTGTTTTCACACATATTAAAAAAATCACCTTTTTAACATTAA

[0185] TTAGCATTGAAATTGACTATTATAACCCTTACTATCTTTTCACATAAATACTCC

[0186] TAACACATACTCTAATGTTATTTACTCCAAGGGCAATGTAGGAAAAAAATAA

[0187] TTAATTCATTATTGAATCTGAAAAAATTTTTTTGGACAAAAAAAAAAA

[0188] AAACAAAAATCACTTATTGGACCCGGAGAGTAATTCTTGTAAAA

[0189] TAATTAAAAAGGTCAATTAWINCTWORTHACGTGGATTTTGTAT

[0190] TAGTGCCTTCATCAAATTCCGAATTAAATAAACAAATAAATAAATATACG

[0191] AAGAAAATCACAGGCTGCTGGAAGTTAAGATATGCAAGCATGCATCTTCCT

[0192] GACATGTTTCTTTTTGGTATTTTTTCTTTTTCTTTTTCACCACAAGG

[0193] TTATACAGAAAGTCTTTGCTCCATATTCTTATAAAATGCTAACCGTAGATGT

[0194] TCATGAACTATCTCACAAAAAAAAAAATTACK

[0195] SEQ ID NO.4:AGAGGAACTTGTTAGCGTAT

[0196] SEQ ID NO.5:AGGAAGATGAAGAGATGG

[0197] SEQ ID NO.6:AGTAATGGCTTCTCTTCCACT;

[0198] SEQ ID NO.7:AGGTGTCAATCAATCCTCAAGC

[0199] SEQ ID NO.8:TTACGAACGATAGCCATGGCTTCTCTTCCACTCC SEQ ID NO.9:TGTTAACAAGGCCTGTCAATCCTCAAGCACAATCTTGACA SEQ ID NO.10:GAAGAGATGGCGTTGGTGGTGGG

[0200] SEQ ID NO.11:ATTTGGAAGTGTTAATAGTTTGG SEQ ID NO.12:

[0201] AATTCGAGCTCGGTACCCGGGGATCCAGAGGAACTTGTTAGCGTATSEQ ID NO.13:

[0202] TTACCCTCAGATCTACCATGGTACTATAATTTTTGCTTGGTTTGAG

[0203] SEQ ID NO.14:AGCAAGTGGATTGATGTGA

[0204] SEQ ID NO.15:GATGATGTTCGCCGTTGTCG

[0205] SEQ ID NO.16:ACTCTTGTTGGTGGTCATC

[0206] SEQ ID NO.17:ATCATCGTCTGGAAGTTGTT

[0207] SEQ ID NO.18:ACCACAAGGTTATACAGAAAAGTCT

[0208] SEQ ID NO.19:ATGTAACAGTACCTGGTGGC

[0209] SEQ ID NO.20:CCACCTACCAACG

[0210] SEQ ID NO.21:CCACCTACCAACG SEQ ID NO.22:CCACCACCAACG.

Claims

1. Application of inhibiting or knocking out tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Specifically, it is used to inhibit tobacco browning, thereby improving tobacco's resistance to ripening and baking.

3. The use according to claim 1 or 2, characterized in that: Specifically, it is used in inhibiting tobacco browning during the tobacco maturation and baking stages.

4. The use according to claim 1, characterized in that: The method for inhibiting the tobacco polyphenol oxidase gene NtPPO12 includes a reagent for inhibiting the expression of the gene; and the method for knocking out the tobacco polyphenol oxidase gene NtPPO12 includes targeted knockout using CRISPR / Cas9 technology.

5. The use according to claim 4, characterized in that: A targeted knockout site of the gene NtPPO12 was designed, and a mutant base was inserted into the gene NtPPO12 to inactivate the expression of the gene NtPPO12.

6. The use according to claim 5, characterized in that: The knockout target sequences of gene NtPPO12 are shown in SEQ ID NO.10 and SEQ ID NO.

11.

7. Application of a protein that inhibits the expression of tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning; its amino acid sequence is shown in SEQ ID NO.2; specifically, application of a protein in inhibiting tobacco browning to thereby improve tobacco's resistance to ripening and baking; more specifically, application of a protein in inhibiting tobacco browning during the tobacco ripening and baking stages.

8. Use of the tobacco polyphenol oxidase gene NtPPO12 according to claim 1 in screening tobacco varieties with low browning.

9. A promoter driving the expression of tobacco polyphenol oxidase gene NtPPO12, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

3.

10. Use of the promoter according to claim 9 in promoting the expression of tobacco polyphenol oxidase gene NtPPO12.

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