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

Abstract

The present application provides an application of inhibiting or knocking out tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning. The present application screens an important gene NtPPO12 related to tobacco browning through transcriptome sequencing and molecular biology experiments, further verifies the promoter activity and gene expression site of NtPPO12, and deeply researches the function of the gene through gene editing and overexpression. The present application proves by molecular biology experiments that the NtPPO12 gene provided by the present application is a key gene related to browning in the mature curing process of tobacco, and by regulating the expression amount of the NtPPO12 gene, the browning degree of tobacco can be controlled, and the tobacco maturity and curing resistance can be improved, which is beneficial to reducing the picking frequency, improving the curing characteristics, and improving the appearance and internal quality of tobacco leaves, so it has a broad market application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tobacco genes, and particularly relates to application of inhibition or knockout of tobacco polyphenol oxidase gene NtPPO12 in inhibition of tobacco browning and improvement of tobacco resistance to maturity and resistance to curing in the mature curing process, and also relates to application of the gene protein, application of the gene in screening of tobacco varieties with low browning, and the gene promoter and application thereof. BACKGROUND

[0002] The curing property of tobacco leaves is an important factor affecting the quality of tobacco leaves, including two aspects of "easy curing" and "curing resistance". "Easy curing" mainly refers to the yellowing property of tobacco leaves in the early curing stage and the ease of color fixation after yellowing. The tobacco leaves that are easy to turn yellow and fix color are considered to be easy to cure. "Curing resistance" mainly refers to the tolerance of tobacco leaves to the curing environment in the color fixation stage in the later curing stage. The tobacco leaves that are not easy to turn brown are considered to have good curing resistance.

[0003] In the curing process, the browning of the color of tobacco leaves is mainly due to the conversion of polyphenol substances in tobacco leaves into dark substances such as quinone 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 cured tobacco leaves. Whether the tobacco leaves are easy to turn brown after curing and entering the color fixation stage is an important indicator for measuring the color fixation property of tobacco leaves. Tobacco enzymatic browning reaction can cause tobacco leaves to turn gray, steam sheet, black dregs, etc., and also cause the tobacco leaves to become thin, poor in elasticity, easy to break, etc. At the same time, curing resistance and maturity resistance have certain correlation. Improving the maturity resistance of tobacco leaves in the mature period can reduce the number of harvests and promote the integration of agricultural machinery and agricultural technology.

[0004] The easy curing and curing resistance of tobacco leaves are affected by genetic factors and are important related to the activity of polyphenol oxidase PPO. Through bioinformatics analysis and expression profile analysis, it is found that 12 PPO family member genes in the tobacco genome, although part of the genes are verified to have reduced PPO enzyme activity through gene knockout or RNA interference experiment, no evidence is found that these PPO genes regulate PPO enzyme activity and inhibit browning in the mature and curing process. Tobacco maturity and curing are two key periods of tobacco production. Only when the maturity and curing quality of tobacco leaves meet the processability and usability requirements of cigarettes, the tobacco leaves have the best commodity value, use value and economic value.

[0005] At present, there is almost no gene that can significantly regulate tobacco enzymatic browning reaction in the tobacco maturity and curing period. There is no related report on the use of genetic engineering means to reduce the activity of polyphenol oxidase in this special period to reduce the occurrence of enzymatic browning reaction. The NtPPO12 gene of the application plays a significant role in the two key periods of tobacco production, i.e. maturity and curing, and has important application value for improving the maturity resistance and curing resistance. SUMMARY

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

[0007] The primer sequence for amplifying the tobacco polyphenol oxidase gene NtPPO12 is shown as SEQ ID NO. 6 and SEQ ID NO. 7.

[0008] The above-mentioned application is specifically used in inhibiting tobacco browning, thereby improving tobacco maturity tolerance and baking tolerance.

[0009] More specifically, the application is used in inhibiting tobacco browning during tobacco maturation and baking.

[0010] Further,

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

[0012] Further,

[0013] The targeted knocking out site of the gene NtPPO12 is designed, and a mutant base is inserted into the gene NtPPO12 to inactivate the expression of the gene NtPPO12.

[0014] The knocking out target sequence of the gene NtPPO12 is shown as SEQ ID NO. 10 and SEQ ID NO. 11.

[0015] The second aspect of the present application is to provide an application of a protein for inhibiting the expression of tobacco polyphenol oxidase gene NtPPO12 in inhibiting tobacco browning, wherein the amino acid sequence is shown as SEQ ID NO. 2.

[0016] The application is specifically used in inhibiting tobacco browning, thereby improving tobacco maturity tolerance and baking tolerance.

[0017] More specifically, the application is used in inhibiting tobacco browning during tobacco maturation and baking.

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

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

[0020] The primer sequence for amplifying the promoter is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0021] The fifth aspect of the present application is to provide the application of the promoter in starting the expression of tobacco polyphenol oxidase gene NtPPO12.

[0022] Compared with the prior art, the advantages and technical effects of the present application are: the present application first screens out the key gene NtPPO12 related to tobacco browning during the maturation and curing period, further explores the activity and expression site of the NtPPO12 promoter by constructing a GUS staining vector, constructs a subcellular localization vector of NtPPO12 and transforms N. benthamiana to detect the expression of GFP protein, and deeply studies the function of the gene through gene editing and overexpression. The present application proves by molecular biology experiments that the NtPPO12 gene provided by the present application is an important gene related to tobacco browning during maturation and curing, and by regulating the expression amount of the NtPPO12 gene, the browning of tobacco can be effectively controlled, the maturation resistance and curing resistance can be improved, and the present application is used for cultivating excellent varieties, improving the quality of cured tobacco leaves, and has broad market prospect and economic benefit.

[0023] The present application will be further described in detail in combination with the drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

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

[0031] Figure 8Figure 1 is a picture of NtPPO12 overexpression line (right) and common tobacco K326 (left) appearance comparison;

[0032] Figure 9 Figure 2 is a picture of appearance changes of middle leaves of control (WT) and NtPPO12 overexpression materials under dark box treatment;

[0033] Figure 10 Figure 3 is a result of PPO activity of middle leaves of NtPPO12 overexpression line and control (WT) line;

[0034] Figure 11 Figure 4 is a picture of appearance changes of middle leaves of control (WT) and NtPPO12 gene editing materials under dark box treatment;

[0035] Figure 12 Figure 5 is a result of PPO activity of middle leaves of NtPPO12 gene editing line and control (WT). DETAILED DESCRIPTION

[0036] In order to improve the understanding of the present application, the present application is further described below through preferred embodiments. It should be understood by those skilled in the art that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

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

[0038] 1. Expression analysis of polyphenol oxidase genes in tobacco leaves under dark box simulation curing

[0039] According to the tobacco industry standard YC / T 311-2009, a dark box environment was constructed to simulate curing. The middle leaves of K326 variety at the mature stage were placed in the paper box, and the leaves were taken out every 12 hours for photography until the leaves turned brown. The samples were taken every 24 hours and stored in a -80°C low-temperature refrigerator for transcriptome sequencing.

[0040] The yellowing and browning phenotype of tobacco leaves under dark treatment stress was observed, as shown in Figure 1 To identify the polyphenol oxidase genes that play a role in the browning of tobacco leaves, the expression amount of 12 PPO genes in tobacco was analyzed. The HTSeq software was used to count the number of reads mapped to each gene, the expression amount was standardized using FPKM value, and a heat map was drawn. The results are shown in Figure 2 Among the 12 NtPPO genes, NtPPO12 was significantly up-regulated with the progress of dark stress, indicating that NtPPO12 gene is involved in the browning reaction of tobacco leaves in the late maturation and curing process.

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

[0042] In order to verify the differential expression of NtPPO12 gene in transcriptome sequencing analysis, the expression of NtPPO12 gene in the samples of 0h, 24h, 48h, 72h and 96h in the dark box was verified by qRT-PCR technology. Figure 3 The experimental results show that the expression amount of NtPPO12 is significantly up-regulated at 72h in the dark box, and the qRT-PCR analysis further shows that NtPPO12 gene is related to tobacco browning in the mature stage and under dark stress.

[0043] Example 2: Expression pattern analysis of NtPPO12 promoter

[0044] 1. Amplification of the promoter of NtPPO12 gene

[0045] In order to study the expression characteristics of NtPPO12 gene, the promoter of NtPPO12 gene was first cloned (as shown in SEQ ID NO. 3), and the cloned sequence was analyzed. Figure 4

[0046] The upstream sequence of 3000bp of NtPPO12 gene was used as a reference sequence, and specific primers ProNtPPO12-F / R were designed:

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

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

[0049] The fragment was obtained from the genome of Nicotiana tabacum K326 using the primers ProNtPPO12-F / R, and the cloning sequencing showed that the length of the amplified promoter fragment ProNtPPO12 was 2074bp (as shown in SEQ ID NO. 3). The sequence was introduced into the PlantCARE database, and the cis-acting elements in the NtPPO12 promoter region were analyzed, and it was found that in addition to a large number of TATA boxes and CAAT boxes possessed by general promoters, and a large number of light response elements (I-box, LTR, G-Box, GA-motif, GATA-motif, GT1-motif, etc.), there are also many specific and stress response elements.

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

[0051] ​After purification of the NtPPO12 promoter amplification product, it is connected to the TA cloning vector, i.e. TA-PPO12-pro. Using the primer GUS-NtPPO12-F / R with a linker, PCR amplification is performed with this template, and the product is recovered by gel cutting. The PCAMBIA1301 vector is digested with BamHI and Ncol to remove the 35S promoter on the vector, and gel recovery is performed. Then the NtPPO12 promoter sequence is connected to the PCAMBIA1301 vector by seamless cloning, and DH5a competent cells are transformed by heat shock method, identified by colony PCR, and the plasmid is extracted by expanding culture for sequencing verification. Finally, the correctly constructed expression vector ProNtPPO12-1301 is used for genetic transformation of tobacco.

[0052] GUS-NtPPO12-F:

[0053] AATTCGAGCTCGGTACCCGGGGATCCAGAGGAACTTGTTAGCGTAT;

[0054] (SEQ ID NO. 12)

[0055] GUS-NtPPO12-R:

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

[0057] Figure 5 The results show that GUS staining is positive in the leaves and flowers of the transformed plants, indicating that the NtPPO12 promoter regulates the expression of the GUS gene in the leaves and flowers, especially the deeper blue color in the leaf vascular bundles and glandular hairs.

[0058] Example 3, Subcellular localization

[0059] To detect the localization of PPO12 protein in tobacco cells, GFP fluorescent protein gene is fused to the C-terminal of PPO12. First, the PCAMBIA35S-EGFP vector is digested with Kpnl, and then the target gene is connected to the vector by seamless cloning method, followed by transformation of E. coli DH5a. After obtaining positive colonies by colony PCR, the plasmid is extracted by expanding culture, and sequencing verification is performed to obtain the PPO12 subcellular localization vector PPO12-PCAMBia35S-EGFP. Through Agrobacterium-mediated transient transformation of tobacco, it is transiently expressed in Nicotiana benthamiana, and the GFP fluorescent signal is detected by laser confocal microscope to verify the subcellular localization, and the results are shown in Figure 6

[0060] Figure 6 ​The GFP signal (GFP), chlorophyll autofluorescence signal (Chlorophyll), and the superimposed signal of the GFP signal and the chlorophyll autofluorescence signal and bright field (Merged) are shown in the figure. The green fluorescence of the PPO12-PCAMBia35S-EGFP fusion protein is mixed with the red fluorescence of the chloroplast autofluorescence to form yellow fluorescence after superimposition, and thus 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 product of the enzyme is located in the thylakoid cavity.

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

[0062] The nucleotide sequence of the NtPPO12 gene provided in the present application is shown in SEQ ID NO. 1, and the corresponding amino acid sequence is shown in SEQ ID NO. 2. The cDNA extracted from the cultivated tobacco K326 after 72h treatment in the dark box experiment is used as a template to amplify the coding sequence of the NtPPO12 gene, and the upstream and downstream primer sequences of the NtPPO12 gene are shown in SEQ ID NO. 6 and SEQ ID NO. 7. The homologous recombination primers PO12-KpnI-F-2.0 and PO12-KpnI-R-2.0 are used to construct the overexpression vector NtPPO12-PCAMBIA35S-eGFP containing the target fragment NtPPO12 gene, and to transform E. coli DH5α. Single colonies are picked, PCR amplified and electrophoresed for identification. The positive strain is cultured, the plasmid is extracted, transformed into Agrobacterium, and the tobacco leaves are infected.

[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 primer was designed as follows, and the regenerated seedling genomic DNA was extracted to perform PCR amplification verification.

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

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

[0071] The genetic transformation of tobacco was performed by using Agrobacterium mediation, and 10 NtPPO12 overexpression (NtPPO12-OE) strains were obtained. The young leaves of T0 generation plants were selected as samples, and DNA was extracted. The extracted DNA was used as a template for PCR reaction. As shown in Figure 7 Figure 7 1, 2, 3, 4, 7, 8, 9, 10, and 11 are samples, and WT is wild-type Nicotiana tabacum K326. The results show that 7 of the 9 PPO12-OE strains can amplify bands, and the negative control wild-type plants cannot amplify bands, indicating that 7 PPO12-OE transgenic seedlings are positive seedlings.

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

[0073] The results show that the expression of 7 NtPPO12 overexpression strains is significantly higher than that of wild-type tobacco, and the expression of strain NtPPO12-OE-3 is the highest Figure 7 .

[0074] 3, phenotype analysis of overexpression strains under dark box treatment

[0075] The NtPPO12 overexpression strains have no obvious difference in plant type and appearance from Nicotiana tabacum K326, as shown in Figure 8 . The middle leaves of T0 generation NtPPO12-OE overexpression positive plants and control K326 plants were selected for dark treatment at the present budding stage, and the leaf browning phenotype was observed. The results are shown in Figure 9 .

[0076] ​Refer to Gao Y, et al. 2023. Quantitative determination of green area, yellow area and brown area percentage of tobacco leaves (Gao Y, Fang M, Wang YH, et al. Effects of harvesting maturity on color changes of cigar tobacco leaves during curing process [J]. China Tobacco Science, 2023, 44(04): 87-93.). The results are shown in Table 1. The green area of the middle leaves gradually decreased, and the yellow area gradually increased during the 0-72h dark box treatment. At 72h, the tobacco leaves were completely yellow, and the NtPPO12-OE line had a yellowing rate of 78.49%, which was higher than the control of 71.95%. The browning of the middle leaves mainly occurred after yellowing (72h), and the browning degree gradually stabilized at 168-192h. The browning degree of the NtPPO12-OE overexpression line was significantly higher than that of the wild type control. At 192h, the browning area of the NtPPO12-OE line was 67.97%, and that of the control was 53.21%.

[0077] Table 1 Percentage of color change area of middle leaves of overexpression materials and wild type tobacco under dark box treatment

[0078]

[0079]

[0080] 4. Determination of polyphenol oxidase activity

[0081] The polyphenol oxidase (PPO) kit (Suzhou Gexisi Biological Technology Co., Ltd., Catalog No. G0113W) was used to determine the PPO activity of the NtPPO12-OE overexpression line and the wild type line under dark box treatment.

[0082] 0.1g of oven-dried sample (the sample was killed at 105℃ for 3min, then oven-dried at 60℃ to constant weight, crushed, and sieved through a 40-60 mesh sieve to obtain the oven-dried sample) was weighed, 1mL of extraction solution was added, and homogenization was performed in an ice bath. The supernatant was taken after centrifugation at 4℃ and 12000rpm for 15min, and the polyphenol oxidase activity was determined.

[0083] The results are shown in Table 1. The PPO activity of the NtPPO12-OE overexpression line was higher than that of the WT at each time point after dark box treatment, and the PPO activity was highest at 72h. Figure 10

[0084] Example 6: Gene editing

[0085] 1. Gene knockout

[0086] ​According to the sequence of tobacco NtPPO12 gene (shown in SEQ ID NO. 1), a knockout target was designed, and the NtPPO12 target sequence design is shown in Table 2; after linearization treatment of the CRISPR / Cas expression vector skeleton, the annealing product was connected, which was introduced into the gRNA upstream region downstream of the U6 promoter, and the vector also enhanced the expression of Cas9 protein through two 35S strong promoters in series.

[0087] Table 2 Target site sequence of gene editing vector

[0088]

[0089] The constructed NtPPO12-CRISPR / Cas9 recombinant plasmid was transformed into tobacco by Agrobacterium mediation. After sampling T0 generation plants, DNA was extracted, PCR amplified and subjected to Sanger sequencing (Shenzhen Huada Gene Co., Ltd.), and the primers used are shown in Table 3;

[0090] Table 3 Detection primers for gene editing

[0091]

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

[0093] Table 4 Homozygous mutation types of gene editing

[0094]

[0095] 2, Phenotype analysis of NtPPO12-GE knockout plants

[0096] The middle leaves of T0 generation NtPPO12 gene editing strains and wild type control plants were selected at the present budding stage and subjected to dark treatment, and the browning phenotype was observed, and the results are shown in Figure 11 Similarly, the proportions of green area, yellow area and brown area of tobacco leaves were determined, and the results showed that during the dark box treatment process, the green area of the leaves of NtPPO12 gene knockout strains and WT gradually decreased with time, the yellow area increased, and the browning degree of tobacco leaves showed an overall upward trend. In 96-120h, the middle leaves of NtPPO12 gene knockout strains were completely yellow, and then 120h tobacco leaves showed obvious browning, and at 192h, the browning degree of WT was 60.5%, and that of NtPPO12-GE was 39.06%. Overall, the browning degree of NtPPO12 gene knockout strains in the middle leaves was significantly lower than that of control WT, indicating that the baking resistance and baking tolerance were significantly improved.

[0097]

[0098]

[0099] Table 5 Percentage of color change area of middle leaves of gene editing material and wild type tobacco under dark treatment

[0100] 4. Analysis of polyphenol oxidase activity of gene editing material

[0101] Further PPO activity determination was performed on dark-treated leaves, and the results are shown in Table 6. Figure 12 As can be seen, the PPO activity of the NtPPO12 gene editing strain was lower than that of the control group WT during the entire dark treatment process; at 96h, the PPO activity of the NtPPO12-GE strain was significantly lower than that of the control.

[0102] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, for those skilled in the art, the technical solutions recorded in the foregoing examples can still be modified, or some technical features therein can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

[0103] SEQUENCE LISTING

[0104] SEQ ID NO. 1: NtPPO12 gene

[0105] ATGGCTTCTCTTCCACTCCCCACCACCAACGCCATCTCTTCTTCATCTTCCT

[0106] CTTCAACTACTTCAACTCTTTCCAATTTGCATTCTTCTACTTTCTTTACGAAG

[0107] ACATCAAAAGTTTCCACTTTAAGAAAGTACGGTAACCATAGTTTCCAAGTC

[0108] TCATGCAAGGGTACAGAAGATGACCAAACTATTAACACTTCCAAATCTTCT

[0109] GATTCTTCAAACAATAAGATCATTGATAGAAGAAACATGCTACTTGGATTAG

[0110] GAGGCATTTATGGTGCTGCTACTCTTGTTGGTGGTCATCCCTTTGCCTTCGC

[0111] GGCTCCTGTGCCCGGACCTGACGTTTCCAAATGTGGCGCTGCAGATTTGCC

[0112] ACCAGGTGCAGCACCAGTCAACTGTTGTCCTCCGACAACGGCGAACATCA

[0113] TCGACTTCCAACTTCCACCACCGTCAGCCACCCTCCGTACACGGCCAGCAG

[0114] CTCATTCCGCCGATAGTGCCTATATAGAGAAATTCAACAGAGCTATTCAGCT

[0115] CATGAAACAACTTCCAGACGATGATCCACGTAGCTTCAGGCAACAAGCAA

[0116] ATGTTCATTGTGCTTACTGTGACGGTGCCTATGACCAACTAGGTTTCCCAAA

[0117] CTCTGAACTCCAAGTTCATTTCTCTTGGCTTTTCCTCCCTTTTCATCGTTGTT

[0118] ATCTCTACTTCTTCGAAAAAATCTTGGGAAGTTTGATAAATGACCCTACTTT

[0119] CGTTATCCCATTTTGGAACTGGGATCATCCTGATGGCATGAGACTTCCTGCC

[0120] ATGTATGCGAACCGTAGTTCTTCTCTCTTTGATCCTCTCCGTGATCGGAGGC

[0121] ATCAGCCTCCGGTCATGGTCGATCTCGACTTCAATGGAACGGATCCTAACAT

[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] CATTTTCCCTCTAATGGAACAAAAAAACAGTATAACGTATAAAAGAATACC

[0165] ACTTTGCAGGGGTAAAAGATAATGATATAGTAGTAACGGCTTGAAGTTGGA

[0166] AAAGAAGAGAGAAATAGTGAATTACCTTATAAAGAATCATACAGTCAATGT

[0167] GCAGTGCATATATAAAATATTTAGCACACACCAACAAAACGACTTTATTAAA

[0168] GTACCAAAGACCACGTACGAAAGTATAGATTTCGTGAATTTAGCAATATATA

[0169] TTGATTTAAAGGTTTTTTTGGAATCATCATTAATAGTTGTTTGGAAAATATTT

[0170] GGTTAAGTTGGAAAAAAAAAGTATTTGAAATTGAAGTTGAAAAAAATACTT

[0171] AGAGCCCGTTTGGATTGACTTATTTTAATAGCCCGTTTGGACATAAGAAATT

[0172] TTTCCCTTTTTAAAAAAAAATTCATTTTTTTTTAAATCAGCATTTGTTTATAA

[0173] AATTTTCCAATTTTTCAATTGAAGATTATTTTGGAATTAGCAAGATTATTAAT

[0174] TTATTACTTTTGTGCATTGGAGAAAGATAAGGAGAGTTTTATTAATTTCCATT

[0175] TATTCTACTCTTTCGACATTATCGCTGAATGGTCGGTTGATGTTATACAAGGC

[0176] TGCACGTCTTGATTCTTTGCCTAACTGGATAAATAAAATAGCACGTACTTGT

[0177] AAACCTGATATACTTGTTAATTATGAGCAATATTTTCCCCTGTTTATTTAAATT

[0178] TCCTGTTACAATATAATATTGGTTAACACCATTATCTTTCTACATGTTGAATAA

[0179] TTGTTGAAAAAATGAAGGCGTTGAAGCTTCATAAACGGCTTCCTATTTGAG

[0180] ATTGCATCGAAACTTGCTGCTATATTGTAATAAAAAGAAGTCTAAACTAATA

[0181] TTAAATTTTGACACGGCAGACATAATATTTGAAAAAATCACTAATTTCTTCT

[0182] AAACCAACCAGCTATATCTAGAACTTGAAGGACAATTCCATTTCTCTGTGCA

[0183] GTGATTATTCATAAAACAAATAAAAGTTTTTGCTCATTCCGGTCCAAAATAA

[0184] GTGATTTTTTGGTTGTTTTCACACATATTAAAAAAATCACCTTTTAACATTAA

[0185] TTAGCATTGAAATTGACTATTATAACCCTTACTATCTTTTCACATAAATACTCC

[0186] TAACACATACTCTAATGTTATTTACTCCAAGGGCAATGTAGGAAAAAAATAA

[0187] TTAATTCATTATTGAAATCTGAAAAAATCATTTATTTTGGACAAAAAGAAAA

[0188] AAACCAAAAAATCACTTATTATGGACCGGAGAGAGTACAATTCTTGTAAAA

[0189] TAAATTAAAAAGGTCATAATTACAACATCTCAGACAAACGTGGATTTTGTAT

[0190] TAGTGCCTTCATCAAATTCCGAATTAAATAAACAAATAAATAAATAAATACG

[0191] AAGAAAATCACAGGCTGCTGGAAGTTAAGATATGCAAGCATGCATCTTCCT

[0192] GACATGTATTTCTTTTTGGTTAGTATTTTTTCTTTTTCTTTTTCACCACAAGG

[0193] TTATACAGAAAAGTCTTTGCTCCATATTCTATATAAATGCTAACCGTAGATGT

[0194] TCATGAACTACATCTCAAACCAAGCAAAAATTATAGTA

[0195] SEQ ID NO. 4: AGAGGAACTTGTTAGCGTAT

[0196] SEQ ID NO. 5: AGGAAGATGAAGAAGAGATGG

[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. Inhibition or knockout of tobacco polyphenol oxidase gene NtPPO12 In the application of inhibiting tobacco browning, the nucleotide sequence is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Specifically, the application is used for inhibiting tobacco browning, and further improving tobacco maturity and baking resistance.

3. Use according to claim 1 or 2, characterized in that, Specifically, the application is used for inhibiting tobacco browning in tobacco maturity and baking period.

4. Use according to claim 1, characterized in that, The knockout tobacco polyphenol oxidase gene NtPPO12 includes targeted knockout using CRISPR / Cas9 technology.

5. Use according to claim 4, characterized in that, Design genes NtPPO12 Targeted knockout sites ， Insertion of a mutant base in a gene NtPPO12 inactivates the expression of the gene NtPPO12 ​ 6. Use according to claim 5, characterized in that, Gene NtPPO12 The knockout target sequence of the gene is shown as SEQ ID NO. 10 and SEQ ID NO.

11.

7. Inhibition of tobacco polyphenol oxidase genes NtPPO12 The use of the expressed protein in the inhibition of tobacco browning; the amino acid sequence is shown as SEQ ID NO.

2.

8. Use according to claim 7, characterized in that, Specifically, the application is used for inhibiting tobacco browning, and further improving tobacco maturity and baking resistance.

9. Use according to claim 7, characterized in that, Specifically, the application is used for inhibiting tobacco browning in tobacco maturity and baking period.

10. A tobacco polyphenol oxidase gene NtPPO12 A promoter for expression characterized in that, The nucleotide sequence is shown as SEQ ID NO.

3.

11. Use of the promoter of claim 10 to initiate expression of a tobacco polyphenol oxidase gene NtPPO12 Table 2.

Images