Ntppo gene homozygous mutant and application thereof

Abstract

The application discloses a NtPPO gene homozygous mutant and application thereof, and the application is: (1) reducing polyphenol oxidase activity of tobacco leaves; (2) reducing browning degree of tobacco in curing; the NtPPO gene mutant comprises mutant types of NtPPO1, NtPPO2, NtPPO3 and NtPPO4; the NtPPO gene mutant comprises mutant types of NtPPO1, NtPPO2, NtPPO3 and NtPPO4; the nucleotide sequence of the NtPPO1 gene mutant type is shown in SEQ ID NO. 1; the nucleotide sequence of the NtPPO2 gene mutant type is shown in SEQ ID NO. 2; the nucleotide sequence of the NtPPO3 gene mutant type is shown in SEQ ID NO. 3; and the nucleotide sequence of the NtPPO4 gene mutant type is shown in SEQ ID NO. 4.

Description

Technical Field

[0001] This invention relates to a homozygous mutant of the NtPPO gene and its applications, belonging to the field of genetic engineering technology. Background Technology

[0002] Tobacco (Nicotiana acuminata L.) is a commercially valuable crop. The quality of tobacco leaves is a crucial indicator of tobacco quality. Browning of tobacco leaves during curing reduces tobacco quality, thus increasing production costs. Curing is a key step in converting fresh tobacco leaves into commercial tobacco, during which the color and grade of the tobacco are significantly affected by the metabolic characteristics of polyphenolic compounds. Studies have shown that as curing progresses, the total polyphenol content in tobacco leaves increases. Under the action of peroxidase (POD) and polyphenol oxidase (PPO), polyphenols are easily oxidized to form quinones and their polymers, ranging from light red to dark brown, causing the tobacco leaves to gradually turn brown. Therefore, polyphenol oxidase plays an important role in regulating the browning of tobacco leaves.

[0003] Polyphenol oxidases (PPOs) are common copper-binding enzymes found in many plant species. Three common conserved domains in PPO genes include the tyrosinase domain (tyrosinase), the C-terminal conserved domain (DWL, KFDV), and the N-terminal transport peptide. The tyrosinase domain contains two copper ion active sites, Cu-A and Cu-B. The N-terminal transport peptide is primarily responsible for the transport of PPO proteins to the chloroplast membrane. The conserved C-terminal domain is typically hydrolyzed upon translocation to the plastid, thus initiating its function. Based on their function, PPOs are currently classified into tyrosinases (TYRs, EC1.14.18.1), laccases (MCOs, EC1.10.3.2), and catechol oxidases (COs, EC1.10.3.1). Tyrosine converts monophenols to catechols, which are further oxidized to o-quinones. Laccases oxidize o-, m-, and p-diphenols. However, catechol oxidase only utilizes its enzymatic activity to oxidize catechol to o-quinone. MCO oxidizes p-diol dioxygenase.

[0004] Polyphenol oxidase (PPO) is primarily found in the chloroplasts of tobacco leaf tissue. PPO catalyzes the decomposition of catechols, gallic acid, chlorogenic acid, and other phenolic compounds present in vesicles. Under normal conditions, PPO, oxygen, and phenolic substances are not readily available. The process of curing tobacco raises the temperature, disrupting membrane systems and leading to the release of large amounts of protoplasm, increasing membrane permeability while gradually decreasing selective permeability. Therefore, tobacco is exposed to oxygen, causing intracellular enzymes and substrates to bind, ultimately resulting in browning. During production, a small amount of polyphenol oxidation can significantly improve tobacco quality, providing a product with a unique aroma. However, tobacco browning is usually caused by excessive polyphenol oxidation during the tobacco curing process. Consequently, the intrinsic quality and appearance of tobacco deteriorate, its industrial usability decreases, and tobacco production is severely hampered.

[0005] Appropriate browning during tobacco processing can improve flavor, while excessive browning, in addition to altering flavor, can reduce nutritional content and shorten shelf life. Therefore, browning is an important consideration in food preparation and storage. PPO catalyzes the oxidation of phenolic compounds to quinones, which are the main source of enzymatic browning. Current PPO inhibition strategies mainly include physical methods, such as reducing or destroying enzyme activity by changing temperature and pressure; chemical methods, which involve adding chemicals and using reduction, chelation, complexation, and acidification to inhibit PPO activity; and plant extracts, which can be used as alternatives to chemical reagents, by competing with substrates for PPO active sites to weaken the browning reaction. Traditional methods for addressing tobacco leaf browning include adjusting tobacco harvesting strategies, using relevant fertilizers, and changing curing procedures. However, all of the above methods have limitations: physical methods can only alleviate color changes, but cannot effectively control browning; chemical inhibitors pose a risk of residue and cannot fundamentally block PPO expression; while plant extracts have the advantage of being naturally sourced, their inhibitory efficiency is often significantly limited by the extract's composition, concentration, and substrate environment; harvesting strategies are affected by field variations; and the curing process makes it difficult to precisely control the microenvironment. Therefore, existing methods cannot fundamentally solve the problem of browning during tobacco curing. Summary of the Invention

[0006] Based on the above, the present invention provides a homozygous mutant of the NtPPO gene and its application, which reduces the activity of polyphenol oxidase in tobacco leaves and thus weakens the browning degree of tobacco leaves after curing.

[0007] The technical solution of this invention is: the application of NtPPO gene homozygous mutants in any one or more of the following;

[0008] (1) Reduces the activity of polyphenol oxidase in tobacco leaves;

[0009] (2) Reduce the degree of browning of tobacco during baking;

[0010] The NtPPO gene mutants include mutants of NtPPO1, NtPPO2, NtPPO3, and NtPPO4. The nucleotide sequence of the NtPPO1 mutant is shown in SEQ ID NO.1, the nucleotide sequence of the NtPPO2 mutant is shown in SEQ ID NO.2, the nucleotide sequence of the NtPPO3 mutant is shown in SEQ ID NO.3, and the nucleotide sequence of the NtPPO4 mutant is shown in SEQ ID NO.4.

[0011] The beneficial effects of this invention are as follows: This invention, through genome mining, identified 13 NtPPO genes (named NtPPO1–NtPPO13) and conducted bioinformatics analysis on them, including studies on gene structure, protein structure, and physicochemical properties. Further tissue-specific expression analysis showed that NtPPO genes were highly expressed in leaves, with NtPPO9 and NtPPO10 also showing high expression levels in flowers. In-depth analysis of the expression patterns of NtPPO genes in leaves revealed that the expression levels of NtPPO1, NtPPO3, NtPPO5, NtPPO6, and NtPPO7 changed significantly during leaf growth, while NtPPO2, NtPPO9, NtPPO10, and NtPPO13 remained stable. NtPPO1 showed the highest expression level during the tobacco leaf maturation stage. Subcellular localization experiments showed that NtPPO1, NtPPO6, NtPPO9, and NtPPO12 were mainly distributed in chloroplasts. By simultaneously performing homozygous mutations on NtPPO1, NtPPO2, NtPPO3, and NtPPO4, the results showed that although the appearance of the mutant strains was almost identical to that of wild-type (WT) tobacco during the growth stage, the polyphenol oxidase activity of the mutant strains was significantly lower than that of WT. In addition, the degree of browning of the tobacco leaves of the mutant strains was significantly reduced after curing. Attached Figure Description

[0012] Figure 1 Evolution, conserved motifs, and exon-intron structures of NtPPOs. (A) Phylogenetic tree of PPOs in Solanaceae (tomato SlyPPOs, potato StPPOs, pepper CaPPOs, and tobacco NtPPOs); (B) Exon and intron structures of the NtPPO gene family; (C) Conserved CDS motifs of NtPPOs (MEME analysis of conserved motifs in CDS sequences of 13 NtPPOs identified 10 conserved motifs); (D) Conserved domains of NtPPO proteins.

[0013] Figure 2Cis-acting elements of NtPPOs. (A) Circular diagram showing the proportion of cis-acting elements from NtPPO1 to NtPPO13 from the outer circle to the inner circle; (B) Defense and stress response elements; (C) Growth and development elements; (D) Plant hormone response elements; (E) Light response elements.

[0014] Figure 3 NtPPOs expression analysis. (A) Tissue-specific expression of NtPPOs (roots, stems, leaves, seeds, and flowers). (B) NtPPOs expression in tobacco leaves at different stages, including the cross-shaped stage (S1), seedling stage (S2), seedling emergence stage (S3), root elongation stage (S4), and maturity stage (S5).

[0015] Figure 4 Phylogenetic tree of tobacco PPOs.

[0016] Figure 5 Subcellular localization of *Tobacco Benedict* leaves was achieved by infecting GV3101 containing 35S:GFP, NtPPO1-GFP, NtPPO9-GFP, NtPPO10-GFP, and NtPPO13-GFP fusion plasmids. Fluorescence signals of 35S:GFP were observed distributed throughout tobacco cells under excitation at 488 nm, and GFP signals of the fusion proteins were observed under chlorophyll autofluorescence at 640–660 nm. Scale bar = 10 μm.

[0017] Figure 6 Construction of expression vectors and target design. (A) Construction of PEGAtCas9PcUBI-H-PPO expression vector; (B) Assembly diagram of CRISPR / Cas9-edited NtPPOs sequence target sites, with four target sequences shown in red.

[0018] Figure 7 Mutant identification results. Mutation patterns in T1 generation transgenic plants. Protospacer adjacent motifs (PAMs) are highlighted in yellow. Red letters indicate inserted nucleotides. Dashed lines indicate deleted nucleotides. - Deletion; + Insertion.

[0019] Figure 8 Polyphenol oxidase activity of KO-18 and WT at tobacco leaf maturity. Note: p≤0.001,***.

[0020] Figure 9 Image of tobacco leaves after roasting. Detailed Implementation

[0021] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] I. Identification and Physicochemical Properties of NtPPOs

[0023] Using PPO protein sequences found on the Solanaceae Genome Website (https: / / solgenomics.net / ) as parent sequences, blastp searches were performed on tobacco genome data (K326, Edwards, 2017 version; https: / / solgenomics.net / organism / Nicotiana_tabacum / genome). Possible tobacco PPO protein sequences were screened using a similarity ≥70% and an E value ≤ e-10 as thresholds. Further, NCBI's CDD tool (https: / / www.ncbi.nlm.nih.gov / ) was used to search these sequences for PPO family-specific domains. Proteins containing typical PPO domains were identified as the final tobacco PPOs, yielding 13 sequences, which were named PPO1–PPO13 sequentially. Their nucleotide sequences are shown in SEQ ID NO. 5–17, and the physicochemical properties of these genes were analyzed.

[0024] The results are shown in Table 1. The molecular weight of NtPPO proteins ranges from 53.03 to 134.11 kDa, the amino acid sequence ranges from 469 to 1202 amino acids, the isoelectric point ranges from 5.89 to 8.74, and the hydrophilicity parameter ranges from -0.116 to -0.554. Signal peptide prediction results show that none of the NtPPO genes contain a signal peptide. Transmembrane structure prediction results show that all genes except NtPPO4 possess transmembrane structures.

[0025] Table 1 Physicochemical properties of NtPPOs

[0026]

[0027]

[0028] II. Systematic Evolution and Gene Sequence Structure Analysis of NtPPOs

[0029] To investigate the evolutionary relationships of the PPO family, a phylogenetic tree was constructed using the amino acid sequences of 6 tomato SlyPPOs, 9 potato StPPOs, 4 pepper CaPPOs, and 13 tobacco NtPPOs via MEGA11. The results showed that the PPO genes in these plants can be divided into four types (Class I, Class II, Class III, and Class IV). Pepper CaPPOs are distributed in Class II and Class III, while tomato SlyPPOs and potato StPPOs are present in all four classes. Tobacco NtPPOs are only present in Class I, Class II, and Class IV, with NtPPOs 5, 6, and 7 belonging to Class I, NtPPOs 1, 2, 3, 4, 8, 11, 12, and 13 belonging to Class II, and NtPPOs 9 and 10 belonging to Class IV. The multiple evolutionary directions of plant PPOs suggest that PPOs may have multiple functions, and PPOs grouped together may have the same or similar functions. Figure 1 A).

[0030] To understand the gene structure of NtPPOs, the DNA sequences of 13 NtPPOs were analyzed. Six NtPPOs lacked introns; four NtPPOs had one intron; two NtPPOs contained two introns; and NtPPO2, containing four introns, was the most unique of the 13 NtPPOs. Among them, NtPPO2 and NtPPO7 both had relatively long intron structures, while the introns of the remaining NtPPOs were relatively short. Interestingly, NtPPO genes with close evolutionary relationships had similar structures. For example, NtPPO12 and NtPPO13, within the same group, both had two exons and one intron, and their lengths were similar. Figure 1 B).

[0031] To further investigate the gene structure of NtPPOs, conserved motif analysis was performed on the CDS sequences of 13 NtPPOs using MEME, and 10 conserved motifs were identified. NtPPO1–7 and NtPPO9–13 each contain 10 motifs, while NtPPO8 contains only 9 motifs. Structurally, motifs 1, 2, 3, 4, and 5 are usually tandemly linked and located at the beginning, while motifs 6, 7, 8, 9, and 10 are tandemly linked and located at the end; a small number of NtPPO genes show motifs 1, 6, 7, 8, 9, and 10 linked together. NtPPO2 has a unique structure, with its sequence including two sets of motifs (1, 2, 3, 4, 5) and motifs (1, 6, 7, 8, 9, 10). Furthermore, NtPPO1, NtPPO3, and NtPPO4 contain two motif 1s; NtPPO12 contains two motif 2s; NtPPO5 and NtPPO6 contain two motif 4s; and NtPPO8 lacks motif 5. Simultaneously, analysis of the NtPPO protein sequences using CCD and SMART databases revealed three conserved domains. These three conserved domains are Tyrosinase, PPO_DWL, and PPO_KFDV, and are sequentially distributed throughout the NtPPO protein sequences. Specifically, NtPPO2 contains two Tyrosinase, PPO_DWL, and PPO_KFDV domains. Figure 1 CD).

[0032] The above analysis shows that: (1) NtPPOs have fewer introns in their DNA, and introns may have been acquired or lost during evolution; (2) The number and arrangement of conserved motifs in the CDS and conserved domains in the protein sequence of NtPPOs are highly similar, indicating high conservation during evolution. However, NtPPO2 shows a double conserved motif and domain in both conserved motif and conserved domain analyses, suggesting that gene fusion may have occurred during NtPPO2's evolution.

[0033] III. Prediction of the sympathetic element of the NtPPOs promoter

[0034] To understand transcriptional regulation mechanisms, the PlantCARE database was used to analyze cisfunctional elements in the NtPPO promoter region, and statistical analyses were performed on light-responsive elements, hormone-responsive elements, stress and defense-responsive elements, and growth and development elements. Figure 2As shown, Defense and stress responsiveness has 6 elements, with the ARE element appearing most frequently (18); Growth and development has 6 elements, with Unnamed_1 appearing most frequently (12); Phytohormone responsiveness has 8 elements, with TGACG-motif and CGTCA-motif appearing most frequently (13); and Light responsiveness has 20 elements, with G-Box appearing most frequently (42). Among these four cis-acting elements, Light responsiveness accounts for 56.5%, containing far more element types and numbers than the other categories. Phytohormone responsiveness and Defense and stress responsiveness both account for 17.4%, second only to Light responsiveness in terms of quantity, but Phytohormone responsiveness has more element types than Defense and stress responsiveness. Growth and development has the fewest elements, at only 8.7%. These results indicate that light is an important factor in the transcriptional regulation of NtPPOs.

[0035] However, some NtPPOs are unique, such as NtPPO8, where the number of Phytohormone responsiveness elements is the same as that of Light responsiveness elements, both accounting for 40.0% of the total. Furthermore, the Light responsiveness, Defense and stress responsiveness, and Growth and development and Phytohormone responsiveness of NtPPO6 are 33.4%, 33.4%, 9.4%, and 23.8%, respectively. These results suggest that NtPPO8 may be primarily regulated by light and plant hormones, while the expression of NtPPO6 may be broadly regulated.

[0036] IV. NtPPOs Expression Analysis

[0037] Using the high-yielding but enzyme-sensitive browning variety K326 as material, its wild-type (WT) was planted at the Fujian Agriculture and Forestry University experimental base. Leaf samples and samples of different tissues (roots, stems, leaves, flowers, and anthers) at five stages (S1, S2, S3, S4, and S5) of the wild-type (WT) tobacco were collected and stored at -80℃. Total RNA was extracted using a Novizan kit. The total RNA was reverse transcribed into cDNA using an oligodt reverse transcription kit and stored at -20℃. Using NtActin as an internal control gene, qRT-PCR primers were designed using the Primer-BLAST online program; primer sequences are shown in Table 2. qRT-PCR amplification was performed using SYBR GreenMasterMix. -ΔΔCt Calculate the relative expression level of the gene to be tested.

[0038] Table 2. Primers for amplification of tobacco PPO gene family members used for qRT-PCR analysis.

[0039]

[0040] The nucleotide sequences of the above primers are shown in SEQ ID NO.18-45.

[0041] To understand the function of NtPPOs, the expression of NtPPOs in five tissues (root, stem, leaf, anther, and flower) of tobacco K326 was analyzed. The results showed that NtPPOs exhibited tissue-specific expression, with different expression patterns for each gene in different organs. NtPPO4, 12, and 13 were expressed significantly higher in root tissues than in other tissues; NtPPO1, 3, 5, 6, 7, 8, and 11 were mainly expressed in leaves; and NtPPO9 and 10 had the highest expression levels in flowers. Furthermore, NtPPO5, 6, 7, 9, and 10 were also highly expressed in stems and anthers, respectively. Notably, NtPPO5, 6, 7, 9, and 10 exhibited high and stable expression patterns in all five tissues, particularly in leaf tissues. Simultaneously, in developmental evolution, NtPPO5, 6, and 7 belonged to one branch, while NtPPO9 and 10 belonged to another. This suggests that NtPPO5, 6, 7, 9, and 10 are more important than other NtPPOs in tobacco. Figure 3 A).

[0042] The expression patterns of NtPPOs in tobacco leaves at five stages (S1, S2, S3, S4, and S5) were further explored. The results showed that the expression patterns of NtPPOs in tobacco leaves differed significantly across stages. The expression levels of NtPPO2, 9, 10, and 13 varied little, but were highest at stage S5. Conversely, the expression levels of NtPPO1, 3, 5, 6, 7, and 12 changed significantly. Specifically, the expression level of NtPPO1 gradually increased, while NtPPO3 and 12 were highest at stage S5. The expression levels of NtPPO5, 6, and 7 showed an irregular "M"-shaped pattern. The expression changes of the remaining NtPPOs were not significant. Notably, the expression level of NtPPO1 was highest at the tobacco leaf maturity stage (S5). Figure 3 B).

[0043] V. Subcellular localization analysis

[0044] In the phylogenetic analysis of the NtPPOs family, all NtPPOs were further divided into four groups ( Figure 4 In the S5 phase, the highest-expressing NtPPO proteins from each group were selected for cellular localization analysis, such as NtPPO1 from group I, NtPPO6 from group II, NtPPO9 from group III, and NtPPO12 from group IV. NtPPO1, NtPPO6, NtPPO9, and NtPPO12 fused with GFP protein and were expressed in tobacco cells. By detecting GFP signaling, we found that NtPPO1, NtPPO6, NtPPO9, and NtPPO12 all co-localized with chloroplast fluorescence signals. Figure 5 The results indicate that NtPPO1, NtPPO6, NtPPO9, and NtPPO12 are located in chloroplasts.

[0045] VI. Construction of mutant materials

[0046] In this study, highly conserved sequences of the NtPPOs gene were used to design target sequences. Specific sgRNA target regions were designed using an online tool (http: / / crispr.hzau.edu.cn / ) according to design criteria, and off-target risks were assessed using CasOT software. Four sgRNA sequences were selected: Target 1 was designed to mutate the second exon of NtPPO1, 2, and 3, and the unique exon of NtPPO4; Target 2 was designed to mutate the first exon of NtPPO5, 6, and 7; Target 3 was designed to mutate the unique exons of NtPP9, 10, and 11, and the second exon of NtPPO8; Target 4 was designed to mutate the first exons of NtPPO12 and 13. Figure 6To construct the sgRNA expression cassette, primers with homologous arms were designed (Table 3), and the sgRNA expression cassette fragments were amplified by overlap PCR and nested PCR. The four amplified fragments were gel-extracted according to the purification kit instructions. Each purified fragment (30 ng) was mixed with 30 ng of pEGCas9Pubi-H empty vector, 1.5 μL of 10×Cut Smart buffer, 1.5 μL of 10 mM ATP, T4 DNA ligase (30 U), Bsa I-HF restriction enzyme (10 U), and an appropriate amount of ddH2O to form a 15 μL reaction system. The mixture was subjected to alternating digestion-ligation cycles at 37 °C and 20 °C for 5 minutes each time, repeated for 15 cycles to obtain the recombinant vector (…). Figure 6 A) The recombinant plasmid was transformed into DH5α *E. coli* cells, positive single colonies were identified, and plasmid DNA was extracted and purified. Sanger sequencing was used to confirm the insertion and integrity of the sequence; the sequencing primers were E9-F / E9-R (Table 3). The recombinant editing vector was transformed into *Agrobacterium* EHA105, positive *Agrobacterium* were screened, and the transformed *Agrobacterium* were then transformed into K326 callus tissue to finally obtain mutant tobacco. PCR amplification and target gene sequencing were used to detect the mutation types of 13 NtPPO genes in each T1 generation of plants; the amplification primers are shown in Table 4.

[0047] Table 3. Amplification primers used to construct NtPPO gene family gene editing vectors.

[0048]

[0049] The nucleotide sequences of the above primers are shown in SEQ ID NO.46~63.

[0050] Table 4 shows the primers used to identify T1 generation mutation types.

[0051]

[0052] The nucleotide sequences of the above primers are shown in SEQ ID NO.64~89.

[0053] Due to the high conservation of the NtPPO gene family, four sgRNAs were designed based on different groups to construct a CRISPR-Cas9 vector, and KO-18 was successfully generated. Next-generation sequencing revealed that this is a tobacco line with simultaneous mutations in NtPPO1, 2, 3, and 4 genes. Figure 7 The mutation types of each NtPPO gene were identified. All genes terminated prematurely and lost conserved domains. The nucleotide sequences of the NtPPO1, NtPPO2, NtPPO3, and NtPPO4 gene mutants are shown in SEQ ID NO. 1–4, respectively.

[0054] VII. PPO Activity Detection and Tobacco Curing

[0055] Using the high-yielding but enzyme-sensitive browning variety K326 as material, its wild-type (WT) and mutant (KO-18) were planted at the Fujian Agriculture and Forestry University experimental base. Leaves at maturity (S5) of both wild-type (WT) and mutant (KO-18) were collected in six biological replicates for PPO activity measurement. PPO activity was detected using the HERUI polyphenol oxidase activity assay kit. Mature tobacco leaves of WT and mutant were cured according to the curing process (Table 5).

[0056] Table 5 Curing Procedures for WT and KO-18 Tobacco Leaves

[0057]

[0058] There was no significant difference in the leaves of wild-type and mutant strains before baking. However, after measuring their polyphenol oxidase activity, it was found that there were significant differences in their polyphenol oxidase activity, with KO-18 showing significantly lower polyphenol oxidase activity than WT. Figure 8 ). Observation of the leaves after baking revealed that the leaves of the mutant plants were more glossy and showed less browning. Figure 9 The results of this experiment indicate that editing the NtPPOs gene effectively reduced polyphenol oxidase activity, thereby significantly alleviating the browning degree of flue-cured tobacco. This demonstrates that editing the NtPPOs gene can effectively reduce polyphenol oxidase activity.

[0059] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of NtPPO gene mutant combinations in any one or more of the following: (1) Reduces the activity of polyphenol oxidase in tobacco leaves; (2) Reduce the degree of browning in the tobacco during baking; The NtPPO gene mutant combination is composed of NtPPO1, NtPPO2, NtPPO3 and NtPPO4. The nucleotide sequence of the NtPPO1 gene mutant is shown in SEQ ID NO.1, the nucleotide sequence of the NtPPO2 gene mutant is shown in SEQ ID NO.2, the nucleotide sequence of the NtPPO3 gene mutant is shown in SEQ ID NO.3, and the nucleotide sequence of the NtPPO4 gene mutant is shown in SEQ ID NO.4.

Images