Application of NtPPO8 gene in tobacco polyphenol content regulation and germplasm improvement

Abstract

The application discloses application of NtPPO8 gene in content regulation of tobacco polyphenols and improvement of germplasm resources, and belongs to the technical field of plant genetic engineering. The application uses cDNA of leaves of common tobacco at different periods as a template, and uses PCR technology to obtain a NtPPO8 gene. Through cloning and analysis of the NtPPO8 gene, it is found that overexpression of the gene in tobacco plants can significantly improve the activity of polyphenol oxidase in leaves. The tobacco polyphenol oxidase coding gene NtPPO8 found in the application enriches the tobacco polyphenol oxidase gene family, and lays a foundation for studying the function of polyphenol oxidase in tobacco. Further detection shows that, compared with a normal control group, the content of chlorogenic acid and rutin in a transgenic strain is significantly reduced. The application lays a foundation for clarifying the molecular regulation mechanism of chlorogenic acid and rutin biosynthesis, and provides a new precise regulation gene for synthesis of chlorogenic acid and rutin in tobacco.

Description

Technical Field

[0001] This invention relates to the application of the NtPPO8 gene in the regulation of tobacco polyphenol content and germplasm resource improvement, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Chlorogenic acid (CGA), also known as coffee tannin, is a phenylpropanoid compound produced by plants via the phenylalanine pathway during aerobic respiration. It is widely distributed in the plant kingdom, primarily belonging to the Caprifoliaceae, Asteraceae, Eucommia, and Solanaceae families. Chlorogenic acid has significant medicinal value. Statistics show that all 170 traditional Chinese medicines listed in the Ministry of Health's "Drug Standards" that possess heat-clearing, detoxifying, antibacterial, and anti-inflammatory properties contain chlorogenic acid as a major component. Furthermore, as a novel antioxidant, chlorogenic acid has important applications in food and fruit preservation, sun protection, and skincare. In tobacco, besides its own pleasant aroma, chlorogenic acid can also be converted into nutty compounds such as pyrazines, pyridines, and pyrroles under enzymatic action, significantly contributing to the elegant aroma and aroma intensity of tobacco products. Therefore, in-depth research into the chlorogenic acid synthesis pathway has significant technical and practical value.

[0003] Rutin (also known as rutin glycoside) is a special class of flavonol compounds and a metabolite of the flavonoid pathway. Due to its high antioxidant activity, it can play an anti-cancer role and effectively reduce the risk of hypertension and hyperlipidemia, making it extremely important in medicine and biopesticides. Rutin is also widely used as a stabilizer, preservative, and natural colorant in the pharmaceutical, nutritional food, and medicated cosmetic industries. However, rutin accumulation in plants is relatively low, with the main source being Sophora japonica buds. The yield of Sophora japonica buds, especially the rutin content, is greatly affected by climate, harvesting, and drying conditions, leading to unstable prices. Furthermore, with the improvement of living standards and awareness in my country, the demand for rutin and its products is constantly increasing, making it difficult for rutin production to meet industrial needs.

[0004] Tobacco (Nicotiana tabacum L.) is an annual or short-lived perennial herbaceous plant belonging to the Solanaceae family. It is one of my country's major economic crops and plays a vital role in the country's national economic development. my country ranks first in the world in both tobacco germplasm area and yield. Research has also found that tobacco is one of the plants with high levels of chlorogenic acid and rutin. Currently, in addition to being processed into flue-cured tobacco, research is being conducted on the edible and medicinal value of tobacco (e.g., tobacco protein content is much higher than soybeans, making it another high-quality protein source in the food industry). Furthermore, statistics show that approximately 25% of waste materials such as tobacco dust are generated annually during tobacco harvesting and production. Therefore, if chlorogenic acid and rutin can be fully extracted from tobacco waste, it can not only turn waste into treasure but also bring significant economic benefits.

[0005] Given the increasing sophistication of tobacco tissue culture technology and the ease with which tobacco can be genetically transformed, the development of tobacco varieties with high chlorogenic acid and rutin content through transgenic technology or other breeding techniques would lay a solid technical foundation for using tobacco as a bioreactor to extract chlorogenic acid and rutin. Therefore, in-depth analysis of the synthesis and regulation of chlorogenic acid and rutin from a genetic engineering perspective, and the identification of regulatory genes that can control the content of chlorogenic acid and rutin in tobacco, are of great significance for the improvement of tobacco germplasm resources and the application of chlorogenic acid and rutin. Summary of the Invention

[0006] The first objective of this invention is to provide the application of the NtPPO8 gene in the regulation of tobacco polyphenol content, providing a precise regulatory gene for the regulation of tobacco chlorogenic acid and rutin in the prior art.

[0007] The second objective of this invention is to provide the application of the NtPPO8 gene in the improvement of tobacco germplasm resources, thereby providing a new available gene resource for the improvement of aroma germplasm resources.

[0008] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtPPO8 gene in the regulation of tobacco polyphenol content is as follows:

[0009] The application of the NtPPO8 gene in the regulation of tobacco polyphenol content, wherein the nucleotide sequence of the NtPPO8 gene is shown in SEQ ID NO.1; the tobacco polyphenols are chlorogenic acid and / or rutin.

[0010] The beneficial effects of the above technical solution are as follows: Tobacco is an important model organism in biological research, and a large number of plant molecular biology studies and genetic engineering experiments use tobacco as the specific subject. Because of this, researchers have neglected the discovery, identification, and utilization of tobacco's own functional genes. Based on previous research, this invention uses cDNA from leaves of common tobacco at different stages as a template and employs PCR technology to clone the homologous gene of tobacco polyphenol oxidase, named NtPPO8. Through the cloning of the NtPPO8 gene and the construction and analysis of its expression vector, this invention found that overexpression of this gene in tobacco plants can significantly increase the activity of polyphenol oxidase in leaves. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO8 in this invention enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidase in tobacco.

[0011] Further testing revealed that the transgenic lines contained significantly higher levels of chlorogenic acid and rutin compared to the normal control group. This invention lays the foundation for elucidating the molecular regulatory mechanisms of chlorogenic acid and rutin biosynthesis and provides new, precise regulatory genes for chlorogenic acid and rutin synthesis in tobacco.

[0012] As a further improvement, overexpression of the NtPPO8 gene significantly reduced the content of chlorogenic acid and rutin in tobacco.

[0013] As a further improvement, the tobacco is K326.

[0014] To achieve the above objectives, the technical solution adopted in the application of the NtPPO8 gene in tobacco germplasm resource improvement of this invention is as follows:

[0015] The application of the NtPPO8 gene in the improvement of tobacco germplasm resources, the nucleotide sequence of the NtPPO8 gene is shown in SEQ ID NO.1.

[0016] The beneficial effects of the above technical solution are as follows: This invention cloned the tobacco NtPPO8 gene, and after overexpressing the NtPPO8 gene in tobacco through Agrobacterium-mediated transformation, it was found that the content of chlorogenic acid and rutin in tobacco was significantly reduced. This indicates that the NtPPO8 gene is involved in regulating the synthesis of chlorogenic acid and rutin in tobacco, laying the foundation for elucidating the molecular regulatory mechanism of chlorogenic acid and rutin biosynthesis.

[0017] Furthermore, chlorogenic acid and rutin can form aroma compounds closely related to human sensory perception during processing. These aroma compounds are important sources of tobacco aroma, therefore, the aroma of tobacco can be controlled by adjusting the content of chlorogenic acid and rutin. Simultaneously, chlorogenic acid and rutin also play a crucial role in regulating plant stress resistance; therefore, the stress resistance of tobacco can also be affected by adjusting their content. In summary, this invention provides new usable genetic resources for the improvement of tobacco germplasm resources.

[0018] As a further improvement, the germplasm resource improvement involves regulating the expression level of the NtPPO8 gene to control the content of polyphenols in tobacco.

[0019] As a further improvement, the amount of polyphenols in tobacco was reduced by overexpressing the NtPPO8 gene.

[0020] As a further improvement, the polyphenolic substance is chlorogenic acid and / or rutin.

[0021] As a further improvement, the overexpression involves constructing an overexpression vector for the NtPPO8 gene and overexpressing the NtPPO8 gene.

[0022] As a further improvement, the tobacco is K326. Attached Figure Description

[0023] Figure 1 This is a gel electrophoresis image of the NtPPO8 gene clone in Example 1 of this invention;

[0024] Figure 2 This is an amino acid sequence analysis and alignment diagram from Example 2 of the present invention;

[0025] Figure 3 This is a diagram showing the expression characteristics of the NtPPO8 gene in different tissues in Example 3 of the present invention.

[0026] Figure 4 This is a subcellular localization map (scale bar, 50 μm) of the NtPPO8 gene in tobacco epidermal cells in Example 5 of the present invention.

[0027] Figure 5 This refers to the PCR detection of NtPPO8 gene expression in NtPPO8 gene overexpressing plants in Example 6 of the present invention.

[0028] Figure 6 This is a qRT-PCR detection of the NtPPO8 gene in plants overexpressing the NtPPO8 gene in Example 6 of the present invention (where Con represents the tobacco plant of normal control, and the rest are different individual plants overexpressing the NtPPO8 gene).

[0029] Figure 7This study analyzes the activity of PPO in the leaves of T2 generation NtPPO8 gene overexpressing plants in Example 7 of the present invention (where Con represents the tobacco plant of normal control, and the rest are different individual plants of NtPPO8 gene overexpression).

[0030] Figure 8 Analysis of chlorogenic acid content in leaves of T2 generation NtPPO8 gene overexpressing plants in Example 8 of this invention;

[0031] Figure 9 Analysis of rutin content in leaves of T2 generation NtPPO8 gene overexpressing plants in Example 8 of this invention;

[0032] Figure 10 This study analyzes the hyoscyamine content in the leaves of T2 generation NtPPO8 gene overexpressing plants in Example 8 of this invention. Detailed Implementation

[0033] Existing technologies for chlorogenic acid and rutin have shown promising applications, but their sources are relatively limited. Tobacco, as an important model organism, has a high planting area and yield in my country, and it also contains high levels of both chlorogenic acid and rutin. Theoretically, it can serve as a plant source of chlorogenic acid and rutin. If tobacco varieties with high chlorogenic acid and rutin content can be cultivated through transgenic technology or other breeding techniques, a solid technical foundation can be laid for using tobacco as a bioreactor to extract chlorogenic acid and rutin. Therefore, identifying regulatory genes that can precisely control the content of chlorogenic acid and rutin in tobacco is of great significance for increasing their content in tobacco.

[0034] Based on previous research, this invention used cDNA from leaves of common tobacco at different growth stages as templates and employed PCR technology to clone a homologous gene for tobacco polyphenol oxidase, named NtPPO8. Through cloning the NtPPO8 gene and constructing and analyzing its expression vector, this invention revealed that overexpression of this gene in tobacco plants significantly increases the activity of polyphenol oxidase in leaves. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO8 in this invention enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidase in tobacco.

[0035] Furthermore, chlorogenic acid and rutin can form aroma compounds closely related to human sensory perception during processing. These aroma compounds are important sources of tobacco aroma, therefore, the aroma of tobacco can be controlled by adjusting the content of chlorogenic acid and rutin. Simultaneously, chlorogenic acid and rutin also play a crucial role in regulating plant stress resistance; therefore, the stress resistance of tobacco can also be affected by adjusting their content. In summary, this invention provides new usable genetic resources for the improvement of tobacco germplasm resources.

[0036] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0037] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0038] In the following examples, primer sequence synthesis and gene sequencing were performed by Beijing Liuhe Huada Biotechnology Co., Ltd.

[0039] Biomaterials:

[0040] Tobacco material: Cultivated tobacco (Nicotiana tabacum) variety K326, kindly provided by Yunnan Tobacco Science Research Institute.

[0041] Vector: Super pCAMBIA1300 is a commonly used plasmid vector in molecular biology and can be obtained from public sources.

[0042] DH5α-sensor cells were purchased from Shanghai Sangon Biotech Co., Ltd.; Agrobacterium strain LBA4404 is a commonly used strain in molecular biology and can be obtained through public channels.

[0043] Experimental reagents:

[0044] DNA / RNA extraction kits were purchased from Gene Answer, gel extraction kits / reverse transcription kits were purchased from Takara Bio Engineering (Dalian) Co., Ltd., and homologous recombination kits were purchased from Novizan.

[0045] Experimental equipment:

[0046] Gel electrophoresis apparatus (Bio-Rad), PCR instrument (Biometra), pipette (Eppendorf), and UVP gel imaging system (GelDoc-It310) are all commonly used instruments and equipment in molecular biology experiments.

[0047] Data processing:

[0048] SPSS 18.0 software was selected, and Duncan's test (P<0.05) was used for significance statistical analysis. Different letters represent significant differences in data, and ND means that no data was detected.

[0049] Specific examples of the application of the NtPPO8 gene in the regulation of tobacco polyphenol content and germplasm resource improvement:

[0050] Example 1

[0051] Based on previous research, upstream and downstream primers were designed using Primer Premier 6 software. Using cDNA from leaves of common tobacco at different growth stages as templates, the NtPPO8 gene was cloned using PCR technology. The specific implementation process is as follows:

[0052] 1. Primer design

[0053] The specific PCR amplification primer sequences for non-coding region amplification are designed as follows:

[0054] NtPPO8-clone-F:5'-ACCAGGCTACTTACCGATT-3' (shown in SEQ ID NO.3);

[0055] NtPPO8-clone-R:5'-TCAACAGCTTCAATGGAGAT-3' (shown in SEQ ID NO.4).

[0056] RNA was extracted from tobacco leaves at different growth and development stages (according to the instructions of the Gene Answer RNA Extraction Kit), and the extracted RNA was reverse transcribed into cDNA according to the instructions of the Takara reverse transcription kit.

[0057] 2. PCR amplification

[0058] Using the cDNA prepared in step 1 as a template, PCR amplification was performed using the designed primers. The 25 μL reaction system for PCR amplification is shown in Table 1, and the reaction procedure is shown in Table 2.

[0059] Table 1 PCR reaction system

[0060]

[0061] Table 2 PCR reaction procedure

[0062]

[0063] PCR amplification products were detected by 1.2% agarose gel electrophoresis. A DL2000 DNA marker was used, and the electrophoresis conditions were 120V / 20 min. The samples were then observed under a UV scanner (e.g., ...). Figure 1 As shown in the figure, the target DNA fragment was recovered using a DNA gel recovery kit (Takara), and after the concentration was determined, it was stored at -20 ℃ for later use or directly used for subsequent experimental operations.

[0064] 3. Sequencing and analyzing the NtPPO8 gene

[0065] The purified and recovered target fragment was ligated into the pMD19-T vector, incubated overnight at 4 °C, and then transformed into competent DH5α cells. White single colonies were picked and incubated on a shaker at 37 °C for approximately 12 hours (200 rpm). A small amount of bacterial culture was used as a template for PCR verification to confirm whether it was a positive clone. The recombinant plasmid containing the target fragment was sequenced by Beijing Liuhe Huada Biotechnology Co., Ltd., obtaining the nucleotide sequence of the tobacco NtPPO8 gene.

[0066] Sequencing results showed that the tobacco NtPPO8 gene comprises 1767 bases, and its specific nucleotide sequence is shown in SEQ ID NO. 1. Analysis of this base sequence revealed that the protein encoded by the tobacco NtPPO8 gene consists of 588 amino acids, and its specific amino acid sequence is shown in SEQ ID NO. 2.

[0067] Example 2

[0068] This embodiment compares the amino acid sequences of the protein encoded by the tobacco NtPPO8 gene with those of PPO from other species. The specific implementation steps are as follows:

[0069] The amino acid sequence of the protein (NtPPO8) encoded by the tobacco NtPPO8 gene was compared with that of PPOs from other species, including wild tobacco, tobacco velvet, wolfberry, potato, and pepper. The results showed a high degree of amino acid sequence similarity between NtPPO8 and PPOs from other species. The sequence similarities with NtomPPO from tobacco velvet, NaPPO from wild tobacco, NsyPPO from forest tobacco, NbPPO from tobacco Benzodiazepines, CbPPO from pepper, CcPPO from yellow lantern pepper, SlPPO from tomato, and AtPPO from scopolamine were 99.83%, 96.27%, 96.27%, 95.06%, 84.69%, 85.71%, 82.20%, and 85.59%, respectively.

[0070] Furthermore, Pfam analysis revealed that NtPPO8 possesses typical domains of PPO oxidase, such as... Figure 2 As shown, it has a Tyrosinase domain (PF00264) at amino acids 166-388, a PPO1_DWL domain (PF12142) at amino acids 393-448, and a PPO1_KFDV domain (PF12143) at amino acids 460-587.

[0071] Example 3

[0072] Using real-time quantitative PCR (BIO-RAD, USA) technology, this example provides a preliminary analysis of the expression pattern of the tobacco NtPPO8 gene in tobacco plants. The specific implementation steps are as follows:

[0073] 1. Primer design for real-time PCR

[0074] For real-time quantitative PCR analysis, L25 was used as an internal reference gene, and the specific primer sequences were designed as follows:

[0075] NtPPO8-QF: 5'-GCTACTGCTGCTCCTATAC-3' (shown in SEQ ID NO.5);

[0076] NtPPO8-QR: 5'-GAACACTGTCCATATCACTTG-3' (shown in SEQ ID NO.6);

[0077] L25-F: 5'-CCCCTCACCACAGAGTCTGC-3' (shown in SEQ ID NO.7);

[0078] L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (shown in SEQ ID NO.8);

[0079] RNA was extracted from lateral roots, fibrous roots, stems, new leaves, old leaves, axillary buds, flower buds, receptacles, sepals, stamens, pistils, and ovaries of K326 during its full bloom period and reverse transcribed into cDNA as template samples for later use.

[0080] 2. Quantitative Real-Time PCR Detection

[0081] For quantitative real-time PCR, the 20 μL reaction system design is shown in Table 3, and the amplification program is shown in Table 4 (the instrument used was a Bio-Rad CFX96 from Bio-Rad Laboratories, USA). The relative expression levels obtained after the reaction were calculated using... The data were analyzed using the standard method, with the relative expression level of the blank control group (Con) set at 1. All data were the average of at least three independent experiments. SPSS 18.0 software was used, and Duncan's test (P < 0.05) was employed for statistical significance analysis.

[0082] Table 3 Reaction System

[0083]

[0084] Table 4 Amplification Procedure

[0085]

[0086] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the analysis reveals that the NtPPO8 gene is highly expressed in the axillary buds, flower buds, and pistils of common tobacco K326, and relatively highly expressed in the receptacle, sepals, and ovary. However, no NtPPO8 gene expression was detected in new leaves, old leaves, and stamens.

[0087] Example 4

[0088] Based on the NtPPO8 gene cloned in Example 1, this embodiment constructs an overexpression vector for the NtPPO8 gene to further verify its function. The specific implementation steps are as follows:

[0089] The upstream primer was designed as 5'-ATACACCAAATTGACTCTAGAATGGCTTCTTCTTCTTCTTC-3' (SEQ ID NO. 9), and the downstream primer was designed as 5'-GCCCTTGCTCACCATGGTACCTTAACAATTGACAAGCTTAA-3' (SEQ ID NO. 10). Homologous recombination adapter sequences were added to both primers. Amplification was performed using the correctly sequenced NtPPO8-T plasmid from Example 1 as a template. The recovered and purified PCR product and the enzyme-digested pCAMBIA1300 empty vector were ligated using a homologous recombination kit (ClonExpress Ultra One Step Cloning Kit, Novizan), and transformed into *E. coli* DH5α. Positive clones were identified by colony PCR, and single positive clones were sent to Beijing BGI Genomics Co., Ltd. for sequencing.

[0090] Example 5

[0091] This embodiment selects the NtPPO8 overexpression vector from Example 4 as the research object to observe the localization of the NtPPO8 gene in tobacco epidermal cells. The specific implementation is as follows:

[0092] The NtPPO8-GFP vector and the pCAMBIA1300 empty vector were used to transform Agrobacterium LBA4404, and then Agrobacterium was injected into the leaves of Nicotiana benthamiana via infiltration. Through Agrobacterium infection, the target gene was integrated into the tobacco cells. After culturing the injected plants for 3 days, the transformed leaves were observed using a confocal microscope (Zeiss LSM900 META, Jena, Germany).

[0093] Depend on Figure 4 It can be seen that the green fluorescence of NtPPO8-GFP coincides with the autofluorescence (purple) of chloroplasts, indicating that NtPPO8-GFP is located in chloroplasts.

[0094] Example 6

[0095] The successfully sequenced single colonies from Example 4 were expanded and cultured, plasmids were extracted, and the overexpression vector was transformed into Agrobacterium LBA4404 using electroporation. The specific implementation method is as follows:

[0096] 1. Preparation of Agrobacterium competent cells

[0097] Single colonies of Agrobacterium LBA4404 were picked and cultured overnight at 28°C in 2 mL LB medium (containing 20 mg / mL Rif). 2 mL of the well-grown culture (containing 25 mg / L Rif) was inoculated into 50 mL LB liquid medium and cultured at 28°C with shaking until the OD600 reached approximately 0.5. The culture was then transferred to 50 mL centrifuge tubes, placed on ice for 30 minutes, and centrifuged to collect the cells (5000 rpm / 4°C, 5 minutes). The cells were gently resuspended in 10 mL of pre-chilled 0.15 M sodium chloride solution and centrifuged again (5000 rpm / 4°C, 5 minutes). The supernatant was discarded, and the cells were resuspended in 20 mL of pre-chilled 20 mM calcium chloride solution. The prepared competent cells were aliquoted into 100 µL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0098] 2 plasmids transformed Agrobacterium

[0099] Take 1 μL of NtPPO8 gene overexpression plasmid and add it to a centrifuge tube containing 100 µL of Agrobacterium competent cells. Place on ice for 30 minutes. Then transfer to liquid nitrogen for 1 minute and incubate at 37 °C for 5 minutes. Add 1 mL of LB liquid medium and incubate at 28 °C with shaking for 3 hours. Centrifuge at 5000 rpm for 1 minute, discard the supernatant, add 200 μL of LB liquid medium, and resuspend the precipitate. Take 200 μL of the resuspended bacterial solution and spread it evenly on LB agar plates containing 20 mg / L Rif and 50 mg / L kanamycin (Kan). Incubate at 28 °C for 2-3 days. After confirming the colony PCR results, preserve the bacterial strain.

[0100] 3. Tobacco Conversion

[0101] Disinfect the vigorously growing tobacco leaves and cut them into 1 cm pieces. 2Small pieces were placed in MS differentiation medium and pre-cultured for 2 days at 28 ℃, light intensity of 2000 Lx, and light duration of 16 h / d. Afterward, they were immersed in engineered bacterial solution for 10-15 min, shaking the solution several times during this period. Excess bacterial solution was then blotted dry with sterile filter paper. The plants were then inoculated into MS differentiation medium and co-cultured at 28 ℃ in the dark for 3-5 days. The co-cultured plants were washed three times with sterile water, blotted dry with sterile paper, and transferred to MS differentiation medium containing hygromycin and carbenicillin for constant temperature culture. The medium was changed every 10 days. When the adventitious shoots reached 1-2 cm in length, the clustered adventitious shoots were cut into individual shoots and transferred to MS rooting medium containing hygromycin (kanamycin for gene editing vectors), carbenicillin, and activated carbon to promote rooting. Once the root system has developed well, remove the tissue culture seedlings, wash the culture medium off the roots with clean water, cut off a small number of lower leaves, transfer them to flowerpots filled with loose, sterile soil, and cultivate them according to conventional management methods.

[0102] 4. Detection of positive super-genetically modified tobacco using PCR and qPCR methods

[0103] Design specific primer pairs for the expression vector, with the upstream primer specifically binding to the NtPPO8 gene and the downstream primer specifically binding to the GFP gene.

[0104] The upstream primer was 5'-GAAGACACTATTGCGGTAAC-3' (SEQ ID NO.11); the downstream primer was 5'-ATGGCGGACTTGAAGAAG-3' (SEQ ID NO.12). PCR amplification was performed using genomic DNA from the control and K326 and T0 generation seedlings as templates. Seedlings that amplified specific bands were considered positive for the T0 generation (e.g.,...). Figure 5 (As shown). Next, the expression level of the NtPPO8 gene in the selected positive seedlings was detected using qPCR. NtPPO8 gene-specific primers were used: NtPPO8-QF: 5'-GCTACTGCTGCTCCTATAC-3' (SEQ ID NO. 5), NtPPO8-QR: 5'-GAACACTGTCCATATCACTTG-3' (SEQ ID NO. 6). The tobacco L25 gene was used as an internal control. The upstream primer was 5'-CCCCTCACCACAGAGTCTGC-3' (SEQ ID NO. 7), and the downstream primer was 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (SEQ ID NO. 8). Amplification was performed using a LightCycler® 96 quantitative PCR instrument (reaction system and amplification program as described in Example 3). After the reaction, based on the obtained CT value, 2... -△△CTMethods to calculate the relative expression level of the NtPPO8 gene (e.g.) Figure 6 (As shown).

[0105] To verify whether the phenotype could be stably inherited to the next generation, seeds of T1 generation positive plants were collected, and T2 generation plants were obtained after further planting. PCR amplification analysis was performed, and the analysis showed that the T1 generation plants could be stably inherited to the T2 generation. At the same time, K326 and T2 generation transgenic plants were planted in a greenhouse.

[0106] Example 7

[0107] This embodiment selects the NtPPO8 overexpression line (T2 generation) from Example 6 as the research object to detect the activity of polyphenol oxidase in the leaves of transgenic tobacco plants at the middle leaf maturity stage. The specific implementation operation is as follows:

[0108] The activity of polyphenol oxidase in the leaves of transgenic tobacco plants at the mid-leaf maturity stage was detected using a polyphenol oxidase assay kit (Beijing Solarbio Science & Technology Co., Ltd.). The enzyme activity assay results are as follows: Figure 7 As shown. By Figure 7 It can be seen that the activity of polyphenol oxidase in the leaves of the overexpressing transgenic plants was significantly increased compared with the control (K326) (P < 0.05). This study preliminarily reveals the function of the NtPPO8 gene, demonstrating that the transgenic NtPPO8 gene regulates the activity of polyphenol oxidase in tobacco leaves.

[0109] Example 8

[0110] This embodiment selects the NtPPO8 overexpression line (T2 generation) from Example 6 as the research object, and detects the content of chlorogenic acid and rutin in the leaves of transgenic tobacco plants at the middle leaf maturity stage. The specific implementation operation is as follows:

[0111] Based on HPLC-MS / MS technology and using the internal standard method, the contents of three major polyphenols in tobacco leaves—chlorogenic acid, rutin, and hyoscyamine—were determined. For the detection, 50 mg of freeze-dried tobacco leaf samples were ground into powder using a grinder and then transferred to 1.5 mL (2 mL centrifuge tube) of pre-cooled ethanol-water (4:1, v / v) extraction solution. The mixture was sonicated at room temperature for 1 h; then centrifuged at 14000 r / min for 12 min, and 1 mL of the supernatant was collected and concentrated into a solid under vacuum. The solid was reconstituted with 200 μL of 80% ethanol (containing 75 ng / mL umbelliferone solution), centrifuged at 20000 r / min for 3 min, and the supernatant was collected for HPLC-MS / MS detection. Analytical conditions: Column: BEH Phenyl column (2.1 mm × 150 mm, 1.7 μm); Mobile phase: 0.1% formic acid aqueous solution (A) and 0.1% formic acid methanol solution (B); Elution gradient: 0–2 min B phase from 5% to 15%, 2–10 min B phase maintained at 15%, 10.01 min–15 min B phase to 100%; Flow rate: 0.3 mL / min; Column temperature: 35 ℃; Injection volume: 1 μL. Electrospray ionization was used; capillary voltage was 4 kV in positive ionization mode; nebulizer gas pressure was 275.6 kPa; drying gas flow rate was 12 L / min; drying gas temperature was 290 ℃; sheath gas flow rate was 11 L / min; sheath gas temperature was 200 ℃; real-time multiple reaction monitoring (DMRM) mode was used for scanning. The retention times of chlorogenic acid, rutin, and hyoscyamine were 4.06, 7.34, and 7.18 / min, respectively. The results indicate that the NtPPO8 overexpression transgenic lines (such as...) are effective against these strains. Figure 8 and Figure 9 As shown in the figure): Compared with the control (K326), the T2 generation transgenic plants (NtPPO8-1-2-5, NtPPO8-1-2-6, NtPPO8-1-2-8, NtPPO8-1-2-19, NtPPO8-7-4-3) showed that the chlorogenic acid content in the middle leaves at maturity was reduced by 49.50%, 58.85%, 77.25%, 61.63%, and 53.99%, respectively, and the rutin content was reduced by 37.51%, 92.61%, 98.01%, 60.77%, and 46.85%, respectively, with significant differences compared to the control (P < 0.05). This study targeted NtPPO8 overexpression transgenic lines ( Figure 10 The content of hyoscyamine, a polyphenol, in tobacco leaves increased to varying degrees compared to the control, with increases of 18.88%, 32.35%, 24.69%, 3.21%, and 11.51%, respectively. This result indicates that the NtPPO8 gene plays an important role in the regulation of chlorogenic acid and rutin during the maturation period of tobacco leaves.

[0112] This result indicates that the NtPPO8 gene is closely related to the metabolism of chlorogenic acid and rutin in tobacco. Based on this result, in practical applications, the expression of the NtPPO8 gene in tobacco can be inhibited by using genetic engineering methods (such as silencing and knockout), thereby inhibiting the activity of polyphenol oxidase in tobacco and increasing the content of chlorogenic acid and rutin in tobacco leaves, thus improving tobacco germplasm resources.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of overexpression of the NtPPO8 gene in reducing the content of tobacco polyphenols, characterized by: The nucleotide sequence of the NtPPO8 gene is shown in SEQ ID NO.1; overexpression of the NtPPO8 gene significantly reduces the content of chlorogenic acid and / or rutin in tobacco; the tobacco polyphenols are chlorogenic acid and / or rutin.

2. The application of NtPPO8 gene overexpression according to claim 1 in reducing tobacco polyphenol content, characterized in that: The tobacco is K326.

3. The application of the NtPPO8 gene in the improvement of tobacco germplasm resources, characterized by: The nucleotide sequence of the NtPPO8 gene is shown in SEQ ID NO.1; the germplasm resource improvement is achieved by overexpressing the NtPPO8 gene to reduce the content of polyphenols in tobacco; the polyphenols are chlorogenic acid and / or rutin.

4. The application of the NtPPO8 gene in tobacco germplasm resource improvement according to claim 3, characterized in that: The overexpression involves constructing an overexpression vector for the NtPPO8 gene and overexpressing the NtPPO8 gene.

5. The application of the NtPPO8 gene in tobacco germplasm resource improvement according to claim 3, characterized in that: The tobacco is K326.

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