Application of NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves and tobacco variety breeding

Abstract

The application discloses application of NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves and tobacco variety cultivation, and belongs to the technical field of plant genetic engineering. The cDNA of leaves of common tobacco at different periods is used as a template, and a homologous gene of polyphenol oxidase of tobacco is cloned by using PCR technology, and is named as NtPPO7. It is found through cloning of the NtPPO7 gene and construction and analysis of an expression vector that overexpression of the gene in tobacco plants can significantly reduce the content of chlorogenic acid in mature tobacco leaves, which indicates that the NtPPO7 gene has an important role in regulation of chlorogenic acid in mature tobacco leaves. The application enriches the polyphenol oxidase gene family of tobacco, and lays a foundation for in-depth study of the polyphenol oxidase gene family in tobacco. Meanwhile, the application lays a foundation for elucidating a molecular regulation mechanism of chlorogenic acid biosynthesis in tobacco leaves, and provides a new precise regulation gene for regulation of the content of chlorogenic acid in tobacco leaves.

Description

Technical Field

[0001] This invention relates to the application of the NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves and in tobacco variety breeding, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an annual or short-lived perennial herbaceous plant belonging to the Solanaceae family. To meet the ever-growing demand for high-quality tobacco leaves from domestic and international cigarette manufacturers, improving the quality and safety of tobacco leaves is a crucial task that the tobacco industry needs to continuously improve.

[0003] Chlorogenic acid (CGA), also known as coffee tannin, is a phenylpropanoid compound produced by plants through the phenylalanine pathway during aerobic respiration. It is widely distributed in the plant kingdom, primarily belonging to the Caprifoliaceae, Asteraceae, Eucommiaceae, 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. Furthermore, chlorogenic acid molecules contain unsaturated double bonds, ester bonds, and polyphenols, making them important natural antioxidants closely related to plant cell resistance to abiotic and biotic factors such as low temperature, ultraviolet radiation, and diseases. However, chlorogenic acid is a secondary metabolite in tobacco, and traditional cultivation techniques cannot effectively regulate its content. Therefore, in-depth analysis of the biosynthesis and accumulation pathways of chlorogenic acid in tobacco from a biochemical perspective, and in-depth analysis of chlorogenic acid synthesis and regulatory genes from a genetic engineering perspective, are of great technical significance for improving tobacco quality and enhancing the stress resistance of tobacco varieties. Summary of the Invention

[0004] The first objective of this invention is to provide the application of the NtPPO7 gene in regulating the chlorogenic acid content in tobacco leaves, thus providing a usable gene resource for the precise regulation of chlorogenic acid in tobacco in the prior art.

[0005] The second objective of this invention is to provide the application of the NtPPO7 gene in tobacco variety breeding, laying a certain technical foundation for tobacco quality regulation and the breeding of new tobacco varieties.

[0006] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves is as follows:

[0007] Application of the NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves, the nucleotide sequence of the NtPPO7 gene is shown in SEQ ID NO.1.

[0008] The beneficial effects of the above technical solution are as follows: The application of the NtPPO7 gene in the regulation of chlorogenic acid content in tobacco leaves is a pioneering invention. Based on previous research, this invention uses cDNA from leaves at different stages of common tobacco growth as a template and employs PCR technology to clone the homologous gene of tobacco polyphenol oxidase, named NtPPO7. Through cloning and constructing and analyzing the expression vector of the NtPPO7 gene, this invention found that overexpression of this gene in tobacco plants can significantly reduce the chlorogenic acid content in mature tobacco leaves, indicating that the NtPPO7 gene plays an important role in the regulation of chlorogenic acid in mature tobacco leaves. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO7 in this invention enriches the tobacco polyphenol oxidase gene family and lays the foundation for in-depth research on the polyphenol oxidase gene family in tobacco. Simultaneously, this invention lays the foundation for elucidating the molecular regulatory mechanism of chlorogenic acid biosynthesis in tobacco leaves and provides a new, precise regulatory gene for the regulation of chlorogenic acid content in tobacco leaves.

[0009] As a further improvement, overexpression of the NtPPO7 gene significantly reduced the chlorogenic acid content in mature tobacco leaves.

[0010] As a further improvement, the overexpression involves constructing an overexpression vector for the NtPPO7 gene and overexpressing the NtPPO7 gene.

[0011] As a further improvement, the tobacco is K326.

[0012] To achieve the above objectives, the technical solution adopted in this invention for the application of the NtPPO7 gene in tobacco variety breeding is as follows:

[0013] The application of the NtPPO7 gene in tobacco variety breeding, wherein the nucleotide sequence of the NtPPO7 gene is shown in SEQ ID NO.1.

[0014] The beneficial effects of the above technical solution are as follows: This invention cloned the tobacco NtPPO7 gene, and after overexpressing the NtPPO7 gene in tobacco through Agrobacterium-mediated transformation, it was found that the content of chlorogenic acid in mature tobacco leaves was significantly reduced. This indicates that the NtPPO7 gene is involved in regulating the synthesis of chlorogenic acid in mature tobacco leaves, laying the foundation for elucidating the molecular regulatory mechanism of chlorogenic acid biosynthesis.

[0015] Furthermore, chlorogenic acid can form aroma compounds closely related to human senses during processing. These aroma compounds are an important source of tobacco aroma, so the aroma of tobacco can be controlled by adjusting the chlorogenic acid content. Simultaneously, chlorogenic acid plays a crucial role in regulating plant stress resistance; therefore, the stress resistance of tobacco can also be affected by adjusting its content. In summary, this invention provides new and usable genetic resources for tobacco variety breeding.

[0016] As a further improvement, the tobacco variety breeding involves regulating the chlorogenic acid content in tobacco leaves by adjusting the expression level of the NtPPO7 gene.

[0017] As a further improvement, the NtPPO7 gene was overexpressed through genetic engineering to screen for transgenic tobacco lines with reduced chlorogenic acid content.

[0018] As a further improvement, the tobacco is K326. Attached Figure Description

[0019] Figure 1 This is a gel electrophoresis image of the NtPPO7 gene clone in Example 1 of this invention;

[0020] Figure 2 This is an amino acid sequence analysis and alignment diagram from Example 2 of the present invention;

[0021] Figure 3 This is a diagram showing the expression characteristics of the NtPPO7 gene in different tissues in Example 3 of the present invention;

[0022] Figure 4 This is a subcellular localization map (scale bar, 50 μm) of the NtPPO7 gene in tobacco epidermal cells in Example 5 of the present invention.

[0023] Figure 5 This refers to the PCR detection of NtPPO7 gene expression in NtPPO7 gene-overexpressing plants in Example 6 of the present invention.

[0024] Figure 6 The NtPPO7 gene was detected by qRT-PCR in plants overexpressing the NtPPO7 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 NtPPO7 gene).

[0025] Figure 7 This study analyzes the activity of PPO in the leaves of T2 generation NtPPO7 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 NtPPO7 gene overexpression).

[0026] Figure 8Analysis of chlorogenic acid content in leaves of T2 generation NtPPO7 gene overexpressing plants in Example 8 of this invention;

[0027] Figure 9 Analysis of rutin content in leaves of T2 generation NtPPO7 gene overexpressing plants in Example 8 of this invention;

[0028] Figure 10 This study analyzes the hyoscyamine content in the leaves of T2 generation NtPPO7 gene overexpressing plants in Example 8 of this invention. Detailed Implementation

[0029] 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. It is precisely because of this that researchers have neglected the discovery, identification, and utilization of tobacco's own functional genes.

[0030] Polyphenol oxidase (PPO) is a copper-binding enzyme widely found in plants, animals, and microorganisms. In plants, PPO genes are not only functionally complex but also mostly exist as gene families with numerous members. For example, 12 PPO genes have been identified in eggplant, 7 in olive, 10 in banana, the poplar PPO gene family contains 18 genes, and 9 in potato. These PPO genes have different functions during plant growth and development. Sequence homology analysis in the common tobacco genome database indicates that there may be 12–14 polyphenol oxidase genes in tobacco. However, due to insufficient research on tobacco polyphenol oxidase gene families, the functions of most polyphenol oxidase genes remain unknown. To address this, this invention provides the application of the NtPPO7 gene in the regulation of chlorogenic acid content in tobacco leaves and in tobacco variety breeding.

[0031] Based on previous research, this invention cloned the homologous gene of tobacco polyphenol oxidase, named NtPPO7, using cDNA from leaves at different stages of common tobacco growth as a template and employing PCR technology. Through cloning and constructing and analyzing the expression vector of the NtPPO7 gene, this invention revealed that overexpression of this gene in tobacco plants significantly reduced the chlorogenic acid content in mature tobacco leaves, indicating that the NtPPO7 gene plays an important role in the regulation of chlorogenic acid in tobacco leaves during the maturation stage. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO7 in this invention enriches the tobacco polyphenol oxidase gene family and lays the foundation for in-depth research on the polyphenol oxidase gene family in tobacco. Simultaneously, this invention lays the foundation for elucidating the molecular regulatory mechanism of chlorogenic acid biosynthesis in tobacco leaves, providing a new and precise regulatory gene for the regulation of chlorogenic acid content in tobacco leaves.

[0032] 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.

[0033] 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.

[0034] In the following examples, primer sequence synthesis and gene sequencing were performed by Beijing Liuhe Huada Biotechnology Co., Ltd.

[0035] Biomaterials:

[0036] Tobacco material: Cultivated tobacco (Nicotiana tabacum) variety K326, kindly provided by Yunnan Tobacco Science Research Institute.

[0037] Vector: Super pCAMBIA1300 is a commonly used plasmid vector in molecular biology and can be obtained from public sources.

[0038] 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.

[0039] Experimental reagents:

[0040] 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.

[0041] Experimental equipment:

[0042] 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.

[0043] Data processing:

[0044] 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.

[0045] Specific embodiments of the application of the NtPPO7 gene in regulating chlorogenic acid content in tobacco leaves and in tobacco variety breeding:

[0046] Example 1

[0047] 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 NtPPO7 gene was cloned using PCR technology. The specific implementation process is as follows:

[0048] 1. Primer design

[0049] The specific PCR amplification primer sequences for non-coding region amplification are designed as follows:

[0050] NtPPO7-clone-F:5'-ATATAAGGCAAAGGTGGAAC-3' (shown in SEQ ID NO.3);

[0051] NtPPO7-clone-R:5'-CATCAAGCTGATTTCAACAC-3' (shown in SEQ ID NO.4).

[0052] 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.

[0053] 2. PCR amplification

[0054] 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.

[0055] Table 1 PCR reaction system

[0056] cDNA 2μL upstream primer 0.4μL Downstream primer 0.4μL PremixTaq 12.5μL <![CDATA[ddH2O]]> 9.7μL

[0057] Table 2 PCR reaction procedures

[0058]

[0059] PCR amplification products were detected by 1.2% agarose gel electrophoresis. The DNA marker used was DL2000, and the electrophoresis conditions were 120V / 20min. 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.

[0060] 3. Sequencing and analyzing the NtPPO7 gene

[0061] 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 cultured on a shaker at 37°C for approximately 12 hours (200 rpm). PCR was performed using a small amount of bacterial culture as a template to verify whether the clone was positive. The recombinant plasmid containing the target fragment was sequenced by Beijing Liuhe Huada Biotechnology Co., Ltd., obtaining the nucleotide sequence of the tobacco NtPPO7 gene.

[0062] Sequencing results showed that the tobacco NtPPO7 gene comprises 1806 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 NtPPO7 gene consists of 601 amino acids, and its specific amino acid sequence is shown in SEQ ID NO. 2.

[0063] Example 2

[0064] This embodiment compares the amino acid sequences of the protein (NtPPO7) encoded by the tobacco NtPPO7 gene with those of PPO from other species. The specific implementation steps are as follows:

[0065] The amino acid sequences of tobacco NtPPO7 were compared with those of other species, including wild tobacco, tobacco velvet, wolfberry, potato, and pepper PPO. The results showed that the amino acid sequences of NtPPO7 and other species PPO were highly similar. The sequence similarity with tobacco velvet NtomPPO, forest tobacco NsyPPO, wild tobacco NaPPO, yellow lantern pepper CcPPO, and Mexican pepper CaPPO were 99.83%, 96.17%, 95.51%, 70.49%, and 73.30%, respectively, indicating a high degree of sequence similarity.

[0066] Furthermore, Pfam analysis revealed that NtPPO7 possesses typical domains of PPO oxidase, such as... Figure 2 As shown, it has a Tyrosinase domain (PF00264) at amino acids 175-388, a PPO1_DWL domain (PF12142) at amino acids 396-443, and a PPO1_KFDV domain (PF12143) at amino acid sequences 463-600.

[0067] Example 3

[0068] Using real-time quantitative PCR (BIO-RAD, USA) technology, this example provides a preliminary analysis of the expression pattern of the tobacco NtPPO7 gene in tobacco plants. The specific implementation steps are as follows:

[0069] 1. Primer design for real-time PCR

[0070] For real-time quantitative PCR analysis, L25 was used as an internal reference gene, and the specific primer sequences were designed as follows:

[0071] NtPPO7-QF: 5'-TTCAACCACCAAGTCTTCTT-3' (shown in SEQ ID NO.5);

[0072] NtPPO7-QR: 5'-ACAACATTAGCAGCACCAT-3' (shown in SEQ ID NO.6);

[0073] L25-F: 5'-CCCCTCACCACAGAGTCTGC-3' (shown in SEQ ID NO.7);

[0074] L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (shown in SEQ ID NO.8);

[0075] 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.

[0076] 2. Quantitative Real-Time PCR Detection

[0077] 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 a 2-1... -△△CT 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.

[0078] Table 3 Reaction System

[0079]

[0080]

[0081] Table 4 Amplification Procedure

[0082]

[0083] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the analysis reveals that the NtPPO7 gene is highly expressed in the lateral roots and stamens of common tobacco K326 during its full bloom period.

[0084] Example 4

[0085] Based on the NtPPO7 gene cloned in Example 1, this embodiment constructs an overexpression vector for the NtPPO7 gene to further verify its function. The specific implementation steps are as follows:

[0086] 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. Using the correctly sequenced NtPPO7-T plasmid from Example 1 as a template, amplification was performed. 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 positive single clones were sent to Beijing BGI Genomics Co., Ltd. for sequencing.

[0087] Example 5

[0088] This embodiment selects the NtPPO7 overexpression vector from Example 4 as the research object to observe the localization of the NtPPO7 gene in tobacco epidermal cells. The specific implementation is as follows:

[0089] The NtPPO7-GFP vector and the pCAMBIA1300 empty vector were used to transform Agrobacterium LBA4404, and then the Agrobacterium was injected into the leaves of Nicotiana benthamiana via an infiltration method. 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).

[0090] Depend on Figure 4 It can be seen that the green fluorescence of NtPPO7-GFP coincides with the autofluorescence (purple) of chloroplasts, indicating that NtPPO7-GFP is located in chloroplasts.

[0091] Example 6

[0092] 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:

[0093] 1. Preparation of Agrobacterium competent cells

[0094] 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.

[0095] 2 plasmids transformed Agrobacterium

[0096] Take 1 μL of NtPPO7 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.

[0097] 3. Tobacco Conversion

[0098] Disinfect the vigorously growing tobacco leaves and cut them into 1cm pieces. 2 Small 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. The cultured plants were then 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 buds reached 1-2 cm in length, the clustered adventitious buds were cut into individual buds and transferred to MS rooting medium containing hygromycin, carbenicillin, and activated carbon to promote rooting. After the root system was well developed, the tissue culture seedlings were removed, the culture medium was washed off the roots with clean water, a few lower leaves were trimmed, and the seedlings were transferred to pots filled with loose, sterile soil and cultured according to standard management practices.

[0099] 4. Detection of positive super-genetically modified tobacco using PCR and qPCR methods

[0100] Design specific primer pairs for the expression vector, with the upstream primer specifically binding to the NtPPO7 gene and the downstream primer specifically binding to the GFP gene.

[0101] The upstream primer was 5'-GTTCAGTTCCTCCAGTTACC-3' (SEQ ID NO.11); the downstream primer was 5'-GTCGTCCTTGAAGAAGATGG-3' (SEQ ID NO.12). PCR amplification was performed using genomic DNA from the control and K326 and T0 generation seedlings as templates. Seedlings amplifying specific bands were considered T0 generation positive (e.g., ...). Figure 5 (As shown).

[0102] Next, the expression level of the NtPPO7 gene in the selected positive seedlings was detected using qPCR. NtPPO7 gene-specific primers were used: NtPPO7-QF: 5'-TTCAACCACCAAGTCTTCTT-3' (SEQ ID NO. 5) and NtPPO7-QR: 5'-ACAACATTAGCAGCACCAT-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). Quantitative PCR was performed using a qPCR instrument. 96 was used for amplification (reaction system and amplification procedure as described in Example 3). After the reaction, based on the obtained CT value, 2 -△△CT Methods for calculating the relative expression level of the NtPPO7 gene (e.g.) Figure 6 (As shown).

[0103] 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.

[0104] Example 7

[0105] This embodiment selects the NtPPO7 overexpression line (T2 generation) from Example 6 as the research object to detect the activity of polyphenol oxidase in the leaves of mid-leaf mature transgenic tobacco plants. The specific implementation operation is as follows:

[0106] The activity of polyphenol oxidase in the leaves of mature transgenic tobacco plants in the middle leaves 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 NtPPO7 gene, demonstrating that the transgenic NtPPO7 gene regulates the activity of polyphenol oxidase in tobacco leaves.

[0107] Example 8

[0108] This embodiment selects the NtPPO7 overexpression line (T2 generation) from Example 6 as the research object, and the specific implementation is as follows: The contents of chlorogenic acid, rutin, and hyoscyamine in the leaves of mature transgenic tobacco plants in the middle leaves are detected:

[0109] Based on HPLC-MS / MS technology and 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 sample was ground into powder using a grinder and transferred to 1.5 mL (2 mL centrifuge tube) of pre-cooled ethanol-water (4:1, v / v) extraction solution. The sample was sonicated at room temperature for 1 h; then centrifuged at 14000 rpm 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 rpm 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 source was used for ionization; 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 NtPPO7 overexpression transgenic lines (such as...) are effective against these strains. Figure 8As shown in the figure): Compared with the control (K326), the chlorogenic acid content in the middle leaves of the T2 generation transgenic plants (NtPPO7-5-1-1, NtPPO7-5-1-22, NtPPO7-5-4-1, NtPPO7-5-4-6, NtPPO7-5-4-17) at the maturity stage was reduced by 36.68%, 26.17%, 54.31%, 46.77%, and 61.63%, respectively, and the differences with the control were all statistically significant (P<0.05). This result indicates that the NtPPO7 gene plays an important role in the regulation of chlorogenic acid at the maturity stage of tobacco leaves. For NtPPO7 overexpression transgenic lines (such as...), further research is needed. Figure 9 As shown in the figure: The rutin content in the middle leaves of T2 generation transgenic plants at maturity was not significantly different from the control (P<0.05). This was observed in NtPPO7 overexpression transgenic lines (…). Figure 10 The content of hyoscyamine, a polyphenol in tobacco leaves, increased to varying degrees compared with the control, increasing by 33.16%, 24.69%, 14.26%, 32.45%, and 26.96%, respectively.

[0110] This result indicates that the NtPPO7 gene is closely related to the content of polyphenols, especially chlorogenic acid, in tobacco leaves. Based on this result, in practical applications, the expression of the NtPPO7 gene in tobacco can be inhibited by using genetic engineering methods (such as silencing or knocking out), thereby inhibiting the activity of polyphenol oxidase in tobacco and increasing the content of chlorogenic acid in tobacco leaves. Then, tobacco varieties with increased chlorogenic acid content can be obtained through screening.

[0111] 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. NtPPO7 The application of genes in regulating the content of hyoscyamine in tobacco leaves is characterized by: The NtPPO7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; overexpression NtPPO7 Genes showed a significant increase in the content of hyoscyamine in mature tobacco leaves.

2. As described in claim 1 NtPPO7 The application of genes in regulating the content of hyoscyamine in tobacco leaves is characterized by: The overexpression is for the construction NtPPO7 Gene overexpression vectors, for NtPPO7 The gene is overexpressed.

3. As described in claim 1 or 2 NtPPO7 The application of genes in regulating the content of hyoscyamine in tobacco leaves is characterized by: The tobacco is K326.

4. NtPPO7 The application of genes in tobacco variety breeding is characterized by: The NtPPO7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the tobacco variety was bred by overexpressing the gene through genetic engineering. NtPPO7 Gene screening was used to obtain transgenic tobacco lines with increased hyoscyamine content.

5. The method according to claim 4 NtPPO7 The application of genes in tobacco variety breeding is characterized by: The tobacco is K326.

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