Tobacco polyphenol oxidase NtPPO5, encoding gene thereof, recombinant expression vector and application thereof

Abstract

The present application discloses a tobacco polyphenol oxidase NtPPO5, a coding gene thereof, a recombinant expression vector and application. The tobacco polyphenol oxidase coding gene NtPPO5 has a nucleotide sequence of (1) a nucleotide sequence shown in SEQ ID NO. 1; or (2) a nucleotide sequence shown in SEQ ID NO. 1, which is substituted and / or deleted and / or added with one or more nucleotides and expresses a same function protein. It is found through detection that overexpression of the NtPPO5 gene can significantly increase the activity of polyphenol oxidase in tobacco plants; moreover, the tobacco plants overexpressing the NtPPO5 gene have increased internode distance, reduced total leaf and bud number, and can effectively improve the plant type of the tobacco plants; in addition, the NtPPO5 gene overexpression can reduce the contents of polyphenols chlorogenic acid and rutin in tobacco leaves in the maturation period.

Description

Technical Field

[0001] This invention relates to tobacco polyphenol oxidase NtPPO5, its encoding gene, recombinant expression vector, and applications, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Polyphenol oxidase (PPO) is a copper-binding enzyme widely found in plants, animals, and microorganisms. Based on substrate specificity and mechanism of action, it can be divided into three categories: tyrosinase (EC 1.14.18.1), catechol oxidase (EC 1.10.3.1), and laccase (EC 1.10.3.2). PPO has multiple functions in organisms. It participates in plant growth and development processes, such as flower opening, fruit ripening, and leaf yellowing. PPO can convert polyphenols (such as butyric acid and catechols) in plants into highly active oxidation products, such as quinoline and quinolinol. This oxidation reaction typically uses molecular oxygen as a substrate, and the resulting oxidation products possess various biological activities, including antioxidant, antibacterial, and antitumor effects. Furthermore, the oxidation of polyphenol substrates by PPO is considered a major cause of browning discoloration in many fruits and vegetables during harvesting, storage, transportation, and processing. Therefore, inhibiting the activity of polyphenol oxidase can improve the freshness and quality of food. Polyphenol oxidase can also be applied in environmental protection, catalyzing the oxidative degradation of organic matter in industrial wastewater and water bodies, thereby reducing water pollution. Furthermore, polyphenol oxidase can be used to treat pesticides and organic pollutants in soil and waste. As the applications of polyphenol oxidase expand, research on it is becoming increasingly in-depth.

[0003] Polyphenol oxidase genes in plants are not only functionally complex but also exist primarily as gene families. Researchers have identified multiple polyphenol oxidases in plants such as potatoes, tomatoes, corn, soybeans, salvia miltiorrhiza, and tobacco. Sequence homology analysis based on the common tobacco genome database indicates that there may be 12-14 polyphenol oxidase genes in tobacco. Chinese invention patent application CN110846431A discloses the application of tobacco polyphenol oxidase genes NtPPO2a and / or NtPPO2b in the breeding of drought-resistant tobacco varieties. Specifically, it discloses the nucleotide sequences of tobacco polyphenol oxidase genes NtPPO2a and NtPPO2b and demonstrates that mutations in these genes reduce the drought resistance of the tobacco variety. However, due to the complex functions of polyphenol oxidase genes in tobacco, research on the tobacco polyphenol oxidase gene family is currently insufficient, and the functions of most polyphenol oxidase genes remain unknown. Summary of the Invention

[0004] The first objective of this invention is to provide the tobacco polyphenol oxidase encoding gene NtPPO5, in order to solve the problem that some members of the tobacco polyphenol oxidase gene family are unknown in the prior art.

[0005] The second objective of this invention is to provide tobacco polyphenol oxidase NtPPO5 to address the problem that some members of the tobacco polyphenol oxidase family are unknown in the prior art.

[0006] The third objective of this invention is to provide a recombinant expression vector to address the problem that the existing research on the tobacco polyphenol oxidase gene family is not in-depth, resulting in the unknown function of polyphenol oxidase genes in tobacco.

[0007] The fourth objective of this invention is to provide the application of the tobacco polyphenol oxidase encoding gene NtPPO5 or a recombinant expression vector in the regulation of tobacco polyphenol content, in order to solve the problem that the existing technology does not have in-depth research on the tobacco polyphenol oxidase gene family, resulting in the unknown function of polyphenol oxidase genes in tobacco.

[0008] The fifth objective of this invention is to provide the application of the tobacco polyphenol oxidase encoding gene NtPPO5 or a recombinant expression vector in the phenotypic optimization of tobacco plants, in order to solve the problem that the existing technology does not have in-depth research on the tobacco polyphenol oxidase gene family, resulting in the unknown function of polyphenol oxidase genes in tobacco.

[0009] To achieve the above objectives, the technical solution adopted by the tobacco polyphenol oxidase encoding gene NtPPO5 in this invention is as follows:

[0010] The nucleotide sequence of the tobacco polyphenol oxidase encoding gene NtPPO5 is as follows:

[0011] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0012] Or (2) the nucleotide sequence shown in SEQ ID NO.1, which is substituted and / or deleted and / or added with one or more nucleotides and expresses the same functional protein.

[0013] The beneficial effects of the above technical solution are as follows: 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 a homologous gene of tobacco polyphenol oxidase, named NtPPO5. Through cloning and constructing and analyzing the expression vector of the NtPPO5 gene, this invention found that overexpression of this gene in tobacco plants can significantly increase the activity of polyphenol oxidase in leaves. The NtPPO5 gene, the tobacco polyphenol oxidase encoding gene discovered in this invention, enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidase in tobacco.

[0014] To achieve the above objectives, the technical solution adopted by the tobacco polyphenol oxidase NtPPO5 of the present invention is as follows:

[0015] Tobacco polyphenol oxidase NtPPO5 has the following amino acid sequence:

[0016] (1) The amino acid sequence shown in SEQ ID NO.2;

[0017] Or (2) a derivative protein with the same function, but with the amino acid sequence shown in SEQ ID NO.2 replaced and / or with one or more amino acid residues deleted and / or added.

[0018] The beneficial effects of the above technical solution are as follows: This invention demonstrates through experiments that overexpression of the tobacco polyphenol oxidase encoding gene NtPPO5 in tobacco plants can significantly increase the activity of polyphenol oxidase in leaves.

[0019] To achieve the above objectives, the technical solution adopted by the recombinant expression vector of the present invention is as follows:

[0020] A recombinant expression vector containing the tobacco polyphenol oxidase encoding gene NtPPO5.

[0021] The beneficial effects of the above technical solution are as follows: by using genetic engineering methods, the NtPPO5 gene is inserted into the pCAMBIA1300 starting vector to construct a recombinant expression vector that overexpresses the NtPPO5 gene. After transformation into tobacco, the expression level of the NtPPO5 gene is significantly increased, providing a good tool for subsequent research on the function of this gene.

[0022] To achieve the above objectives, the technical solution adopted by the present invention for the application of the tobacco polyphenol oxidase encoding gene NtPPO5 or the recombinant expression vector in the regulation of tobacco polyphenol content is as follows:

[0023] Application of the tobacco polyphenol oxidase encoding gene NtPPO5 or recombinant expression vector in the regulation of tobacco polyphenol oxidase content, wherein the polyphenols include chlorogenic acid and rutin.

[0024] The beneficial effects of the above technical solution are as follows: This invention constructs a recombinant expression vector through genetic engineering, transforms it into tobacco, and overexpresses the NtPPO5 gene, obtaining NtPPO5 gene-overexpressing tobacco plants. Detection revealed a significant increase in polyphenol oxidase activity in the leaves of the obtained NtPPO5 gene-overexpressing tobacco plants. Compared with the normal control group, the content of polyphenols chlorogenic acid and rutin in the mature leaves of NtPPO5 gene-overexpressing tobacco plants was significantly reduced. This invention enriches the polyphenol oxidase system in tobacco and provides a new research subject for the study of polyphenol oxidase function in tobacco.

[0025] As a further improvement, overexpression of the NtPPO5 gene reduced the content of chlorogenic acid and rutin in tobacco.

[0026] To achieve the above objectives, the technical solution adopted in the application of the tobacco polyphenol oxidase encoding gene NtPPO5 or its recombinant expression vector in tobacco plant phenotyping is as follows:

[0027] Application of the tobacco polyphenol oxidase encoding gene NtPPO5 or recombinant expression vector in tobacco plant phenotypic optimization.

[0028] The beneficial effects of the above technical solution are as follows: This invention constructs tobacco plants overexpressing the NtPPO5 gene. Observations show that after NtPPO5 gene overexpression, the internode length of tobacco plants increases, while the total number of leaves and flower buds decreases. Tobacco leaves are relatively large, and the plants are relatively tall, resulting in poor light transmission to the lower leaves. Furthermore, the large leaf openings of tobacco plants also affect ventilation in tobacco fields. Currently, research on tobacco plant type, especially internode traits, is relatively limited. This invention demonstrates that the NtPPO5 gene can regulate the internode length and leaf number of tobacco plants. This provides an important research direction for improving the internode length of tobacco, thereby improving light transmission and ventilation, ultimately increasing the photosynthetic efficiency and total yield of tobacco. It also provides genetic materials and theoretical basis for improving tobacco leaf quality and the genetic improvement of tobacco varieties.

[0029] As a further improvement, overexpression of the NtPPO5 gene increased the internode distance of tobacco plants and reduced the total number of leaves and flower buds.

[0030] The beneficial effects of the above technical solution are as follows: Experiments have shown that the NtPPO5 gene can regulate the phenotype of tobacco plants. Overexpression of this gene in tobacco can increase the internode length and improve the plant type. Therefore, the NtPPO5 gene, which encodes tobacco polyphenol oxidase, has broad application prospects in the field of plant type breeding and has great potential for economic benefits. Attached Figure Description

[0031] Figure 1 This is a gel electrophoresis image of the NtPPO5 gene clone in Experimental Example 1 of this invention;

[0032] Figure 2 This is a comparison diagram of amino acid sequence analysis in Experimental Example 2 of the present invention;

[0033] Figure 3 This is a diagram showing the expression characteristics of the NtPPO5 gene in different tissues in Experimental Example 3 of this invention.

[0034] Figure 4 This is a PCR detection of NtPPO gene expression in NtPPO5 overexpressing plants in Experimental Example 5 of this invention.

[0035] Figure 5 The NtPPO gene was detected by qRT-PCR in plants overexpressing the NtPPO5 gene in Experiment Example 5 of this invention (where Con represents the tobacco plant of normal control, and the rest are different individual plants overexpressing the NtPPO5 gene).

[0036] Figure 6 This is a phenotypic diagram of the T1 generation NtPPO5 gene overexpressing plants in Experiment Example 6 of this invention;

[0037] Figure 7 This study analyzes the activity of PPO in the leaves of T1 generation NtPPO5 gene overexpressing plants in Experiment Example 6 of the present invention (where Con represents the tobacco plant of normal control, and the rest are different individual plants of NtPPO5 gene overexpression).

[0038] Figure 8 This study analyzes the chlorogenic acid content in the leaves of T1 generation NtPPO5 gene overexpressing plants in Experiment Example 7 of this invention (where Con represents the tobacco plant of normal control, and the rest are different individual plants of NtPPO5 gene overexpression).

[0039] Figure 9 This study analyzes the rutin content in the leaves of T1 generation NtPPO5 gene overexpressing plants in Experiment Example 7 of this invention (where Con represents the normal control tobacco plant, and the rest are different individual plants overexpressing the NtPPO5 gene). Detailed Implementation

[0040] 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 NtPPO5. Through cloning the NtPPO5 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 NtPPO5 enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidases in tobacco.

[0041] 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.

[0042] 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.

[0043] Tobacco: Tobacco K326, a common tobacco material.

[0044] Vector: Super pCAMBIA1300 is a commonly used plasmid vector in molecular biology and can be obtained from public sources.

[0045] DH5α-sensor cells were purchased from Shanghai Sangon Biotech Co., Ltd.; Agrobacterium strain LBA4404 is a commonly used strain in molecular biology and is publicly available.

[0046] Primer sequence synthesis and gene sequencing were completed by Beijing Liuhe Huada Biotechnology Co., Ltd.

[0047] Experimental reagents:

[0048] 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.

[0049] Experimental equipment:

[0050] 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.

[0051] Example 1 of the tobacco polyphenol oxidase encoding gene NtPPO5

[0052] The nucleotide sequence of the tobacco polyphenol oxidase encoding gene NtPPO5 in this embodiment is as shown in SEQ ID NO.1.

[0053] Example 1 of tobacco polyphenol oxidase NtPPO5

[0054] The amino acid sequence of tobacco polyphenol oxidase NtPPO5 in this embodiment is as shown in SEQ ID NO.2.

[0055] Example 1 of recombinant expression vector

[0056] The recombinant expression vector of this embodiment contains the tobacco polyphenol oxidase encoding gene NtPPO5, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0057] Example 1: Application of the tobacco polyphenol oxidase encoding gene NtPPO5 or recombinant expression vector in the regulation of tobacco polyphenol content.

[0058] In this embodiment, tobacco plants were transformed with a recombinant expression vector containing the tobacco polyphenol oxidase encoding gene NtPPO5 to construct tobacco plants overexpressing the NtPPO5 gene. The activity of polyphenol oxidase in the leaves of this line was significantly increased, and the content of chlorogenic acid and rutin in the middle leaves was significantly reduced during the maturity period of the middle leaves.

[0059] Example 1: Application of the tobacco polyphenol oxidase encoding gene NtPPO5 or recombinant expression vector in tobacco plant phenotypic optimization

[0060] In this embodiment, the tobacco polyphenol oxidase encoding gene NtPPO5 was overexpressed in tobacco through genetic engineering, resulting in increased internode spacing and reduced total number of leaves and flower buds in tobacco plants.

[0061] Example 1: Cloning of the NtPPO5 gene encoding tobacco polyphenol oxidase

[0062] Based on previous research, this invention utilizes Primer Premier 6 software to design upstream and downstream primers. Using cDNA from leaves at different growth stages of common tobacco as templates, the homologous gene of tobacco polyphenol oxidase, named NtPPO5, was cloned using PCR technology. The specific implementation steps for cloning the tobacco NtPPO5 gene are as follows:

[0063] 1. Primer design

[0064] The specific primer sequences for PCR amplification are designed as follows:

[0065] NtPPO5-F: 5'-ATGGCTTCTCTTCCACTCCC-3' (shown in SEQ ID NO.3),

[0066] NtPPO5-R: 5'-TCAATCCTCAAGCACAATCT-3' (shown in SEQ ID NO.4).

[0067] RNA was extracted from leaves of tobacco K326 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 reverse transcription kit (Takara).

[0068] 2. PCR amplification

[0069] Using the cDNA prepared in step 1 above as a template, PCR amplification was performed using the designed primers. The 25 μL reaction system for PCR amplification was designed as follows: 2 μL cDNA, 0.4 μL upstream primer, 0.4 μL downstream primer, 12.5 μL PremixTaq, and 9.7 μL ddH2O. The reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 10 min, and incubation at 4℃.

[0070] PCR amplification products were detected by 1.0% agarose gel electrophoresis. A DL2000 DNA marker was used, and the electrophoresis conditions were 120V / 20min. The results were then observed under a UV scanner. The length of the target DNA fragment was as follows: Figure 1 As shown, DNA was recovered using a DNA gel recovery kit (Takara), and after concentration determination, it was stored at -20℃ for later use or directly used for subsequent experimental operations.

[0071] 3. Sequencing and analyzing the NtPPO5 gene

[0072] 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 NtPPO5 gene.

[0073] Sequencing results showed that the tobacco NtPPO5 gene consists of 1821 bases, and the specific base sequence is shown in SEQ ID NO.1.

[0074] Analysis of the base sequence showed that the polyphenol oxidase encoded by the tobacco NtPPO5 gene consists of 604 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.2.

[0075] Experimental Example 2: Amino acid sequence analysis of tobacco polyphenol oxidase NtPPO5

[0076] The amino acid sequences of tobacco polyphenol oxidase NtPPO5 were compared with those of PPO from other species, including wild tobacco, tobacco fluff, wolfberry, potato, and pepper. The results showed that NtPPO5 exhibited high amino acid sequence similarity to PPO from other species. Specifically, it showed sequence similarity of 95.72% with NaPPO from wild tobacco, 92.28% with NtomPPO from tobacco fluff, 79.42% with LfPPO from wolfberry, 73.98% with SvPPO from potato, and 95.72% with CaPPO from pepper.

[0077] Furthermore, Pfam analysis revealed that NtPPO5 possesses typical domains of PPO oxidases, such as... Figure 2 As shown, it has a Tyrosinase domain (PF00264) in the amino acid sequence from 181 to 388, a PPO1_DWL domain in the amino acid sequence from 395 to 446, and a PPO1_KFDV domain between the amino acid sequences from 475 to 603.

[0078] Example 3: Analysis of the expression pattern of the tobacco polyphenol oxidase gene NtPPO5 in tobacco.

[0079] Using real-time quantitative PCR (BIO-RAD, USA) technology, this experiment conducted a preliminary analysis of the expression pattern of the NtPPO5 gene in tobacco plants. The specific procedures are as follows:

[0080] 1. Primer design and sample preparation for real-time PCR

[0081] For real-time quantitative PCR analysis, L25 was used as an internal reference gene, and the specific primer sequences were designed as follows:

[0082] NtPPO5-QF: 5'-ACCATAGTTTCCAAGTCTCA-3' (shown in SEQ ID NO.5);

[0083] NtPPO5-QR: 5'-ATGCCTCCTAATCCAAGTAG-3' (shown in SEQ ID NO.6);

[0084] L25-F: 5'-CCCCTCACCACAGAGTCTGC-3' (shown in SEQ ID NO.7);

[0085] L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (shown in SEQ ID NO.8);

[0086] RNA was extracted from old leaves, old leaf veins, new leaves, new leaf veins, lateral roots, fibrous roots, stems, axillary buds, flower buds, calyxes, stamens, pistils, and ovaries of tobacco K326 during its peak flowering period, and reverse transcribed into cDNA as template samples for later use.

[0087] 2. Quantitative Real-Time PCR Detection

[0088] The instrument used was a Bio-Rad CFX96 from Bio-Rad Laboratories, USA. The reaction mixture consisted of 20 μL of: 10 μL SYBR PremixEx Taq™, 1 μL forward primer, 1 μL reverse primer, 2 μL cDNA, and 6 μL ddH2O. The amplification program was: 94℃ for 30 s pre-denaturation; 94℃ for 5 s denaturation; 60℃ for 20 s annealing; 72℃ for 20 s extension, for 45 cycles. The relative expression levels obtained after the reaction were plotted using a 22 -△△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.

[0089] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the NtPPO5 gene is highly expressed in the receptacle and pistil, and is also expressed in older leaves. No expression is found in the veins of older leaves, new leaves, veins of new leaves, fibrous roots, stems, axillary buds, flower buds, stamens, and ovaries, indicating that the expression of the NtPPO5 gene is tissue-specific.

[0090] Example 4: Construction of Recombinant Expression Vector

[0091] An NtPPO5 overexpression vector was constructed using cloning primers with homologous recombination adapter sequences added to both primers. The upstream primer was 5'-AAATTGACTCTAGAAAGCTTATGGCTTCTCTTCCACTCCC-3' (SEQ ID NO. 9), and the downstream primer was 5'-CCCTTGCTCACCATGGTACCTCAATCCTCAAGCACAATCT-3' (SEQ ID NO. 10). The NtPPO5-T plasmid, correctly sequenced in Example 1, was used as a template for amplification. The recovered and purified PCR product and the 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.

[0092] Experiment Example 5: Construction of tobacco plants through Agrobacterium transformation and overexpression of the tobacco polyphenol oxidase encoding gene NtPPO5.

[0093] The single colonies successfully sequenced in Experiment 4 were expanded and cultured, plasmids were extracted, and the recombinant expression vector was transformed into Agrobacterium LBA4404 using electroporation. This transformed vector was then transferred into tobacco plants to obtain tobacco plants overexpressing the NtPPO5 gene. The specific implementation method is as follows:

[0094] 1. Preparation of Agrobacterium competent cells:

[0095] Single colonies of Agrobacterium LBA4404 were picked and cultured overnight at 28°C in 2 mL of LB liquid medium (containing 20 mg / mL Rif). 2 mL of well-grown bacterial culture (containing 25 mg / L Rif) was inoculated into 50 mL of LB liquid medium and cultured at 28°C with shaking until the OD600 reached approximately 0.5. The bacterial culture was transferred to 50 mL centrifuge tubes, placed on ice for 30 minutes, and then 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.

[0096] 2. Plasmid transformation of Agrobacterium:

[0097] Take 1 μL of the recombinant expression vector and add it to a centrifuge tube containing 100 μL of Agrobacterium competent cells. Place the tube on ice for 30 minutes. Then, transfer the tube 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. Spread 200 μL of the resuspended bacterial solution evenly on an LB agar plate containing 20 mg / L Rif and 50 mg / L kanamycin (Kan), and incubate at 28°C for 2-3 days. After confirming the colony PCR results, preserve the bacterial strain.

[0098] 3. Tobacco Conversion:

[0099] Disinfect the leaves of the vigorously growing tobacco K326 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 pieces were then immersed in engineered bacterial solution for 10-15 minutes, 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 (kanamycin for gene editing vectors) and carbenicillin, and cultured at a constant temperature. 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, 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.

[0100] 4. Detection of tobacco plants with positive NtPPO5 gene overexpression using PCR and qPCR methods:

[0101] Specific primer pairs for the expression vector were designed, with the upstream primer specifically binding to the NtPPO5 gene and the downstream primer specifically binding to the GFP gene. The upstream primer was 5'-CGTGCCACATCATAGTCAT-3' (SEQ ID NO.11); the downstream primer was 5'-CGTCGTCCTTGAAGAAGAT-3' (SEQ ID NO.12). PCR amplification was performed using genomic DNA from control K326 and T0 generation seedlings as templates. The seedlings that amplified specific bands were T0 generation positive NtPPO5 gene overexpressing tobacco. The expression level of the NtPPO5 gene in the selected positive seedlings was then detected using qPCR. Using NtPPO5 gene-specific primers, NtPPO5-QF: 5'-ACCATAGTTTCCAAGTCTCA-3' (SEQ ID NO. 5), NtPPO5-QR: 5'-ATGCCTCCTAATCCAAGTAG-3' (SEQ ID NO. 6); and using the tobacco L25 gene as an internal control, L25-F: 5'-CCCCTCACCACAGAGTCTGC-3' (SEQ ID NO. 7), L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (SEQ ID NO. 8), quantitative PCR was performed. Amplification was performed using Roche (http: / / www.roche.com / index.htm). After the reaction, based on the obtained CT values, 2... -△△CT The method calculates the relative expression level of the NtPPO5 gene.

[0102] 5. Validation of tobacco plants overexpressing the NtPPO5 gene

[0103] To verify whether the phenotype could be stably inherited by the next generation, seeds of T0 generation positive plants were collected, and T1 generation plants were obtained after further planting. PCR amplification analysis was then performed, and the results are as follows: Figure 4 As shown in the figure, the T0 generation plants can be stably inherited into the T1 generation. At the same time, K326 and T1 generation transgenic plants were grown in a greenhouse.

[0104] The results of relative gene expression levels in T1 generation transgenic plants were obtained using quantitative PCR. Figure 5 As shown in the figure, the relative expression level of the NtPPO5 gene in the T1 generation transgenic plants was significantly higher than that in the control K326.

[0105] Experiment Example 6: Phenotypic Analysis and Polyphenol Oxidase Activity Analysis of Tobacco Plants Overexpressing the NtPPO5 Gene

[0106] 1. Phenotypic observation:

[0107] Phenotypic observation results of T1 generation tobacco plants overexpressing the NtPPO5 gene during full bloom are as follows: Figure 6 As shown, tobacco plants overexpressing the NtPPO5 gene had increased internode length, decreased total number of leaves, and significantly fewer flower buds compared to K326.

[0108] 2. Polyphenol oxidase activity detection:

[0109] The polyphenol oxidase activity in leaves of T1 generation tobacco plants overexpressing the NtPPO5 gene during the vigorous growth period was detected using a polyphenol oxidase assay kit (Beijing Solarbio Science & Technology Co., Ltd.). The enzyme activity 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 plants overexpressing the NtPPO5 gene was significantly increased compared with the control (K326) (P<0.01). This study preliminarily reveals the function of the NtPPO5 gene and proves that the transgenic NtPPO5 gene regulates the activity of polyphenol oxidase in tobacco leaves.

[0110] Experiment Example 7: Detection of chlorogenic acid and rutin content in tobacco plants overexpressing the NtPPO5 gene

[0111] This invention uses GC-MS analysis to detect the chlorogenic acid and rutin content in tobacco plants overexpressing the NtPPO5 gene. The HPLC-UV absolute quantitative analysis conditions are as follows: Symmetry C18 column (4.6×250mm, 5μm), detection wavelength 340nm, injection volume 10μL, and flow rate 1.0mL / min.

[0112] Mobile phase A is water / methanol / acetic acid (44 / 5 / 1, v / v / v), and mobile phase B is methanol / water / acetic acid (44 / 5 / 1, v / v / v).

[0113] Gradient elution:

[0114] 0~15.0min, 10%B-30%B;

[0115] 15.0~26.0min, 30%B-90%B;

[0116] 26.0–28.0 min, 90% B;

[0117] 28.1-35.0 min, 10% B.

[0118] The results are as follows Figure 8 and Figure 9 As shown, by Figure 8 It can be seen that, compared with the control (K326), the chlorogenic acid content in the middle leaves of tobacco plants overexpressing the NtPPO5 gene in the T1 generation (PPO-3, PPO-4, PPO-12, PPO-14, PPO-18, PPO-19) at the maturity stage decreased by 40.03%, 26.34%, 31.91%, 16.53%, 22.81%, and 34.38%, respectively, and the differences with the control were all statistically significant (p<0.05). Figure 9 It can be seen that, compared with the control (K326), the rutin content in the middle leaves of tobacco plants overexpressing the NtPPO5 gene in the T1 generation (PPO-3, PPO-4, PPO-12, PPO-14, PPO-18, PPO-19) at the maturity stage was reduced by 40.14%, 31.21%, 26.61%, 34.31%, 14.91%, and 23.07%, respectively, and the differences with the control were all statistically significant (p<0.05). This result indicates that the NtPPO5 gene plays an important role in the regulation of polyphenol (including chlorogenic acid and rutin) content in tobacco leaves at the maturity stage.

[0119] 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. A gene encoding tobacco polyphenol oxidase NtPPO5 Its application in tobacco plant phenotypic optimization is characterized by: overexpression NtPPO5 Genes that increase internode distance and decrease the total number of leaves and flower buds in tobacco plants; the gene encoding the tobacco polyphenol oxidase. NtPPO5 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The tobacco polyphenol oxidase encoding gene according to claim 1 NtPPO5 Its application in tobacco plant phenotypic optimization is characterized by: The tobacco variety is K326.

3. The application of a recombinant expression vector in tobacco plant phenotypic optimization, characterized in that: overexpression NtPPO5 The gene increases the internode distance in tobacco plants and reduces the total number of leaves and flower buds; the recombinant expression vector contains the gene encoding tobacco polyphenol oxidase. NtPPO5 The gene encoding the tobacco polyphenol oxidase NtPPO5 The nucleotide sequence is shown in SEQ ID NO.

1.

4. The application of the recombinant expression vector according to claim 3 in tobacco plant phenotypic optimization, characterized in that: The tobacco variety is K326.

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