Tobacco polyphenol oxidase NtPPO6 and its encoding gene, gene editing vector and applications
By cloning and gene editing the tobacco polyphenol oxidase NtPPO6 gene, a gene editing vector was constructed, and the NtPPO6 gene was successfully knocked out. This solved the problem of insufficient research on the tobacco polyphenol oxidase family, achieving reduced polyphenol oxidase activity and increased chlorogenic acid content, thereby improving the phenotype and quality of tobacco varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHENGZHOU TOBACCO RES INST OF CNTC
- Filing Date
- 2023-12-25
- Publication Date
- 2026-05-26
AI Technical Summary
Research on the tobacco polyphenol oxidase gene family is not in-depth enough, the functions of some members are unknown, and there is a lack of effective research tools, resulting in the unknown function of polyphenol oxidase genes in tobacco, which affects variety breeding and plant phenotypic optimization.
The tobacco polyphenol oxidase NtPPO6 gene was cloned and validated. A gene editing vector was designed, and the NtPPO6 gene was knocked out using gene editing technology to construct gene-edited tobacco plants. Changes in polyphenol oxidase activity and chlorogenic acid content were then detected.
It significantly reduces the activity of polyphenol oxidase in tobacco leaves, increases chlorogenic acid content, regulates plant height, leaf number and size, improves light transmittance and ventilation in tobacco, provides genetic material and theoretical basis, and lays the foundation for tobacco variety improvement.
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Figure CN117757818B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to tobacco polyphenol oxidase NtPPO6, its encoding gene, gene editing vector, and applications, belonging to the field of plant genetic engineering technology. Background Technology
[0002] Polyphenol oxidases (PPOs) are a class of copper-binding enzymes widely found in nature, including plants, animals, and microorganisms. Based on their substrate specificity and mechanism of action, they can be divided into three classes: tyrosinases (EC 1.14.18.1), catechol oxidases (EC 1.10.3.1), and laccases (EC 1.10.3.2).
[0003] Polyphenol oxidase has multiple functions: (1) It participates in the growth and development of plants, such as flower opening, fruit ripening, and leaf yellowing; (2) It converts polyphenolic substances (such as butyric acid and catechol) in plants into highly active oxidation products, such as quinoline and quinolinol. This oxidation reaction usually uses molecular oxygen as a substrate, and the resulting oxidation products have a variety of biological activities, including antioxidant, antibacterial, and antitumor effects; (3) The oxidation of polyphenol substrates by PPO is considered to be the main reason for the browning of 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; (4) Polyphenol oxidase can also be applied in the field of environmental protection, catalyzing the oxidative degradation of organic matter in industrial wastewater and water bodies, thereby reducing water pollution; (5) Polyphenol oxidase can also be used to treat soil and waste containing pesticides and organic pollutants. It is precisely because the application fields of polyphenol oxidase are gradually expanding that people's research on polyphenol oxidase is becoming more and more in-depth.
[0004] Polyphenol oxidase genes in plants are not only functionally complex but also often exist in gene families. Researchers have identified multiple polyphenol oxidases in plants such as potatoes, tomatoes, corn, soybeans, salvia miltiorrhiza, and tobacco. For example, six PPO genes were found in eggplant, a cash crop in the Solanaceae family; seven in tomatoes; and two in potatoes. At least six PPO genes were found in red clover, and Cai et al. found eight different PPO genes in sorghum, but only one in grapevines. Sequence homology analysis in the common tobacco genome database indicates that there may be 12–14 polyphenol oxidase genes in tobacco. Chinese invention patent application CN107653256A discloses a tobacco polyphenol oxidase gene NtPPO1 and its site-directed mutagenesis method and application, specifically disclosing the nucleotide sequence of the tobacco polyphenol oxidase gene NtPPO1 and demonstrating its association with tobacco browning. However, due to the complex functions of polyphenol oxidase genes in tobacco, the research on the tobacco polyphenol oxidase gene family is not yet in-depth, and the functions of most polyphenol oxidase genes are unknown. Summary of the Invention
[0005] The first objective of this invention is to provide the tobacco polyphenol oxidase encoding gene NtPPO6, in order to solve the problem that some members of the tobacco polyphenol oxidase gene family are unknown in the prior art.
[0006] The second objective of this invention is to provide tobacco polyphenol oxidase NtPPO6 to address the problem that some members of the tobacco polyphenol oxidase family are unknown in the prior art.
[0007] The third objective of this invention is to provide a gene editing vector to address the problem that there are no suitable research tools in the prior art to study the function of the NtPPO6 gene.
[0008] The fourth objective of this invention is to provide the application of the tobacco polyphenol oxidase encoding gene NtPPO6 or a gene editing vector in tobacco variety breeding, in order to solve the problem that the existing technology does not have sufficient in-depth research on the tobacco polyphenol oxidase gene family, resulting in the unknown function of polyphenol oxidase genes in tobacco.
[0009] The fifth objective of this invention is to provide the application of the tobacco polyphenol oxidase encoding gene NtPPO6 or gene editing 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.
[0010] To achieve the above objectives, the technical solution adopted by the tobacco polyphenol oxidase encoding gene NtPPO6 in this invention is as follows:
[0011] The nucleotide sequence of the tobacco polyphenol oxidase encoding gene NtPPO6 is as follows:
[0012] (1) The nucleotide sequence shown in SEQ ID NO.1;
[0013] 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.
[0014] 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 NtPPO6. Through cloning the NtPPO6 gene, analyzing the amino acid sequence of tobacco polyphenol oxidase NtPPO6, analyzing the expression pattern of the NtPPO6 gene, and constructing NtPPO6 gene-edited tobacco plants, this invention found that knocking out the NtPPO6 gene in tobacco plants significantly reduces the activity of polyphenol oxidase in leaves, indicating that it is indeed a member of the polyphenol oxidase gene family. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO6 in this invention enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidase in tobacco.
[0015] To achieve the above objectives, the technical solution adopted by the tobacco polyphenol oxidase NtPPO6 of the present invention is as follows:
[0016] Tobacco polyphenol oxidase NtPPO6 has the following amino acid sequence:
[0017] (1) The amino acid sequence shown in SEQ ID NO.2;
[0018] 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.
[0019] The beneficial effects of the above technical solution are as follows: By analyzing the amino acid structure of tobacco polyphenol oxidase NtPPO6, this invention predicts that it is a member of the tobacco polyphenol oxidase family. By constructing tobacco plants with NtPPO6 gene editing, and by detecting the activity of polyphenol oxidase in their leaves, it was found that after knocking out the NtPPO6 gene, the content of polyphenol oxidase in tobacco leaves decreased significantly, proving that tobacco polyphenol oxidase NtPPO6 does indeed participate in regulating the activity of polyphenol oxidase in tobacco.
[0020] To achieve the above objectives, the technical solution adopted by the gene editing vector of this invention is as follows:
[0021] A gene editing vector containing a target site knockout sequence designed based on the NtPPO6 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0022] The beneficial effects of the above technical solution are as follows: by using gene editing technology, a knockout sequence was designed based on the NtPPO6 gene, a gene editing vector was constructed, and after transformation into tobacco, the NtPPO6 gene was successfully knocked out. This indicates that the gene editing vector constructed in this invention can effectively knock out the NtPPO6 gene, providing a good tool for subsequent research on the function of this gene.
[0023] As a further improvement, the knockout primer sequence designed based on the target site knockout sequence is as follows:
[0024] NtPPO6-CF: 5'-GATTGTCCCCTACTCTTACACAATG-3';
[0025] NtPPO6-CR: 5'-AAACATTGTGTAAGAGTAGGGGAC-3'.
[0026] To achieve the above objectives, the technical solution adopted in this invention for the application of the tobacco polyphenol oxidase encoding gene NtPPO6 or the gene editing vector in tobacco variety breeding is as follows:
[0027] Application of the tobacco polyphenol oxidase encoding gene NtPPO6 or gene editing vector in tobacco variety breeding.
[0028] The beneficial effects of the above technical solution are as follows: This invention constructs a gene-editing vector using gene-editing technology, which is then transferred into tobacco to successfully knock out the NtPPO6 gene, resulting in NtPPO6 gene-knockout tobacco plants. Subsequent testing revealed that the chlorogenic acid content in the leaves of the NtPPO6 gene-knockout tobacco plants was significantly increased, laying the foundation for obtaining tobacco varieties with increased chlorogenic acid content.
[0029] As a further improvement, it is applied to tobacco varieties that have increased chlorogenic acid content.
[0030] To achieve the above objectives, the technical solution adopted in the application of the tobacco polyphenol oxidase encoding gene NtPPO6 or the gene editing vector in tobacco plant phenotyping is as follows:
[0031] Application of the tobacco polyphenol oxidase encoding gene NtPPO6 or gene editing vector in tobacco plant phenotypic optimization.
[0032] The beneficial effects of the above technical solution are as follows: This invention constructs tobacco plants with the NtPPO6 gene knocked out. Observations show that after NtPPO6 gene knockout, the tobacco plant height increases, the average number of leaves decreases, and the width of the largest leaf in the middle, the width of the top leaf, and the length of the top leaf increase. However, the larger tobacco leaves and taller plant height result in poor light transmission to the lower leaves, and the larger leaf openings also affect ventilation in the tobacco field. Currently, research on tobacco plant type, especially internode traits, is relatively limited. This invention demonstrates that the NtPPO6 gene can regulate the plant height and number of leaves in tobacco plants. This provides an important research direction for improving the light transmission and ventilation of tobacco, ultimately increasing the photosynthetic utilization rate and total yield. It also provides genetic materials and theoretical basis for improving tobacco leaf quality and the genetic improvement of tobacco varieties. The tobacco polyphenol oxidase encoding gene NtPPO6 of this invention has broad application prospects in the field of plant type breeding, with significant economic potential.
[0033] As a further improvement, suppressing the expression of the NtPPO6 gene increased the height of tobacco plants, reduced the average number of leaves, and increased the width of the largest leaf in the middle, the width of the top leaf, and the length of the top leaf. Attached Figure Description
[0034] Figure 1 This is a gel electrophoresis image of the NtPPO6 gene clone in Experiment Example 1 of the present invention (where M is Marker 2000; I is the NtPPO6 gene amplification product);
[0035] Figure 2 This is a comparison diagram of amino acid sequence analysis in Experimental Example 2 of the present invention;
[0036] Figure 3 This is a diagram showing the expression characteristics of the NtPPO6 gene in different tissues in Experimental Example 3 of this invention.
[0037] Figure 4 This is a schematic diagram of the target site for NtPPO6 gene knockout in Experiment Example 4 of the present invention (the PAM region is after the 20bp target site, + indicates the positive strand, and asterisks indicate the relative position of SgRNA).
[0038] Figure 5 This is the sequencing result of the knockout target site in the T0 generation NtPPO6 gene knockout tobacco plant in Experiment Example 5 of this invention.
[0039] Figure 6 Phenotypic diagram of the T2 generation NtPPO6 gene knockout tobacco plant in Experiment Example 6 of this invention;
[0040] Figure 7This is a diagram showing the activity analysis of polyphenol oxidase in the leaves of T2 generation NtPPO6 gene knockout tobacco plants in Experiment Example 6 of the present invention (where Con represents the normal control tobacco plant, and the rest are different individual plants with NtPPO6 gene knockout).
[0041] Figure 8 This is a graph showing the chlorogenic acid content in the middle leaves of the T2 generation NtPPO6 gene knockout tobacco plants at the maturity stage in Experiment Example 7 of this invention (where Con represents the normal control tobacco plants, and the rest are different individual plants with NtPPO6 gene knockout). Detailed Implementation
[0042] 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 of tobacco polyphenol oxidase, named NtPPO6. Analysis of the cloning and expression patterns of the NtPPO6 gene revealed that inhibiting its expression in tobacco plants significantly increased chlorogenic acid activity in leaves. The discovery of the tobacco polyphenol oxidase encoding gene NtPPO6 enriches the tobacco polyphenol oxidase gene family and lays the foundation for studying the function of polyphenol oxidases in tobacco.
[0043] 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.
[0044] 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.
[0045] Tobacco: Tobacco K326, a common tobacco material.
[0046] Vector: CRISPER / Cas9 plasmid, a common and frequently used gene editing plasmid vector in existing molecular biology research experiments, which can be obtained from public channels. The plasmid (pORE-Cas9 / gRNA) used in the examples was kindly provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University.
[0047] 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.
[0048] Primer sequence synthesis and gene sequencing were completed by Beijing Liuhe Huada Biotechnology Co., Ltd.
[0049] Experimental reagents:
[0050] DNA / RNA extraction kits were purchased from Gene Answer, and gel extraction / reverse transcription kits were purchased from Takara Bio Engineering (Dalian) Co., Ltd.
[0051] Experimental equipment:
[0052] 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.
[0053] Example 1 of the tobacco polyphenol oxidase encoding gene NtPPO6
[0054] The nucleotide sequence of the tobacco polyphenol oxidase encoding gene NtPPO6 in this embodiment is as shown in SEQ ID NO.1.
[0055] Example 1 of tobacco polyphenol oxidase NtPPO6
[0056] The amino acid sequence of tobacco polyphenol oxidase NtPPO6 in this embodiment is as shown in SEQ ID NO.2.
[0057] Example 1 of gene editing vector
[0058] The gene editing vector of this embodiment contains a target site knockout sequence designed based on the NtPPO6 gene, the nucleotide sequence of which is shown in SEQ ID NO.1. The knockout primer sequences designed based on the target site knockout sequence are shown below:
[0059] NtPPO6-CF: 5'-GATTGTCCCCTACTCTTACACAATG-3';
[0060] NtPPO6-CR: 5'-AAACATTGTGTAAGAGTAGGGGAC-3'.
[0061] Example 1: Application of the tobacco polyphenol oxidase encoding gene NtPPO6 or gene editing vector in tobacco variety breeding
[0062] In this embodiment, tobacco plants were transformed with a gene editing vector containing a target site knockout sequence designed based on the NtPPO6 gene to construct NtPPO6 gene knockout tobacco plants, and the chlorogenic acid content in the leaves of this line was significantly increased.
[0063] Example 1: Application of the tobacco polyphenol oxidase encoding gene NtPPO6 or gene editing vector in tobacco plant phenotypic optimization
[0064] In this embodiment, the gene encoding tobacco polyphenol oxidase NtPPO6 was knocked out in tobacco through genetic engineering. This resulted in an increase in tobacco plant height, a decrease in the average number of leaves, and an increase in the width of the largest leaf in the middle, the width of the top leaf, and the length of the top leaf.
[0065] Example 1: Cloning of the NtPPO6 gene encoding tobacco polyphenol oxidase
[0066] 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 NtPPO6, was cloned using PCR technology. The specific implementation steps for cloning the tobacco NtPPO6 gene are as follows:
[0067] 1. Primer design
[0068] The specific primer sequences for PCR amplification are designed as follows:
[0069] NtPPO6-F: 5'-ATGGCTTCTTCATTTGTTC-3' (shown in SEQ ID NO.3),
[0070] NtPPO6-R: 5'-TTAACAAGGGACCAACTG-3' (shown in SEQ ID NO.4).
[0071] 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).
[0072] 2. PCR amplification
[0073] 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℃.
[0074] 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 PCR electrophoresis results are shown below. Figure 1 As shown. The target DNA fragment was recovered using a DNA gel extraction kit (Takara), and after concentration determination, it was stored at -20℃ for later use or directly used for subsequent experimental procedures.
[0075] 3. Sequencing and analyzing the NtPPO6 gene
[0076] 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 NtPPO6 gene.
[0077] Sequencing results showed that the tobacco NtPPO6 gene contains 1737 bases, and the specific base sequence is shown in SEQ ID NO.1.
[0078] Analysis of the base sequence showed that the polyphenol oxidase encoded by the tobacco NtPPO6 gene consists of 578 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.2.
[0079] Experimental Example 2: Amino acid sequence analysis of tobacco polyphenol oxidase NtPPO6
[0080] The amino acid sequences of tobacco polyphenol oxidase NtPPO6 were compared with those of other species, including Nicotiana flavescens, yellow lantern pepper, wolfberry, tomato, and Nicotiana benthamiana. The results showed that NtPPO6 had a high degree of similarity to the amino acid sequences of PPO from other species, with sequence similarities of 93.77% with NtomPPO from Nicotiana flavescens, 57.41% with CcPPO from yellow lantern pepper, 56.51% with LfPPO from wolfberry, 58.02% with SlPPO from tomato, and 56.68% with NbPPO from Nicotiana benthamiana.
[0081] Furthermore, Pfam analysis revealed that NtPPO6 possesses typical domains of PPO oxidase, such as... Figure 2 As shown, it has a Tyrosinase domain (PF00264) in the amino acid sequence from position 160 to 365, a PPO1_DWL domain (PF12142) in the amino acid sequence from position 372 to 423, and a PPO1_KFDV domain (PF12143) between the amino acid sequences from position 445 to 575.
[0082] Example 3: Analysis of the expression pattern of the tobacco polyphenol oxidase gene NtPPO6 in tobacco.
[0083] Using real-time quantitative PCR (BIO-RAD, USA) technology, this experiment conducted a preliminary analysis of the expression pattern of the tobacco NtPPO6 gene in tobacco plants. The specific procedures are as follows:
[0084] 1. Primer design and sample preparation for real-time PCR
[0085] For real-time quantitative PCR analysis, L25 was used as an internal reference gene, and the specific primer sequences were designed as follows:
[0086] NtPPO6-QF: 5'-TTCAAGCCACAACCAAGA-3' (shown in SEQ ID NO.5);
[0087] NtPPO6-QR: 5'-TCACATCCAATTCCACATTC-3' (shown in SEQ ID NO.6);
[0088] L25-F: 5'-CCCCTCACCACAGAGTCTGC-3' (shown in SEQ ID NO.7);
[0089] L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3' (shown in SEQ ID NO.8);
[0090] 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 K326 during its full bloom period and reverse transcribed into cDNA as template samples for later use.
[0091] 2. Quantitative Real-Time PCR Detection
[0092] For quantitative real-time PCR, the instrument used was a Bio-Rad CFX96 from Bio-Rad Laboratories, USA. The reaction system consisted of 20 μL of the following components: 10 μL of LSYBR Premix Ex Taq™, 1 μL of forward primer, 1 μL of reverse primer, 2 μL of cDNA, and 6 μL of ddH2O.
[0093] The amplification program was as follows: 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 2... -△△CTThe 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.
[0094] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the NtPPO6 gene is most highly expressed in the axillary buds of common tobacco K326 during its full bloom stage, and its expression level is also relatively high in flower buds. Its expression level is low in other tissues, and it is not expressed in the veins of old leaves, stems, and ovaries.
[0095] Example 4: Construction of gene editing vector
[0096] Based on the genomic and coding region sequences of the NtPPO6 gene obtained from sequencing in Example 1, and referring to the principles of gene editing target site design, a target site of approximately 20 bp was designed on the first exon of the NtPPO6 gene, such as... Figure 4 As shown, the knockout primer sequences are designed as follows:
[0097] NtPPO6-CF: 5'-GATTGTCCCCTACTCTTACACAATG-3' (shown in SEQ ID NO. 9);
[0098] NtPPO6-CR: 5'-AAACATTGTGTAAGAGTAGGGGAC-3' (shown in SEQ ID NO. 10).
[0099] First, PCR amplification was performed to obtain the double-stranded DNA at the target site; the 20 μL reaction system was prepared as follows: 4 μL each of upstream and downstream primers (50 μmol / L), 4 μL of Annealing Buffer for DNA Oligos (5×), and ddH2O was added to bring the total to 20 μL.
[0100] Reaction procedure: 95℃ for 5 min; decrease the temperature by 0.1℃ every 8 s until it reaches 25℃; store the reaction product at 4℃ for later use.
[0101] Second, the annealing product obtained from the above PCR reaction was ligated with the plasmid pORE-Cas9 / gRNA (pre-digested with BsaI). The 10 μL ligation system consisted of 3 μL of digested vector, 2 μL of annealing product, and 5 μL of Solution I. The reaction conditions were 16℃ for 30 min.
[0102] Third, the above-mentioned ligation product was transformed into DH5α competent cells and subjected to resistance screening culture (cultured at 37℃ for about 12 hours).
[0103] Finally, after selecting positive clones for colony PCR identification, the plasmids that were correctly identified were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing identification to ensure that the plasmid recombination was correct.
[0104] It should be noted that, in colony PCR identification, the primer pair nucleotide sequence used for identification of the NtPPO6 gene editing vector is as follows:
[0105] U26-jiance-F: 5'-TTAGGTTTACCGCCAATA-3' (shown in SEQ ID NO.11),
[0106] NtPPO6-CR: 5'-AAACATTGTGTAAGAGTAGGGGAC-3' (shown in SEQ ID NO. 10).
[0107] Experiment 5: Construction of tobacco plants transformed with Agrobacterium and edited with the NtPPO6 gene
[0108] The single colony successfully sequenced in Experiment 4 was expanded and cultured, plasmids were extracted, and the gene-editing vector was transferred into Agrobacterium LBA4404 using electroporation. This vector was then used to infect tobacco plants to obtain gene-edited tobacco plants with the NtPPO6 gene knocked out. The specific implementation method is as follows:
[0109] 1. Preparation of Agrobacterium competent cells:
[0110] (1) Select a single colony of Agrobacterium LBA4404 and incubate it overnight at 28°C in 2 mL LB (containing 20 mg / mL Rif);
[0111] (2) Take 2 mL of well-grown bacterial culture (containing 25 mg / L Rif) and inoculate it into 50 mL of LB liquid medium. Incubate at 28°C with shaking until OD600 = 0.5.
[0112] (3) Transfer the bacterial culture to a 50mL centrifuge tube, place it on ice for 30 minutes, and then centrifuge at 4℃ and 5000rpm for 5 minutes to collect the bacterial cells;
[0113] (4) After lightly suspending the bacterial cells in 10 mL of 0.15 M pre-cooled sodium chloride solution, collect the bacterial cells by centrifugation at 5000 rpm for 5 min at 4 °C;
[0114] (5) Add 20 mL of pre-cooled 20 mM calcium chloride solution to suspend the bacterial cells and complete the preparation of competent cells. After aliquoting the prepared competent cells into 100 μL / tubes, store at -80℃ for later use.
[0115] 2. Plasmid transformation of Agrobacterium:
[0116] (1) Take 1 μL of gene editing vector, add it to a centrifuge tube containing 100 μL of Agrobacterium competent cells, place it on ice for 30 minutes, then transfer it to liquid nitrogen for 1 minute, and then incubate it at 37°C for 5 minutes.
[0117] (2) Add 1 mL of LB liquid medium to the competent cells in step (1) and culture at 28°C with shaking for 3 hours; after the culture is completed, centrifuge at 5000 rpm for 1 minute, discard the supernatant (medium), add 200 μL of LB liquid medium, and resuspend the precipitate.
[0118] (3) Spread 200 μL of the resuspended bacterial solution evenly onto an LB agar plate containing 20 mg / L Rif and 50 mg / L kanamycin (Kan). Incubate at 28°C for 2-3 days. Select positive plasmids for amplification and perform colony PCR identification to ensure correct plasmid transformation. Store the correctly transformed recombinant strains for later use. Further amplify the correctly transformed strains by shaking, collect the bacterial cells by centrifugation, and then resuspend them in MSO to prepare the transfection solution for later use.
[0119] 3. Tobacco Conversion:
[0120] (1) Take vigorous K326 tobacco leaves, disinfect and sterilize them, and cut them into 1cm pieces. 2 After pre-culturing the small pieces in the culture medium for 2 days, place them in the infection solution prepared in step (2) and fully infect them for about 10-15 minutes.
[0121] (2) After the infected leaf pieces were placed in the culture medium and cultured in the dark for 4 days, they were transferred to the culture medium to induce differentiation in order to induce the formation of clustered adventitious buds (during this period, the culture medium was changed every 10 days as needed, and antibiotics were added to the culture medium for resistance screening).
[0122] (3) When the adventitious buds grow to 1-2cm, the clustered adventitious buds are divided into individual buds and further transferred into the culture medium to induce rooting;
[0123] (4) After the root system has developed and grown, the tissue culture seedlings are taken out and transferred to flower pots filled with loose, sterile soil for routine management and cultivation.
[0124] 4. PCR detection of whether the Cas9 gene fragment is inserted into the tobacco plant:
[0125] For the transgenic regenerated seedlings, a suitable amount of leaves from the regenerated tobacco plants were collected as samples, and their genomic DNA was extracted. The PCR method was used to detect and confirm whether the exogenous DNA fragment was inserted into the plant genome.
[0126] For PCR amplification verification, the primers were designed as follows:
[0127] Cas9-F:5'-GGGACCCTAAGAAGTACGGC-3' (shown in SEQ ID NO.12);
[0128] Cas9-R:5'-TATTCTCGGCCTGCTCTCTG-3' (shown in SEQ ID NO.13).
[0129] The 25 μL reaction system was designed as follows: 1 μL each of upstream and downstream gene primers (10 μmol / L), 1 μL DNA template (100 ng / μL), and 10×Taq Buffer (Mg). 2+ Add 2.5 μL of dNTPs (2.5 mmol / L), 0.25 μL of Taq DNA polymerase, and ddH2O to a final volume of 25 μL.
[0130] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 20 s, 30 cycles; 72℃ extension for 10 min; store at 4℃.
[0131] After PCR, the PCR amplification products were detected by 1% agarose gel electrophoresis to determine whether the exogenous DNA fragment (Cas9 protein gene) was inserted into the plant genome.
[0132] 5. Further validation of NtPPO6 gene knockout tobacco plants
[0133] For the NtPPO6 gene knockout positive plants identified in the preliminary PCR screening in step 4, further testing and identification were conducted to further determine whether the target gene was successfully edited and mutated, and to specifically confirm the mutation type. The details are briefly described below.
[0134] (1) Based on the NtPPO6 gene sequence and target site location, the following primer pairs were designed for detection:
[0135] NtPPO6-BJ-F:5'-ggagtgagtacggtgtgcTTCCAAGTATCATGCAACCA-3' (shown in SEQ ID NO. 14),
[0136] NtPPO6-BJ-R:5'-gagttggatgctggatggGGATAGGAGGACAACAAGTG-3' (shown in SEQ ID NO. 15).
[0137] (2) Using the DNA from the above-mentioned Cas9-positive transgenic plants as a template, PCR amplification was performed:
[0138] The 50 μL amplification system was designed as follows: 2 μL each of upstream and downstream primers (10 μmol / L), 2 μL of DNA template (100 ng / μL), and 10×Taq Buffer (Mg). 2+ Add 5 μL of dNTPs (2.5 mmol / L), 0.5 μL of Taq DNA polymerase, and ddH2O to a final volume of 50 μL.
[0139] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, 35 cycles; 72℃ extension for 10 min, store at 4℃.
[0140] After electrophoresis detection of the PCR amplification products, the PCR products were recovered and purified, and sent to Xi'an Qingxue Biotechnology Co., Ltd. for Hi-TOM sequencing. The mutation rate of the genes was analyzed by comparison based on the sequencing results.
[0141] Partial sequencing results as follows Figure 5 As shown in the figure. Analysis results indicate that in the 20 T0 generation tobacco plants with NtPPO6 gene knockout, two types of gene mutations were observed: a one-base insertion and a one-base insertion followed by a one-base deletion. These base mutations all occurred at the knockout target site, while no mutations were detected in the NtPPO6 gene of the wild-type plants. This demonstrates that the T0 generation plants have successfully mutated the NtPPO6 gene through either base deletion or insertion.
[0142] To verify whether the relevant phenotype could be stably inherited to the next generation, seeds of T0 generation positive plants were collected, and T2 generation plants were obtained after further planting and analysis. The results showed that T0 generation plants could be stably inherited to the T2 generation.
[0143] Experimental Example 6: Phenotypic Analysis and Polyphenol Oxidase Activity Analysis of NtPPO6 Gene Knockout Tobacco Plants
[0144] 1. Phenotypic observation:
[0145] The results of the observation of the mid-leaf maturity phenotype of NtPPO6 gene knockout T2 generation tobacco plants are as follows: Figure 6 As shown, compared with the control K326, the NtPPO6 gene knockout tobacco plants had an average increase of 7 cm in plant height, an average decrease of 3 leaves, an increase of 4.7 cm in the width of the largest leaf in the middle, an increase of 5.7 cm in the width of the top leaf, and an increase of 6.1 cm in the length of the top leaf. Other parameters, such as stem circumference and the length of the largest leaf in the middle, did not change significantly.
[0146] 2. Polyphenol oxidase activity detection:
[0147] The activity of polyphenol oxidase 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 mature leaves of the middle leaves of NtPPO6 gene knockout T2 generation tobacco plants was significantly reduced compared with the control (K326). Figure 7 The levels were reduced by 39.55%, 24.50%, 36.38%, 30.31%, and 26.38%, respectively. This study preliminarily reveals the function of the NtPPO6 gene, demonstrating that inhibiting NtPPO6 gene expression through gene editing can downregulate polyphenol oxidase activity.
[0148] Experimental Example 7: Detection of chlorogenic acid content in NtPPO6 gene knockout tobacco plants
[0149] Using GC-MS analysis, this invention detected the chlorogenic acid content in NtPPO6 gene knockout tobacco plants. The HPLC-UV absolute quantitative analysis conditions were as follows: Symmetry C18 column (4.6×250mm, 5μm), detection wavelength 340nm, injection volume 5μL, and flow rate 1.0mL / min.
[0150] 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).
[0151] Gradient elution:
[0152] 0~15.0min, 10%B-30%B;
[0153] 15.0~26.0min, 30%B-90%B;
[0154] 26.0–28.0 min, 90% B;
[0155] 28.1-35.0 min, 10% B.
[0156] Test results as follows Figure 8 As shown in the figure, compared with the control (K326), the chlorogenic acid content in the middle leaves of the T2 generation NtPPO6 gene knockout tobacco plants (7-6-9, 7-6-10, 7-6-12, 7-6-15, 7-6-18) at the leaf maturity stage increased by 27.64%, 41.11%, 32.35%, 42.66%, and 44.95%, respectively, and all differences compared with the control were statistically significant (p<0.01). This result indicates that the NtPPO6 gene plays an important role in the regulation of chlorogenic acid during the leaf maturity stage of tobacco.
[0157] 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 NtPPO6 Its application in tobacco plant phenotypic optimization is characterized by: inhibition NtPPO6 Gene expression leads to increased plant height, decreased average leaf size, and increased maximum leaf width in the middle section, width of the top leaf, and length of the top leaf; NtPPO6 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The tobacco polyphenol oxidase encoding gene according to claim 1 NtPPO6 Its application in tobacco plant phenotypic optimization is characterized by: The tobacco variety is K326.
3. The application of a gene-editing vector in tobacco plant phenotypic optimization, characterized in that: inhibition NtPPO6 Gene expression leads to increased plant height, decreased average leaf size, and increased maximum leaf width in the middle section, width of the top leaf, and length of the top leaf; NtPPO6 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The gene-editing vector contains according to NtPPO6 Knockout sequences designed to target gene sites.
4. The application of the gene editing vector according to claim 3 in tobacco plant phenotypic optimization, characterized in that: The knockout primer sequences designed based on the target site knockout sequence are shown below: NtPPO6-CF: 5'-GATTGTCCCCTACTCTTACACAATG-3'; NtPPO6-CR: 5'-AAACATTGTGTAAGAGTAGGGGAC-3'.
5. The application of the gene editing vector according to claim 3 in tobacco plant phenotypic optimization, characterized in that: The tobacco variety is K326.