Application of NtBZR6 gene in tobacco chlorogenic acid regulation and germplasm resource improvement

Abstract

The invention relates to the technical field of biology, in particular to application of an NtBZR6 gene in tobacco chlorogenic acid regulation and germplasm resource improvement. Through cloning of the NtBZR6 gene and construction and analysis of an expression vector, it is found that the expression quantity of the NtBZR6 gene in leaves can be remarkably increased through overexpression of the gene in tobacco plants. Furthermore, through detection, compared with a normal control group, the content of chlorogenic acid in a transgenic line is remarkably increased, the height of a tobacco plant is increased, the internode spacing is increased, and a new gene resource is provided for tobacco breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the NtBZR6 gene in the regulation of chlorogenic acid in tobacco and the improvement of germplasm resources. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an annual or short-lived perennial herbaceous plant belonging to the Solanaceae family. Polyphenols are widely present in tobacco, and their biosynthesis is mainly completed through the phenylpropanoid metabolic pathway, finely regulated by various transcription factors. The main polyphenolic components in tobacco include chlorogenic acid, rutin, hyoscyamine, as well as neochlorogenic acid, 4-O-caffeoquinic acid, and kaempferol glucoside. Among these, chlorogenic acid is the most abundant polyphenolic compound, typically accounting for over 46% of the total polyphenols. Studies have also found that tobacco is one of the plant resources with a high content of chlorogenic acid. Chlorogenic acid, also known as caffeotannin, has significant medicinal value. In the 175 kinds of traditional Chinese medicine preparations for clearing heat and detoxifying, and for antibacterial and anti-inflammatory purposes listed in the Ministry of Health's "Drug Standards," chlorogenic acid is one of the main active ingredients. Furthermore, as a novel natural antioxidant, chlorogenic acid also shows broad application potential in food preservation, fruit storage, and sun protection and skin care. In the tobacco system, chlorogenic acid not only has a fresh aroma, but it can also be converted into aroma components such as pyrazine, pyridine and pyrrole in enzymatic reactions, giving tobacco products a unique nutty aroma and effectively improving their overall aroma quality.

[0003] Currently, besides its primary use in flue-cured tobacco production, tobacco's potential in edible and medicinal applications is gradually gaining attention. During tobacco harvesting and processing, approximately 25% of waste tobacco dust and other byproducts are generated annually but remain unutilized. Efficiently extracting chlorogenic acid from these byproducts could not only achieve resource recovery from waste but also generate significant economic benefits. With the increasing maturity of tobacco tissue culture technology and its ease of genetic transformation, it has become possible to cultivate tobacco varieties with high chlorogenic acid content through transgenic or other breeding methods. This lays the technological foundation for the large-scale extraction of these two active ingredients using tobacco as a bioreactor. Therefore, in-depth analysis of the synthesis and regulatory mechanisms of chlorogenic acid from a genetic engineering perspective, and the identification of its key regulatory genes, are of great significance for the improvement of tobacco germplasm resources and the further development and utilization of chlorogenic acid. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the application of the NtBZR6 gene in the regulation of chlorogenic acid in tobacco and the improvement of germplasm resources.

[0005] This invention provides the application of NtBZR6 protein in regulating plant chlorogenic acid content and / or improving plant germplasm resources;

[0006] The amino acid sequence of the NtBZR6 protein is shown in SEQ ID NO:2.

[0007] The plants described in this invention include plants from the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, and / or Solanaceae families; further, the Solanaceae plants include tobacco; and even further, the tobacco is K326.

[0008] The regulation includes raising and / or lowering.

[0009] The germplasm resource improvement includes: increasing plant height and / or increasing internode spacing.

[0010] This invention provides plant breeding or assisted breeding products, comprising at least one of the following: A) to F):

[0011] A) Amplification primers, detection primers, and / or targeting primers using nucleic acids encoding the NtBZR6 protein as templates;

[0012] B) gRNA targeting NtBZR6;

[0013] C) Interference fragments targeting the NtBZR6;

[0014] D) A recombinant vector containing nucleic acid encoding the NtBZR6, or gRNA as described in B), or interfering fragment as described in C;

[0015] E) Transformation or transfection of host cells with the recombinant vector described in D);

[0016] F) A mixture obtained by culturing host cells as described in E).

[0017] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0018] In a specific embodiment of the present invention, the nucleic acid is DNA, and its nucleotide sequence is shown in SEQ ID NO:1.

[0019] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism or for performing a desired operation. In this invention, the backbone of the recombinant vector is derived from bacteria, fungi, yeast, plants, and / or viruses; this invention is not limited to these sources. In some embodiments of this invention, the backbone of the recombinant vector used to obtain the gene sequence is derived from a bacterial cloning vector, specifically pMD19-T. In other embodiments of this invention, the vector backbone of the recombinant vector is derived from a plant, specifically pCAMBIA1300, used for overexpressing the nucleic acid in plants.

[0020] In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0021] The host cell described in this invention includes plant cells, and the plant includes plants of the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, or Solanaceae families. Further, in a specific embodiment of this invention, the plant is a Solanaceae plant, and further, the Solanaceae plant is tobacco.

[0022] This invention provides a kit comprising the product and excipients described herein.

[0023] Furthermore, the excipients include at least one of the following: DNA extraction reagent, dNTP, DNA reverse transcription reagent, culture medium, antibiotic and / or buffer.

[0024] This invention provides the application of the product or kit described herein in increasing the chlorogenic acid content of plants and / or in plant breeding or assisted plant breeding.

[0025] This invention provides a method for increasing the chlorogenic acid content of plants and / or for plant breeding or assisted plant breeding, comprising using the products or kits described herein.

[0026] Tobacco is an important model organism in biological research, and a large number of plant molecular biology studies and genetic engineering experiments use tobacco as the specific subject. Because of this, researchers have neglected the discovery, identification, and utilization of tobacco's own functional genes. Based on previous research, this invention uses cDNA from leaves of common tobacco at different growth stages as a template and employs PCR technology to clone the NtBES1 gene, named NtBZR6. Through cloning and constructing and analyzing the expression vector of the NtBZR6 gene, this invention found that overexpression of this gene in tobacco plants significantly increases the expression level of the NtBZR6 gene in leaves. Further detection revealed that, compared with the normal control group, the chlorogenic acid content in the transgenic lines was significantly increased. This invention lays the foundation for elucidating the molecular regulatory mechanism of chlorogenic acid biosynthesis and provides a new, precise regulatory gene for chlorogenic acid synthesis in tobacco.

[0027] Chlorogenic acid can form aroma compounds closely related to human senses during processing, and these aroma compounds are an important source of tobacco aroma. Therefore, 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 usable genetic resources for the improvement of tobacco germplasm resources.

[0028] This invention, through the cloning and construction and analysis of the NtBZR6 gene and its expression vector, revealed that overexpression of this gene in tobacco plants significantly increases the expression level of the NtBZR6 gene in leaves. Further analysis showed that, compared with the normal control group, the transgenic lines exhibited significantly higher chlorogenic acid content, increased plant height, and increased internode spacing, providing new genetic resources for tobacco breeding. Attached Figure Description

[0029] Figure 1 Gel electrophoresis image of the NtBZR6 gene clone;

[0030] Figure 2 The amino acid sequence analysis comparison diagram of NtBZR6 and other BZRs is shown.

[0031] Figure 3 A diagram showing the expression characteristics of the NtBZR6 gene in different tissues;

[0032] Figure 4 Subcellular localization map of NtBZR6 gene in tobacco epidermal cells (scale bar, 50 μm);

[0033] Figure 5 Gel electrophoresis image of PCR products used to verify the DNA level of the NtBZR6 gene;

[0034] Figure 6The NtBZR6 gene was detected by qRT-PCR in plants overexpressing the NtBZR6 gene (where Con represents the normal control tobacco plant, and the rest are different individual plants overexpressing the NtBZR6 gene).

[0035] Figure 7 Representative typology analysis of T1 plants overexpressing NtBZR6;

[0036] Figure 8 Analysis of chlorogenic acid content in leaves of T1 generation NtBZR6 gene overexpressing plants. Detailed Implementation

[0037] This invention provides the application of the NtBZR6 gene in the regulation of chlorogenic acid and the improvement of germplasm resources in tobacco. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] Chlorogenic acid has shown promising applications in existing technologies, but its sources are relatively limited. Tobacco, as an important model organism, has a high planting area and yield in my country, and its chlorogenic acid content is also high. Theoretically, it can serve as a plant source of chlorogenic acid. If tobacco varieties with high chlorogenic acid content can be cultivated through transgenic technology or other breeding techniques, a solid technical foundation can be laid for using tobacco as a bioreactor to extract chlorogenic acid. Therefore, identifying regulatory genes that can precisely control the chlorogenic acid content in tobacco is of great significance for increasing its concentration in tobacco.

[0039] Based on previous research, this invention uses cDNA from leaves of common tobacco at different growth stages as a template and employs PCR technology to clone a transcription factor gene, named NtBZR6. Through cloning the NtBZR6 gene and constructing and analyzing its expression vector, this invention reveals that overexpression of this gene in tobacco plants significantly increases the chlorogenic acid content in the leaves.

[0040] 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 usable genetic resources for the improvement of tobacco germplasm resources.

[0041] 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.

[0042] In the following examples, primer sequence synthesis and gene sequencing were performed by Beijing Liuhe Huada Biotechnology Co., Ltd.

[0043] Biomaterials:

[0044] Tobacco material: Cultivated tobacco (Nicotiana tabacum) variety K326, kindly provided by Guizhou Tobacco Science Research Institute.

[0045] Vector: pCAMBIA1300 is a commonly used plasmid vector in molecular biology and can be obtained from public sources.

[0046] 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.

[0047] Experimental reagents:

[0048] DNA / RNA extraction kits were purchased from Beijing Bio-Labs Technology Co., Ltd., gel extraction kits / reverse transcription kits were purchased from Shanghai Boson Biotechnology Co., Ltd., and homologous recombination kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[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] Data processing:

[0052] The significance of the data was analyzed using Duncan's test (P<0.05). Different letters represent significant differences in the data, with ND indicating that the data were not detected.

[0053] Specific examples of the application of the NtBZR6 gene in the regulation of chlorogenic acid content and germplasm improvement in tobacco:

[0054]

[0055] NtBZR6 Amino acid sequence: MMWEGGGSPATSSAGGGGAGGAMGSSSSSGRRKPSWRERENNRRRERRRRAIAAKIYAGLRAQGNYNLPKHCDNNEVLKALCAEAGWIVEPDGTTYRKGCSRPTPMEIGGTSANITPSSSRHPSPPSSYFASPIPSYQPSPTSSSFPSPSRGDANMSSHPFAF LHSSIPSSLPPLRISNSAPVTPPLSSPTRLPKQTFNLETLARESMSALNIPFFAASAPTSPTRGQRFTPATIPECDESDSSTIDSGQWMSFQKYATNGVPTSPTFNLIKPAAQRIPSNDMIIDKGKSVEFDFENVSVKAAWEGEKIHEVGLDDLELTLGSGSGRM (SEQ ID NO:2);

[0056] Chlorogenic acid, belonging to the caffeoylquinic acid family, specifically 3-caffeoylquinic acid, not only helps plants enhance their resistance to various biotic and abiotic stresses, but also plays an important role in human health, possessing antibacterial and anti-inflammatory properties, and preventing obesity and cardiovascular diseases.

[0057] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0058] Example 1: Cloning and Analysis of the NtBZR6 Gene

[0059] 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 NtBZR6 gene was cloned using PCR technology. The specific implementation process is as follows:

[0060] 1. Primer design

[0061] The specific PCR amplification primer sequences for non-coding region amplification are designed as follows:

[0062] NtBZR6-clone-F:5'-atgatgtgggaaggtggagg-3' (shown in SEQ ID NO:3);

[0063] NtBZR6-clone-R:5'-tccagatcatctaaaccaacct-3' (shown in SEQ ID NO:4).

[0064] RNA was extracted from roots of tobacco plants at different growth and development stages (according to the instructions of the Bio-Rad RNA Extraction Kit), and the extracted RNA was reverse transcribed into cDNA according to the instructions of the Takara Reverse Transcription Kit.

[0065] 2. PCR amplification

[0066] 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.

[0067] Table 1. PCR reaction system

[0068]

[0069] Table 2. PCR reaction procedure

[0070]

[0071] PCR amplification products were detected by 1.2% agarose gel electrophoresis. A DL2000 DNA marker was used, and the electrophoresis conditions were 120V / 20 min. The samples were then observed under a UV scanner (e.g., ...). Figure 1 As shown in the figure, the target DNA fragment was recovered using a DNA gel recovery kit (Boersen Biotechnology), and after the concentration was determined, it was stored at -20 ℃ for later use or directly used for subsequent experimental operations.

[0072] 3. Sequencing and analyzing the NtBZR6 gene

[0073] 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). A small amount of bacterial culture was used as a template for PCR verification to confirm whether it was a positive clone. The recombinant plasmid containing the target fragment was sequenced by Beijing Liuhe Huada Biotechnology Co., Ltd., obtaining the nucleotide sequence of the tobacco NtBZR6 gene.

[0074] Sequencing results showed that the tobacco NtBZR6 gene comprises 984 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 NtBZR6 gene consists of 328 amino acids, and its specific amino acid sequence is shown in SEQ ID NO:2.

[0075] 4. Amino acid sequence alignment of NtBZR6 with BZR from other species

[0076] This embodiment compares the amino acid sequences of the protein encoded by the tobacco NtBZR6 gene with those of BZR from other species. The specific implementation steps are as follows:

[0077] The proteins encoded by the tobacco NtBZR6 gene are highly conserved compared to the AtBES1 / BZR1 family, and each protein contains a characteristic BES1_N domain (Pfam: PF05687). Figure 2 ).

[0078] Example 2: Analysis of the expression pattern of the NtBZR6 gene in tobacco

[0079] Using real-time quantitative PCR (BIO-RAD, USA) technology, this example provides a preliminary analysis of the expression pattern of the tobacco NtBZR6 gene in tobacco plants. The specific implementation steps are as follows:

[0080] 1. Primer design 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] NtBZR6-QF: 5'-gaacctgatggcactactta-3' (shown in SEQ ID NO: 5);

[0083] NtBZR6-QR: 5'-ttggatgtcgtgaagaactt-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 the roots, stems, young leaves, mature leaves, senescent leaves, flower buds, and mature flowers of K326 during its full bloom period and reverse transcribed into cDNA as template samples for later use.

[0087] 2. Quantitative Real-Time PCR Detection

[0088] 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... -△△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.

[0089] Table 3. Reaction System

[0090]

[0091] Table 4. Amplification Procedure

[0092]

[0093] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the NtBZR6 gene is highly expressed in mature flowers, lower leaves, and middle leaves of common tobacco K326, followed by stems, upper leaves, and flower buds, and is least expressed in roots.

[0094] Example 3 Construction of NtBZR6 gene overexpression vector

[0095] An NtBZR6 overexpression vector was constructed. The cloning primers were used: upstream primer: 5'-aaattgactctagaaagcttatgatgtgggaaggtggagg-3' (SEQ ID NO. 9), and downstream primer: 5'-cccttgctcaccatggtacctccagatcatctaaaccaacct-3' (SEQ ID NO. 10). Homologous recombination adapter sequences were added to both primers. Amplification was performed using the NtBZR6-T plasmid (which was correctly sequenced in Example 1) as a template. 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 each positive clone was sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing; the sequencing results were correct.

[0096] Example 4: Localization of the NtBZR6 gene in tobacco epidermal cells

[0097] This embodiment selects the NtBZR6 overexpression vector from Example 3 as the research object to observe the localization of the NtBZR6 gene in tobacco epidermal cells. The specific implementation is as follows:

[0098] The pCAMBIA1300-NtBZR6 vector and the empty pCAMBIA1300 vector were transformed into Agrobacterium LBA4404, and then the Agrobacterium was injected into the leaves of Nicotiana benthamiana via infiltration. Through infection by Agrobacterium, 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).

[0099] Depend on Figure 4 It can be seen that the fluorescence signal of pCAMBIA1300-NtBZR6 overlaps with that of the nuclear localization gene Ghd7-RFP, indicating that NtBZR6 is located in the cell nucleus.

[0100] Example 5: Construction and phenotypic study of NtBZR6 gene overexpression plants.

[0101] The successfully sequenced single colonies from Example 3 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:

[0102] 1. Preparation of Agrobacterium competent cells

[0103] 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.

[0104] 2. Granule-transformed Agrobacterium

[0105] Take 1 μL of the NtBZR6 gene overexpression plasmid 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. Take 200 μL of the resuspended bacterial solution and spread it evenly on an LB agar plate containing 20 mg / L Rif and 50 mg / L kanamycin (Kan). Incubate at 28°C for 2 to 3 days. After confirming the colony PCR results, preserve the bacterial strain.

[0106] 3. Tobacco Conversion

[0107] 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℃, 2000 Lx light intensity, and 16 h / d light duration. The plants were then immersed in engineered bacterial solution for 10–5 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.

[0108] 4. Detection of positive super-genetically modified tobacco using PCR and qPCR methods

[0109] Design specific primer pairs for the expression vector, with the upstream primer specifically binding to the NtBZR6 gene and the downstream primer specifically binding to the GFP gene.

[0110] Upstream primer: 5'-ctgattcgtccactattgattc-3' (SEQ ID NO.11); Downstream primer: 5'-gtcgtccttgaagaagatgg-3' (SEQ ID NO.12). PCR amplification was performed using genomic DNA from controls and K326 and T0 generation seedlings as templates. Seedlings amplifying specific bands were considered T0 generation positive (e.g., Figure 5(As shown). To verify whether the phenotype could be stably inherited to the next generation, seeds of T0 generation positive plants were collected and planted to obtain T1 generation plants. Then, the expression level of the NtBZR6 gene in the selected positive seedlings was detected by qPCR. NtBZR6 gene-specific primers were used: NtBZR6-QF: 5'-gaacctgatggcactactta-3' (SEQ ID NO:5) and NtBZR6-QR: 5'-ttggatgtcgtgaagaactt-3' (SEQ ID NO:6). The tobacco L25 gene was used as an internal control for the reaction. The upstream primer was 5'-cccctcaccacagagtctgc-3' (SEQ ID NO:7) and the downstream primer was 5'-aagggtgttgttgtcctcaatctt-3' (SEQ ID NO:8). Amplification was performed using a LightCycler® 96 quantitative PCR instrument (reaction system and amplification program are shown in Tables 3 and 4). After the reaction is complete, based on the obtained CT value, use 2 -△△CT Methods for calculating the relative expression level of the NtBZR6 gene (e.g., Figure 6 (As shown). Analysis shows that T0 generation plants can stably inherit traits to T1 generation.

[0111] 5. Phenotypic analysis of T1 generation NtBZR6 transgenic tobacco

[0112] During the vigorous growth period (40-60 days after transplanting), a systematic phenotypic analysis was performed on the T1 generation NtBZR6 overexpressing transgenic lines and the wild-type control K326. The results showed that, compared with K326, the NtBZR6 overexpressing plants (BZR6-OE) exhibited significant morphological changes: they were taller and had greater internode distances, but fewer leaves. Notably, despite the reduced number of leaves, the length and width of individual leaves were significantly greater than the control. Figure 7 ).

[0113] Example 6: Detection of chlorogenic acid expression in NtBZR6 gene overexpressing plants

[0114] This embodiment selects the NtBZR6 overexpression line (T1 generation) from Example 5 as the research object, and detects the chlorogenic acid content in the leaves of transgenic tobacco plants at the middle leaf maturity stage. The specific implementation operation is as follows:

[0115] Take 50 mg of frozen tobacco leaf sample and grind it into powder in liquid nitrogen. Transfer it to 1.5 mL of pre-cooled methanol-water (4:1, V / V) extraction solution, sonicate at room temperature for 30 min, and let stand at 4℃ for 12 h; then centrifuge at 4℃ and 20000 r / min for 10 min, and collect the supernatant. The extracted supernatant was analyzed for chlorogenic acid content using an Agilent ultra-high performance liquid chromatography-triple quadrupole mass spectrometer. Absolute quantitative analysis conditions: column ACQUITY uplc HSS T3 (2.1×50 mm, 1.8 µm), injection volume 10 µL, flow rate 0.4 mL / min, mobile phase A: water (0.1% formic acid), mobile phase B: methanol (0.1% formic acid), gradient elution: 0 min, B%: 5; 1 min, B%: 60; 3 min, B%: 70; 3.01 min, B%: 95; 4 min, B%: 95; 4.01 min, B%: 5; 5 min, B%: 5.

[0116] The results showed that, targeting NtBZR6 overexpressing transgenic lines (such as...) Figure 8 As shown in the figure): Compared with the control (K326), the chlorogenic acid content in the middle leaves of the T1 generation transgenic plants (OE-2-1, OE-2-2, OE-3-1, OE-3-3, OE-5-1, OE-5-3) at the maturity stage increased by 21.05%, 52.63%, 28.94%, 23.68%, 36.84%, and 34.21%, respectively, and all differences from the control were statistically significant (P < 0.05). This result indicates that the NtBZR6 gene plays an important role in the regulation of chlorogenic acid during the maturity stage of tobacco leaves.

[0117] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of NtBZR6 protein in regulating chlorogenic acid content and / or improving plant germplasm resources; The amino acid sequence of the NtBZR6 protein is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The plant mentioned includes tobacco.

3. The application according to claim 2, characterized in that, The regulation includes raising and / or lowering.

4. The application according to claim 3, characterized in that, The germplasm resource improvement includes: increasing plant height and / or increasing internode spacing.

5. Products for plant breeding or assisted breeding, characterized in that, Includes at least one of the following: A) to F) A) Amplification primers, detection primers, and / or targeting primers using nucleic acids encoding the NtBZR6 protein as templates; B) gRNA targeting NtBZR6; C) Interference fragments targeting the NtBZR6; D) A recombinant vector containing nucleic acid encoding the NtBZR6, or gRNA as described in B), or interfering fragment as described in C; E) Transformation or transfection of host cells with the recombinant vector described in D); F) A mixture obtained by culturing host cells as described in E).

6. The product according to claim 5, characterized in that, The skeleton of the recombinant vector is derived from pCAMBIA1300.

7. A reagent kit, characterized in that, Includes the product and excipients as described in claim 5 or 6.

8. The reagent kit according to claim 7, characterized in that, The excipients include at least one of the following: DNA extraction reagent, dNTP, DNA reverse transcription reagent, culture medium, antibiotic and / or buffer.

9. The use of the product of claim 5 or 6 or the kit of claim 7 or 8 in plant breeding or assisted plant breeding.

10. A method for plant breeding or assisted plant breeding, characterized in that, This includes plant breeding using the product of claim 5 or 6 or the kit of claim 7 or 8.

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