Application of NtBZR7 gene in the regulation of chlorogenic acid in tobacco and improvement of tobacco germplasm

Abstract

The present application relates to the field of biotechnology, and more particularly to the application of NtBZR7 gene in the regulation of chlorogenic acid in tobacco and improvement of germplasm resources. The present application mines tobacco NtBZR7 gene, which can improve the content of chlorogenic acid in tobacco when overexpressed in tobacco, and can reduce the content of chlorogenic acid in tobacco when silenced, thereby providing a new available gene resource for the improvement of tobacco germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the NtBZR7 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 crop belonging to the Solanaceae family. It is an important economic crop in my country, occupying a significant position in the national economy. China's tobacco planting scale and total output rank among the world's top. Polyphenols are an important class of secondary metabolites in tobacco, mainly biosynthesized via the phenylpropanoid metabolic pathway. The entire metabolic process is precisely regulated by numerous transcription factors. The main polyphenolic components of tobacco include chlorogenic acid, rutin, and hyoscyamine, as well as derivatives such as neochlorogenic acid, 4-O-caffeoylquinic acid, and kaempferol glucoside. Chlorogenic acid is the most abundant polyphenol in tobacco, accounting for more than 50% of the total polyphenol content.

[0003] Chlorogenic acid, also known as caffeine tannin, possesses significant pharmacological activity. All 175 traditional Chinese medicines listed in the Ministry of Health's drug standards for clearing heat and detoxifying, and for antibacterial and anti-inflammatory purposes use chlorogenic acid as a core active ingredient. Furthermore, as a natural antioxidant, chlorogenic acid also shows broad application prospects in food preservation, fruit and vegetable storage, and skincare and sun protection. In tobacco, chlorogenic acid itself releases a refreshing aroma and can be enzymatically degraded into volatile substances such as pyrazine, pyridine, and pyrrole, imparting a unique nutty aroma to cigarettes and significantly improving the overall flavor and taste quality of tobacco leaves.

[0004] The tobacco industry currently focuses on flue-cured tobacco production, but its edible and medicinal value is also gradually gaining attention. The tobacco harvesting and processing process generates approximately 25% of byproducts, such as waste tobacco leaves, which remain largely untapped. Extracting chlorogenic acid from waste tobacco leaves can not only achieve resource recycling of processing waste but also generate considerable economic benefits. Tobacco tissue culture systems are mature, and genetic transformation is simple. Creating new tobacco materials with high chlorogenic acid content using gene editing and transgenic breeding technologies is practically feasible and provides technical support for developing tobacco into a bioreactor for large-scale chlorogenic acid production. In conclusion, elucidating the biosynthesis and regulatory molecular mechanisms of chlorogenic acid in tobacco leaves and identifying key regulatory genes has significant theoretical and practical value for improving tobacco germplasm quality and for the in-depth development and utilization of chlorogenic acid resources. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the application of the NtBZR7 gene in the regulation of chlorogenic acid in tobacco and the improvement of germplasm resources.

[0006] This invention provides the application of NtBZR7 in the improvement of plant germplasm resources, and the amino acid sequence of NtBZR7 is shown in SEQ ID NO.2.

[0007] Furthermore, the germplasm resource improvement includes regulating the content of chlorogenic acid.

[0008] The regulation of chlorogenic acid content includes: increasing the chlorogenic acid content and / or decreasing the chlorogenic acid content.

[0009] The increase in chlorogenic acid content is achieved by overexpressing NtBZR7 in plants; The overexpression of NtBZR7 in plants is achieved by transfecting the nucleic acid encoding NtBZR7 into the plant; the nucleotide sequence of the nucleic acid encoding NtBZR7 is shown in SEQ ID NO.1.

[0010] The reduction of chlorogenic acid content is achieved by using VIGS-mediated gene silencing to target the nucleic acid encoding NtBZR7.

[0011] The target fragment for VIGS-mediated gene silencing is the 134bp to 514bp region of the nucleic acid encoding NtBZR7 shown in SEQ ID NO.1.

[0012] This invention provides a method for regulating the chlorogenic acid content in plants, comprising using at least one of the following methods (A) to (G) to regulate the chlorogenic acid content in plants: A) Overexpression of NtBZR7; B) VIGS target fragments targeting the nucleic acid encoding the NtBZR7; C) gRNA targeting NtBZR7; D) Expression cassettes containing the VIGS target fragment as described in B), or the gRNA as described in C); E) A recombinant vector containing the VIGS target fragment as described in B), or the gRNA as described in C), or the expression cassette as described in D); F) Transformation or transfection of host cells with the recombinant vector described in E); G), a mixture obtained by culturing host cells as described in F).

[0013] Furthermore, the nucleotide sequence of the nucleic acid encoding the NtBZR7 is shown in SEQ ID NO.1; The VIGS target fragment is the 134bp to 514bp region of the nucleic acid encoding NtBZR7 shown in SEQ ID NO.1.

[0014] This invention provides a product for improving plant germplasm resources, comprising at least one of the following: A) to G) A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding NtBZR7 as a template; B) VIGS target fragments targeting the nucleic acid encoding the NtBZR7; C) gRNA targeting NtBZR7; D) Expression cassettes containing the VIGS target fragment as described in B), or the gRNA as described in C); E) A recombinant vector containing the VIGS target fragment as described in B), or the gRNA as described in C), or the expression cassette as described in D); F) Transformation or transfection of host cells with the recombinant vector described in E); G), a mixture obtained by culturing host cells as described in F).

[0015] Furthermore, the nucleotide sequence of the nucleic acid encoding the NtBZR7 is shown in SEQ ID NO.1; The VIGS target fragment is the 134bp to 514bp region of the nucleic acid encoding NtBZR7 shown in SEQ ID NO.1.

[0016] In this invention, the plant includes tobacco.

[0017] The NtBZR7 gene described in this invention can regulate the content of chlorogenic acid in plants. Specifically, through the cloning of the NtBZR7 gene and the construction and analysis of its expression vector, this invention found that overexpression of this gene in tobacco plants significantly increases the expression level of the NtBZR7 gene in leaves, indicating that the gene can be effectively overexpressed in tobacco, laying the foundation for subsequent functional studies at the expression level. Furthermore, through detection, it was found that the chlorogenic acid content in the transgenic lines was significantly increased compared with the normal control group. This result indicates that there is a significant positive correlation between the expression level of the NtBZR7 gene and the accumulation of chlorogenic acid, that is, NtBZR7 has a positive regulatory effect on the synthesis or accumulation of chlorogenic acid. Chlorogenic acid is a polyphenolic compound widely found in plants and an important component of plant secondary metabolites. It plays a role in disease resistance, enhancing plant stress resistance, and improving plant quality. Specifically, in enhancing disease resistance, chlorogenic acid can directly inhibit the growth of pathogens, thereby reducing the severity of diseases, minimizing pathogen infection and damage to plant tissues, and helping plants maintain normal growth and development. Regarding improving plant quality, especially in tobacco, chlorogenic acid is the most abundant polyphenol in tobacco leaves, and its content is often used as an important indicator of tobacco quality. Specifically, chlorogenic acid can form compounds similar to those found in humans during processing. Aroma compounds closely related to sensory perception are important sources of tobacco aroma, playing a positive role in improving the quality and quantity of tobacco aroma, thus affecting the sensory quality and industrial usability of tobacco. Therefore, the aroma of tobacco can be controlled by adjusting the content of chlorogenic acid. Simultaneously, chlorogenic acid plays a crucial role in regulating plant stress resistance, especially in tobacco. Chlorogenic acid can participate in the plant's response to abiotic stresses such as drought, salinity, and low temperature, enhancing the plant's adaptability to adverse environments and mitigating the adverse effects of stress on tobacco growth. Therefore, the stress resistance of tobacco can also be affected by adjusting the content of chlorogenic acid. In summary, this invention provides new usable genetic resources for the improvement of tobacco germplasm resources.

[0018] Furthermore, in the NtBZR7 silencing experiment, this invention utilized virus-induced gene silencing technology to study the loss of function of NtBZR7. During the experiment, photobleaching occurred in the blank control group of the VIGS experiment, indicating that the VIGS system can effectively function in tobacco plants, providing a reliable experimental basis for subsequent analysis of gene silencing effects. After NtBZR7 silencing, the plants showed "good growth status," which means that the loss of NtBZR7 function did not interfere with the normal developmental process of tobacco, indicating that this gene is not an essential key regulatory factor in the normal growth and development of tobacco, and its loss of function will not have a significant adverse effect on the overall growth of the plant.

[0019] This invention has discovered the tobacco NtBZR7 gene, which, when overexpressed in tobacco, can increase the chlorogenic acid content, while silencing it can decrease the chlorogenic acid content, providing a new usable gene resource for the improvement of tobacco germplasm resources. Attached Figure Description

[0020] Figure 1 This shows a gel electrophoresis diagram of the NtBZR7 gene clone in Example 1 of the present invention; Figure 2 The amino acid sequence of NtBZR7 is shown in the comparison diagram. Figure 3 A diagram showing the expression characteristics of the NtBZR7 gene in different tissues; Figure 4 Subcellular localization map of NtBZR7 gene in tobacco epidermal cells (scale bar, 50 μm); Figure 5 The phenotype of tobacco plants silenced with VIGS-NtBZR7 is shown; where con is the control group; VIGS-PDS is the control group; and VIGS-BZR7 is the NtBZR7 gene silencing group. Figure 6 The relative expression level of NtBZR7 gene in VIGS-BZR7 plants was detected by qRT-PCR; where con was the control group; #1 to #4 were different individual plants of the NtBZR7VIGS line. Figure 7 The determination of chlorogenic acid content in VIGS-BZR7 tobacco plants is shown; where con is the control group; #1~#4 are different individual plants of the NtBZR7 VIGS strain. Figure 8 Gel electrophoresis images of PCR products used to verify the DNA level of the NtBZR7 gene; where 1-8 represent the detection results of different individuals in the T0 generation; Figure 9 The NtBZR7 gene was detected by qRT-PCR in plants overexpressing the NtBZR7 gene; where Con represents the normal control tobacco plant, and the rest are different individual plants overexpressing the NtBZR7 gene. Figure 10 Analysis of chlorogenic acid content in leaves of T1 generation NtBZR7 gene overexpressing plants. Detailed Implementation

[0021] This invention provides the application of the NtBZR7 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.

[0022] 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.

[0023] 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 NtBZR7. Through cloning the NtBZR7 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.

[0024] 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.

[0025] 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.

[0026] In the following examples, primer sequence synthesis and gene sequencing were performed by Beijing Liuhe Huada Biotechnology Co., Ltd.

[0027] Biomaterials: Tobacco material: Cultivated tobacco (Nicotiana tabacum) variety K326, kindly provided by Guizhou Tobacco Science Research Institute.

[0028] Vectors: Super pCAMBIA1300, PTRV1 and PTRV2 are commonly used plasmid vectors in molecular biology and can be obtained from public sources.

[0029] 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.

[0030] Experimental reagents: 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.

[0031] Experimental equipment: 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.

[0032] Data processing: 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.

[0033] NtBZR7 CDS: atgatgtgggaaggtggagggtcgccggcgacatcttctgccggtggcggaggcggtggagctgccgtgggaagtagtagtagtgggaggaggaagccatcttggagagaaagagagaataataggaggagagagaggaggagaagagcaatagcagctaaaatatatgcaggattaagagcacaggggaattataatttgcctaaacactgtgataataatgaagttcttaaagctctttgtgctgaagctggttggatcgttgaacctgatggcactacttatcgcaagggatgcagcaggccagctccaatggagattggaggcacttcagccaacattacgccaagttcttcacgacatccaagtcctccgtcttcatactttgctagcccaattccatcttatcaacccagtccaacgtcctcgtctttcccaagtccatctcgtggcgatgctaacatgtcatcacaccctttcgcatttctccataattccattccttcgtcactacctccattacgaatatcaaacagcgcccctgtaacaccacctctttcatcacctactagactccctaagcagacgttcaatttggagactttggccagagaatcaatgtctgctttaaatatccctttctttgctgcttcagccccgacaagcccgactcggggtcagcgattcactcctgccactataccagaatgcgatgaatctgattcgtccactattgattctggccagtggatgagctttcaaaagtatgcaaccaacggtgtccctacttctccgacttttaatcttattaaacctgcagctcagagaattccttctaatgatatgatcatcgataagggtaagagcatagagtttgactttgagaatgtatcagtgagggcggcatgggaaggagaaaagattcatgaggttggtttagatgatctggagctcactctcggaagtgggagcggtcgcatgtga(SEQ ID NO.1); NtBZR7 protein: MMWEGGGSPATSSAGGGGGGGAAVGSSSSGRRKPSWRERENNRRRERRRRAIAAKIYAGLRAQGNYNLPKHCDNNEVLKALCAEAGWIVEPDGTTYRKGCSRPAPMEIGGTSANITPSSSRHPSPPSSYFASPIPSYQPSPTSSSFPSPSRGDANMSSHPFA FLHNSIPSSLPPLRISNSAPVTPPLSSPTRLPKQTFNLETLARESMSALNIPFFAASAPTSPTRGQRFTPATIPECDESDSSTIDSGQWMSFQKYATNGVPTSPTFNLIKPAAQRIPSNDMIIDKGKSIEFDFENVSVRAAWEGEKIHEVGLDDLELTLGSGSGRM (SEQ ID NO.2); The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments: Example 1 Cloning of the NtBZR7 gene 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 NtBZR7 gene was cloned using PCR technology. The specific implementation process is as follows: 1. Primer design The specific PCR amplification primer sequences for non-coding region amplification are designed as follows: NtBZR7-clone-F:5'-atgatgtgggaaggtggagg-3' (shown as SEQ ID NO.3); NtBZR7-clone-R:5'-catgcgaccgctcccacttcc-3' (shown in SEQ ID NO.4).

[0034] 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.

[0035] 2. PCR amplification

[0036] 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.

[0037] Table 1. PCR reaction system

[0038]

[0039] Table 2. PCR reaction procedure

[0040]

[0041] 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.

[0042] 3. Sequencing and analyzing the NtBZR7 gene

[0043] The purified and recovered target fragment was ligated into the pMD19-T vector, incubated overnight at 4 °C, and then transformed into competent DH5α cells. White single colonies were picked and incubated on a shaker at 37 °C for approximately 12 h (200 r / min). 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 NtBZR7 gene.

[0044] Sequencing results showed that the tobacco NtBZR7 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 NtBZR7 gene consists of 328 amino acids, and its specific amino acid sequence is shown in SEQ ID NO.2.

[0045] 4. Comparison of the amino acid sequences of the protein encoded by the tobacco NtBZR7 gene with those of BZR genes from other species.

[0046] The proteins encoded by the tobacco NtBZR7 gene are highly conserved compared to the AtBES1 / BZR1 family, and each protein contains a characteristic BES1_N domain (Pfam: PF05687), as shown in the results. Figure 2 As shown.

[0047] Example 2: Identification of the expression pattern of the tobacco NtBZR7 gene

[0048] Using real-time quantitative PCR (BIO-RAD, USA) technology, this example provides a preliminary analysis of the expression pattern of the tobacco NtBZR7 gene in tobacco plants. The specific implementation steps are as follows: 1. Primer design for real-time PCR For real-time quantitative PCR analysis, L25 was used as an internal reference gene, and the specific primer sequences were designed as follows: NtBZR7-QF: 5'-gaacctgatggcactactta-3' (shown in SEQ ID NO.5); NtBZR7-QR: 5 '-ttggatgtcgtgaagaactt-3 ' (shown in SEQ ID NO.6); L25-F: 5'-cccctcaccacagagtctgc-3' (shown in SEQ ID NO.7); L25-R: 5'-aagggtgttgttgtcctcaatctt-3' (shown in SEQ ID NO.8); 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.

[0049] 2. Quantitative Real-Time PCR Detection

[0050] For quantitative real-time PCR, the 20 μL reaction system design is shown in Table 3, and the amplification program is shown in Table 4 (the instrument used was a Bio-Rad CFX96 from Bio-Rad Laboratories, USA). The relative expression levels obtained after the reaction were calculated using a 2-1... -△△CT The data were analyzed using the standard method, with the relative expression level of the blank control group (Con) set at 1. All data were the average of at least three independent experiments. SPSS 18.0 software was used, and Duncan's test (P < 0.05) was employed for statistical significance analysis.

[0051] Table 3. Reaction System

[0052]

[0053] Table 4. Amplification Procedure

[0054]

[0055] The results of quantitative fluorescence detection are as follows Figure 3 As shown in the figure, the analysis reveals that NtBZR7 expression varies significantly across different tobacco tissues. The gene is most highly expressed in immature flowers, significantly higher than in other tissues. Expression can be detected in roots, stems, mature flowers, and senescent flowers. However, gene expression was not detected in young leaves, mature leaves, or senescent leaves.

[0056] Example 3 Construction of NtBZR7 overexpression vector

[0057] This embodiment is obtained by cloning in Example 1. NtBZR7 Based on genes, it was constructed NtBZR7 The specific implementation steps for gene overexpression vectors are as follows: The upstream primer was designed as 5'-atacaccaaattgactctagaatgatgtgggaaggtggagg-3' (SEQ ID NO. 9), and the downstream primer was designed as 5'-gcccttgctcaccatggtaccatgcgaccgctcccacttcc-3' (SEQ ID NO. 10). Homologous recombination adapter sequences were added to both primers. Using the NtBZR7-T plasmid correctly sequenced in Example 1 as a template, amplification was performed. The recovered and purified PCR product and the enzyme-digested pCAMBIA1300-GFP empty vector were ligated using a homologous recombination kit (ClonExpress Ultra One Step Cloning Kit, Novizan), and transformed into *E. coli* DH5α. Positive clones were identified by colony PCR, and positive single clones were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing verification. ， The plasmid was extracted after verifying the correct colony composition, and the NtBZR7-pCAMBIA1300-GFP vector was obtained. 。

[0058] Example 4: Expression localization of the NtBZR7 gene in tobacco epidermal cells

[0059] This study used the NtBZR7 overexpression vector NtBZR7-pCAMBIA1300-GFP constructed in Example 3 as the research object to observe the localization of NtBZR7 protein in tobacco epidermal cells. The specific implementation operation is as follows: The NtBZR7-pCAMBIA1300-GFP vector and the empty pCAMBIA1300-GFP vector were transformed into Agrobacterium LBA4404, and then Agrobacterium was injected into the leaves of Nicotiana benthamiana via infiltration. Through Agrobacterium infection, the target gene was integrated into the tobacco cells. After culturing the injected plants for 3 days, the transformed leaves were observed using a confocal microscope (Zeiss LSM900 META, Jena, Germany).

[0060] As shown in Figure 4, the fluorescence signal of NtBZR7-GFP highly overlaps with the fluorescence signal of the nuclear marker DAPI, indicating that the NtBZR7 protein is located in the cell nucleus.

[0061] Example 5: Construction of the VIGS silencing vector for the NtBZR7 gene

[0062] Based on the NtBZR7 gene cloned in Example 1, a VIGS vector for the NtBZR7 gene was constructed. The specific implementation steps are as follows: The upstream primer was designed as 5'-gtgagtaaggttaccgaattcagaggaggagaagagcaata-3' (SEQ ID NO. 11), and the downstream primer was designed as 5'-cgtgagctcggtaccggatccgtagtgacgaaggaatggaa-3' (SEQ ID NO. 12). Homologous recombination adapter sequences were added to both primers. Using the correctly sequenced NtBZR7-T plasmid from Example 1 as a template, amplification was performed. The recovered and purified PCR product and the enzyme-digested empty pTRV2 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 Liuhe BGI Genomics Co., Ltd. for sequencing to obtain the recombinant vector pTRV2-NtBZR7 for NtBZR7 gene silencing.

[0063] Experiment 6: Construction of tobacco plants with NtBZR7 gene silence

[0064] The NtBZR7 gene VIGS silencing vector constructed in Example 5 was used to infect Nicotiana benthamiana plants via Agrobacterium-mediated infection. After gene silencing, leaf samples were collected, and the chlorogenic acid content of the leaves was measured. The specific procedures are as follows: pTRV2-NtBZR7 and empty vector pTRV2 (control group, Con) were transformed into Agrobacterium LBA4404 competent cells, respectively; pTRV1 strain was prepared simultaneously. Positive Agrobacterium clones were picked and cultured in a shake culture. The cells were collected, resuspended in infection buffer, and the OD of the culture was adjusted. 600The concentration was increased to 0.6-0.8. pTRV1 bacterial suspension was mixed with pTRV2 empty vector bacterial suspension and pTRV2-NtBZR7 bacterial suspension at a volume ratio of 1:1 to obtain control group and gene-silenced group bacterial suspensions. Nicotiana benthamiana seedlings with uniform growth at the 4-5 leaf stage were selected, and Agrobacterium infection was performed on the leaves using a syringe-based non-invasive infiltration method to obtain control group (empty vector pTRV2, Con), positive control group plants pTRV2-PDS (VIGS-PDS), and NtBZR7 gene-silenced group plants (VIGS-BZR7, or NtBZR7 VIGS). Plants were cultured under normal light conditions and their phenotypes were observed 14 days after infection. The leaves of the pTRV2-PDS positive control plants (control plants, VIGS-PDS) showed obvious photobleaching and albino phenotypes, proving the effectiveness of this VIGS infection system. Compared with the normal control plants (Con), VIGS-NtBZR7 tobacco (NtBZR7 gene-silenced group plants, VIGS-BZR7) did not show obvious developmental abnormalities, albino morphology, or other abnormalities, and the overall growth status of the plants was good, suitable for subsequent sample collection and physiological index determination. Figure 5 ).

[0065] Leaves from the same functional parts were selected, and leaf samples were collected from pTRV2 empty control plants (Con) and pTRV2-NtBZR7 silent plants (VIGS-BZR7), respectively. After being flash-frozen in liquid nitrogen, the samples were stored in a freezer at -80 ℃.

[0066] Example 7: Detection of NtBZR7 VIGS Silent Line Expression

[0067] Gene expression was detected in the NtBZR7 VIGS lines obtained in Example 6. Real-time quantitative PCR results showed that, compared with the control group (Con), the transcription levels of the NtBZR7 gene in the four VIGS-silenced lines #1, #2, #-3, and #-4 were significantly reduced (different lowercase letters represent significant differences, P<0.05). However, there was no significant difference in NtBZR7 expression levels among the four independent silenced lines (VIGS-BZR7, #1~#4). These results confirm that the VIGS vector constructed in this experiment can effectively inhibit the expression of the endogenous NtBZR7 gene in tobacco, obtaining NtBZR7 gene-silenced positive plants, which can be used for subsequent gene function analysis. Figure 6 )

[0068] The specific procedures for detecting the chlorogenic acid content in the leaves of pTRV2-NtBZR7 tobacco plants are as follows: The chlorogenic acid content in tobacco leaves was determined using HPLC-MS / MS with the internal standard method. For the test, 50 mg of freeze-dried tobacco leaf sample was ground into powder and transferred to 1.5 mL (2 mL centrifuge tube) of pre-cooled ethanol-water (4:1, v / v) extraction solution. The sample was sonicated at room temperature for 1 h, then centrifuged at 14000 r / min for 12 min. 1 mL of the supernatant was collected and concentrated into a solid under vacuum. The solid was reconstituted with 200 μL of 80% ethanol (containing 75 ng / mL umbelliferone solution), centrifuged at 20000 r / min for 3 min, and the supernatant was collected for HPLC-MS / MS analysis. Analytical conditions: Column: BEH Phenyl column (2.1 mm × 150 mm, 1.7 μm); Mobile phase: 0.1% formic acid aqueous solution (A) and 0.1% formic acid methanol solution (B); Elution gradient: 0–2 min B phase from 5% to 15%, 2–10 min B phase maintained at 15%, 10.01 min–15 min B phase to 100%; Flow rate: 0.3 mL / min; Column temperature: 35℃; Injection volume: 1 μL. Electrospray ionization was used; capillary voltage was 4 kV in positive ionization mode; nebulizer gas pressure was 275.6 kPa; drying gas flow rate was 12 L / min; drying gas temperature was 290℃; sheath gas flow rate was 11 L / min; sheath gas temperature was 200℃; real-time multiple reaction monitoring (DMRM) mode was used for scanning. The retention time of chlorogenic acid was 4.06 min.

[0069] The results are shown in Figure 7: Compared with the control plants (Con), the chlorogenic acid content of the four VIGS-NtBZR7 silent lines (VIGS-BZR7, #1~#4) was significantly reduced, with decreases of 13.72%, 15.68%, 16.66%, and 14.71%, respectively (P < 0.05). This result indicates that the NtBZR7 gene positively regulates the accumulation of chlorogenic acid in tobacco leaves.

[0070] Example 8: Detection of NtBZR7 gene overexpression lines

[0071] The NtBZR7 gene overexpression vector NtBZR7-pCAMBIA1300-GFP from Example 3 was transformed into Agrobacterium LBA4404. The specific implementation method is as follows: 1. Preparation of Agrobacterium competent cells 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.

[0072] 2. Plasmid transformation of Agrobacterium

[0073] Take 1 μL of NtBZR7 gene overexpression plasmid and add it to a centrifuge tube containing 100 µL of Agrobacterium competent cells. Place on ice for 30 minutes. Then transfer to liquid nitrogen for 1 minute and incubate at 37 °C for 5 minutes. Add 1 mL of LB liquid medium and incubate at 28 °C with shaking for 3 hours. Centrifuge at 5000 rpm for 1 minute, discard the supernatant, add 200 μL of LB liquid medium, and resuspend the precipitate. Take 200 μL of the resuspended bacterial solution and spread it evenly on LB agar plates containing 20 mg / L Rif and 50 mg / L kanamycin (Kan). Incubate at 28 °C for 2-3 days. After confirming the colony PCR results, preserve the bacterial strain.

[0074] 3. Tobacco Conversion

[0075] Disinfect the vigorously growing tobacco leaves and cut them into 1 cm 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. Afterward, they were immersed in engineered bacterial solution for 10-15 min, shaking the solution several times during this period. Excess bacterial solution was then blotted dry with sterile filter paper. The plants were then inoculated into MS differentiation medium and co-cultured at 28 ℃ in the dark for 3-5 days. The co-cultured plants were washed three times with sterile water, blotted dry with sterile paper, and transferred to MS differentiation medium containing hygromycin and carbenicillin for constant temperature incubation. 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. 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.

[0076] 4. Detection of positive super-genetically modified tobacco using PCR and qPCR methods

[0077] Design specific primer pairs for the expression vector, with the upstream primer specifically binding to the NtBZR7 gene and the downstream primer specifically binding to the GFP gene.

[0078] Upstream primer: 5'-gggtaagagcatagagtttga-3' (SEQ ID NO.13); Downstream primer: 5'-gtcgtccttgaagaagatgg-3' (SEQ ID NO.14). PCR amplification was performed using genomic DNA from controls and K326 and T0 generation seedlings as templates. Seedlings amplifying specific bands were T0 generation positive seedlings (e.g., Figure 8 As shown, 1-10 represent different T0 generation positive seedlings. To verify whether the phenotype can be stably inherited to the next generation, seeds of T0 generation positive plants were collected, and T1 generation plants were obtained after further planting. Then, the expression level of the NtBZR7 gene in the selected positive seedlings was detected by qPCR. The NtBZR7 gene-specific primers NtBZR7-QF: 5'-gaacctgatggcactactta-3' (shown in SEQ ID NO.5) and NtBZR7-QR: 5'-ttggatgtcgtgaagaactt-3' (shown in SEQ ID NO.6) were used. The tobacco L25 gene was used as an internal control in 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 as described in Example 3). After the reaction, based on the obtained CT value, 2... -△△CT Methods for calculating the relative expression level of the NtBZR7 gene (e.g.) Figure 9 As shown in the figure, con represents the blank control group, and BZR7-2-1, BZR7-8-3, BZR7-9-2, BZR7-13-5, BZR7-14-1, BZR7-15-2, and BZR7-18-4 are T1 generation transgenic plants. The analysis shows that T0 generation plants can stably be inherited into the T1 generation.

[0079] 5. Chlorogenic acid content analysis of T1 generation NtBZR7 transgenic cells

[0080] In this embodiment, the NtBZR7 overexpression line (T1 generation) was selected as the research object to detect the chlorogenic acid content in the leaves of transgenic tobacco plants at the middle leaf maturity stage. The specific implementation operation is as follows: 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 20,000 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), 3 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. The results showed that chlorogenic acid was effective against NtBZR7 overexpressing transgenic lines (such as...). Figure 10 As shown in the figure: Compared with the control (K326), the chlorogenic acid content in the middle leaves of the T1 generation transgenic plants (BZR7-2-1, BZR7-8-3, BZR7-9-2, BZR7-13-5, BZR7-14-1, BZR7-15-2, BZR7-18-4) increased by 99.71%, 110.39%, 96.62%, 73.08%, 54.86%, 253.42%, and 145.97% respectively during the vigorous growth period, and the differences with the control were all statistically significant (P < 0.05). This result indicates that the NtBZR7 gene plays an important role in the regulation of chlorogenic acid during the vigorous growth period of tobacco.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of NtBZR7 in plant germplasm resource improvement, characterized in that, The amino acid sequence of NtBZR7 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The improvement of germplasm resources includes regulating the content of chlorogenic acid.

3. The application according to claim 2, characterized in that, The regulation of chlorogenic acid content includes: increasing the chlorogenic acid content and / or decreasing the chlorogenic acid content.

4. The application according to claim 3, characterized in that, The increase in chlorogenic acid content is achieved by overexpressing NtBZR7 in plants; The reduction of chlorogenic acid content is achieved by using VIGS-mediated gene silencing to target the nucleic acid encoding NtBZR7.

5. The application according to claim 4, characterized in that, The overexpression of NtBZR7 in plants is achieved by transfecting the nucleic acid encoding NtBZR7 into the plant; the nucleotide sequence of the nucleic acid encoding NtBZR7 is shown in SEQ ID NO.

1.

6. The application according to claim 4, characterized in that, The target fragment for the VIGS-mediated gene silencing is the 134bp to 514bp region of the nucleic acid encoding NtBZR7 shown in SEQ ID NO.

1.

7. A method for regulating the chlorogenic acid content in plants, characterized in that, This includes using at least one of the following methods (A) to (G) to regulate the chlorogenic acid content in plants: A) Overexpression of NtBZR7; B) VIGS target fragments targeting the nucleic acid encoding the NtBZR7; C) gRNA targeting NtBZR7; D) Expression cassettes containing the VIGS target fragment as described in B), or the gRNA as described in C); E) A recombinant vector containing the VIGS target fragment as described in B), or the gRNA as described in C), or the expression cassette as described in D); F) Transformation or transfection of host cells with the recombinant vector described in E); G), a mixture obtained by culturing host cells as described in F).

8. A product for improving plant germplasm resources, characterized in that, Includes at least one of the following: A) to G) A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding NtBZR7 as a template; B) VIGS target fragments targeting the nucleic acid encoding the NtBZR7; C) gRNA targeting NtBZR7; D) Expression cassettes containing the VIGS target fragment as described in B), or the gRNA as described in C); E) A recombinant vector containing the VIGS target fragment as described in B), or the gRNA as described in C), or the expression cassette as described in D); F) Transformation or transfection of host cells with the recombinant vector described in E); G), a mixture obtained by culturing host cells as described in F).

9. The method according to claim 7 or the product according to claim 8, characterized in that, The nucleotide sequence of the nucleic acid encoding NtBZR7 is shown in SEQ ID NO.1; The VIGS target fragment is the 134bp to 514bp region of the nucleic acid encoding NtBZR7 shown in SEQ ID NO.

1.

10. The application according to claims 1-6, the method according to claim 7, or the product according to claim 8, characterized in that, The plant mentioned includes tobacco.

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