Application of tobacco Nta21g00780 gene in increasing content of chlorogenic acid in tobacco

Abstract

The invention belongs to the technical field of tobacco breeding, and mainly relates to application of a tobacco Nta21g00780 gene in increasing the content of chlorogenic acid in tobacco. According to the invention, a gRNA sequence of CRISPR / Cas9 is designed by taking a tobacco Nta21g00780 gene as a target, a DNA fragment containing the gRNA is connected into a CRISPR / Cas9 carrier, and then tobacco is transformed, so that an editing material of the tobacco Nta21g00780 gene is obtained, and it is found that the editing material can significantly increase the content of chlorogenic acid in tobacco.

Description

Technical Field

[0001] This invention belongs to the field of tobacco breeding technology, and mainly relates to the application of the tobacco Nta21g00780 gene in increasing the chlorogenic acid content of tobacco. Background Art

[0002] Tobacco leaves contain a variety of valuable chemical components, among which chlorogenic acid is one of the most abundant phenolic substances, accounting for approximately 75% to 90% of the total polyphenols. Chlorogenic acid, also known as caffeine tannin, is a phenylpropanoid compound produced by plants through the shikimic acid pathway during aerobic respiration. Tobacco leaves contain 3% or higher of chlorogenic acid, making it the most abundant polyphenolic compound in tobacco and the only tannin found in tobacco. It has a significant impact on tobacco growth and development, leaf color, and aroma and flavor, thus this polyphenol cannot be ignored in research aimed at improving tobacco quality.

[0003] Current research indicates that plants primarily utilize three secondary metabolic pathways: phenylpropanoid metabolism, isoprene metabolism, and alkaloid synthesis. The main substances affecting tobacco aroma (phenolic compounds, terpenoids, and alkaloids) are also formed through these three pathways. In the phenylpropanoid metabolism pathway, shikimic acid from the shikimic acid pathway undergoes transamination via branched prephenylpropionic acid to form phenylalanine, which then enters the phenylpropanoid metabolic pathway. This increases the chlorogenic acid content in tobacco, thus improving its quality. However, current research lacks studies on editing the Nta21g00780 gene to increase chlorogenic acid content in tobacco. Summary of the Invention

[0004] The main technical problem solved by this invention is: how to use genetic engineering to edit the Nta21g00780 gene in tobacco to increase the chlorogenic acid content in tobacco and improve the quality of tobacco.

[0005] This invention discloses the application of the Nta21g00780 gene in increasing the chlorogenic acid content of tobacco, and the nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO: 1.

[0006] Preferably, the amino acid sequence of the Nta21g00780 gene is shown in SEQ ID NO.2.

[0007] The present invention also discloses a method for increasing the chlorogenic acid content in tobacco, wherein the method comprises: performing site-directed mutation on the Nta21g00780 gene in tobacco using gene editing, the nucleotide sequence of the Nta21g00780 gene being shown in SEQ ID NO: 1.

[0008] Preferably, the specific method of gene editing is as follows: using the tobacco Nta21g00780 gene as a target, designing a CRISPR / Cas9-based sgRNA nucleotide sequence, inserting a DNA fragment containing the above-mentioned sgRNA into a CRISPR / Cas9 vector to transform tobacco, thereby achieving targeted editing of the tobacco Nta21g00780 gene and obtaining strains with low or no expression of the Nta21g00780 gene.

[0009] Preferably, the sgRNA nucleotide sequence is as shown in SEQ ID NO:3-4.

[0010] Preferably, the tobacco is cultivated tobacco.

[0011] This invention also discloses a method for cultivating a tobacco mutant with high chlorogenic acid content. The method for obtaining the tobacco mutant with high chlorogenic acid content is as follows: a vector for the target gene Nta21g00780 is constructed using a CRISPR / Cas9 gene knockout system; the expression vector is transformed into tobacco callus tissue using an Agrobacterium-mediated transformation method; and the Nta21g00780 gene is knocked out at a specific site to obtain the tobacco mutant. The nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO: 1; the amino acid sequence encoded by the Nta21g00780 gene is shown in SEQ ID NO: 2.

[0012] The beneficial effects of this invention are:

[0013] This invention targets the tobacco Nta21g00780 gene, designs a CRISPR / Cas9 gRNA sequence, inserts a DNA fragment encoding the gRNA into a CRISPR / Cas9 vector, and then transforms tobacco to obtain tobacco Nta21g00780 gene editing material, and finds that it can increase the chlorogenic acid content in tobacco.

[0014] The inventors previously obtained the Nta21g00780 tobacco mutant, planted F1 hybrids of the Nta21g00780 tobacco mutant and K326 tobacco, and when the plants reached the 5-6 leaf stage, tobacco tissues from the same leaf position were sent to the company. The chlorogenic acid content was detected by liquid chromatography, and it was found that the chlorogenic acid content of the Nta21g00780 tobacco mutant was significantly higher than that of K326 tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Evolutionary analysis of the Agenet domain protein family genes.

[0016] Figure 2 Identification of Nta21g00780 gene mutant materials; deletion sites of Nta21g00780 tobacco mutants.

[0017] Figure 3 Bar chart and liquid chromatography-mass spectra of chlorogenic acid content in Nta21g00780 tobacco mutant and K326 tobacco. DETAILED DESCRIPTION

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1: Editing the Nta21g00780 gene using CRISPR / Cas9

[0020] The tobacco ecotype was K326, the Agrobacterium strain was EHA105, and the vector was pCBSG012-slu-DSG. Major reagents included: restriction endonucleases from Thermo Fisher Scientific, DNA polymerase and infusion ligase from Novizan; reverse transcription kits from Thermo Fisher Scientific; RNA extraction kits from Tiangen Biotech; plasmid extraction kits and DNA recovery kits from Tiangen Biotech; quantitative PCR reagents from Taraka Biotech; MS medium, agar powder, agarose, ampicillin, kanamycin, rifampin, and other antibiotics were purchased from Sigma-Aldrich; all other chemical reagents used in the examples were imported or domestically produced analytical grade reagents; primer synthesis and sequencing were performed by Beijing Qingke Biotechnology Co., Ltd.

[0021] Members of the Agnet domain protein family were screened from tobacco databases using BLAST alignment. Sequences were analyzed using MEGA12 software, and a phylogenetic tree was constructed. Evolutionary analysis of the Agnet domain protein family members yielded the following results: Figure 1 shown.

[0022] The nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO.1; the amino acid sequences encoded by the Nta21g00780 gene are shown in SEQ ID NO.2.

[0023] SEQ ID NO.1

[0024]

[0025] SEQ ID NO.2

[0026] make NQALGRFPALEMHSMSEGRKDSQATAGSSSHLSSPTNLYANSLPGSEETKDFKTTGLVEQSSIIDLPKEAANTSKKVSSRSDINSGSQLGDKLEKEASWSDNGSTSCIAIDPAKTAETTENFYLSNCPLKKFRTSDGVRLHSMGSDSMEAAILDLEELANKIKWLKGLLEFGKPVSNASRPSWKFVEHHASSGNK

[0027] Based on the Nta21g00780 gene sequence, a pair of gRNAs was designed using the Huazhong Agricultural University CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to select targets with high target scores, low off-target rates, and suitable locations. The gRNA sequences are shown in SEQ ID NO.3-4.

[0028] SEQ ID NO:3 (Knockout target 1)

[0029] TTGGGCAATCCGCGGAAGCGAGG

[0030] SEQ ID NO:4 (Knockout target 2)

[0031] AAATGCCCCCATATAACATTGGG

[0032] (1) Construction of CRISPR / Cas9 editing vector

[0033] The 50 μL PCR system consisted of: 20 μL Nuclease-free Water, 25 μL Pfu PCR Mix, 2 μL each of 100 μM forward and reverse primers, and 1 μL template. The PCR was performed at 94℃ for 5 min, 94℃ for 30 s, 50℃ for 45 s, and 72℃ for 6 s, for a total of 35 cycles. Electrophoresis was performed on a 1.5% agarose gel at 5 V / cm for 20 min. The electrophoretic fragments were excised under UV light and separated into their respective systems for sol-gel recovery. The recovered DNA was dissolved in 30 μL of water, and after verification, ligation was performed with the vector.

[0034] The PCR-amplified fragment was ligated into the vector using the following ligation system: 1 μL Nuclease-free Water, 2 μL Recombination Buffer, 4 μL PCR Product, 2 μL Linearized Vector Template, and 1 μL Recombinase. The PCR instrument was run on the recombination program at 37°C for 30 min, and then stored on ice or at 4°C.

[0035] (2) Transformation of Escherichia coli

[0036] Add at least 10 μl of ligation product to 100 μl of competent E. coli cells. After removing the competent DH5α cells from the refrigerator, immediately place them on ice. After 5 minutes, wait for the bacterial block to thaw before adding the ligation product. Incubate on ice for 25 minutes, then heat-shock at 42°C for 45 seconds. Place on ice for 2 minutes (do not shake). Add 100 μl of antibiotic-free LB broth. Incubate at 37°C, 200 rpm for 1 hour. Plate the culture, add LB broth and bacterial antibiotics, and incubate at 37°C for one day. Select colonies for colony PCR identification; positive colonies are then sequenced for identification.

[0037] (3) Agrobacterium-mediated transformation

[0038] 1: 20 μl Agrobacterium (EHA105-WM) + 1 μl plasmid, incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and incubate on ice for 5 min. Add 100 μl of antibiotic-free LB, shake at 200 rpm for 2 h at 28℃, and directly plate onto a plate containing the corresponding bacterial antibiotic + rifampin, and incubate at 28℃ for two days.

[0039] 2: Pick a single clone of bacteria and shake it:

[0040] Two days later, select one monoclonal antibody and place it in a 5ml sterile EP tube. Add 2ml of the corresponding bacterial antibiotic and rifampin beforehand, and shake overnight.

[0041] 3. Preservation of Glycerin Bacteria: Add 100 μL of 75% sterile glycerol to 400 μL of bacterial culture, label the mixture, and store at -70°C. Extract plasmid from the remaining bacterial culture and transform it into E. coli (1 μL plasmid + 20 μL competent E. coli cells, incubate on ice for 30 min, heat shock at 42°C for 35 s, incubate on ice for 2 min, add 100 μL of antibiotic-free LB, shake at 37°C and 200 rpm for 1 h, plate the culture, add LB and the corresponding bacterial antibiotic, and incubate at 37°C for one day).

[0042] 4: Select a single clone, shake the culture in the morning, and send the bacterial culture directly for sequencing in the evening, selecting a portion for sequencing.

[0043] 5. Sequencing feedback is correct, verifying that there are no problems with Agrobacterium, and the prepared Agrobacterium can then be used for subsequent transformation experiments.

[0044] (4) Nta21g00780 editing carrier conversion

[0045] Activated Agrobacterium was picked and cultured in 50 mL of Kan+Rif resistant liquid LB medium at 28°C with shaking at 180 rpm until OD reached. 600 When the OD value approaches 0.6, transfer the bacterial culture to a pre-sterilized and pre-chilled 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in 20 mL of pre-chilled MS liquid medium, centrifuge again at 4000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in pre-chilled MS liquid medium to OD value. 600 The concentration was increased to 0.6, and then AS was added to a final concentration of 20 mg / L, ready for infection. In a clean bench, the edges of sterile leaves were trimmed with scissors, and the leaves were cut into 1.0 × 1.0 cm leaf discs along the midrib. The discs were then placed in the Agrobacterium infection solution for 5 minutes. The infected leaves were removed, and the Agrobacterium solution was blotted dry on sterile filter paper. The leaves were then laid flat, leaf-side down, on co-culture medium (G) and placed in a climate chamber (temperature 26℃, humidity 40%) for incubation in the dark for 3 days.

[0046] S1 subculture. After co-culturing for 3 days, the leaves were transferred face up to S1 differentiation medium. The explants were washed with sterile water containing 500 mg / L Cef, blotted dry with sterile filter paper, and then transferred to S1 medium. 5-8 explants were placed in each dish and cultured in the dark in an artificial climate chamber for about 1 week. Then they were placed under light to continue culturing until clusters of buds appeared at the leaf margins and the buds reached a length of about 0.5 cm.

[0047] S2 subculture. Use tweezers to transfer the shoot clusters from S1 to S2 differentiation medium. Shoot clusters that can be broken off can be directly inoculated; those that cannot be broken off can be inoculated along with their leaves, removing any leaves that have not yet developed shoot clusters. Culture in light for 1-2 weeks until the shoot clusters develop into seedlings.

[0048] S3 subculture. Transfer the seedlings from S2 to S3 differentiation medium and culture under light for 1-2 weeks. Rooting culture. Remove the swollen parts at the base and the yellowing leaves from the lower part of the healthy seedlings from S3, and then inoculate them into tissue culture bottles containing rooting medium R. Culture under light for 1-2 weeks to encourage the seedlings to root as quickly as possible.

[0049] Obtaining genetically modified tobacco. When the seedlings have 3-10 roots, each about 2-3 cm long, open the culture bottle to harden them off. After about 3 days, transplant the seedlings into nutrient pots filled with sterile soil and cover them with plastic film to retain moisture. About a week later, remove the plastic film depending on the seedlings' growth, allowing them to grow rapidly under natural conditions.

[0050] (5) Positive detection of genetically transformed materials

[0051] Six T0 generation transgenic plants transformed with the obtained Nta21g00780 editing vector were tested. After PCR amplification, they were sent to Qingke Company for sequencing. The results are as follows: Figure 2 As shown, several plants with mutations detected in the T0 generation were harvested and sown to plant the T1 generation.

[0052] Chlorogenic acid detection in genetically modified tobacco

[0053] Nta21g00780 tobacco and wild-type K326 tobacco, which had been germinated for one week, were planted in culture pots and placed in a nursery at 28℃ with 16 hours of light / 8 hours of darkness. They were watered once a week and no fertilizer was applied. After four weeks, samples were taken and sent to Shanghai Zhuocai Biotechnology Co., Ltd. for liquid chromatography analysis. The results are as follows: Figure 3 As shown, the chlorogenic acid content in Nta21g00780 tobacco was significantly increased.

Claims

1. Application of the Nta21g00780 gene in increasing the chlorogenic acid content of tobacco, characterized in that, The nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The amino acid sequence of the Nta21g00780 gene is shown in SEQ ID NO.

2.

3. A method for increasing the chlorogenic acid content in tobacco, characterized in that, The method is as follows: site-directed mutagenesis of the Nta21g00780 gene in tobacco is carried out by gene editing, and the nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, wherein The specific method of the gene editing is as follows: using the tobacco Nta21g00780 gene as a target, designing the sgRNA nucleotide sequence based on CRISPR / Cas9, ligating the DNA fragment containing the encoding sgRNA into the CRISPR / Cas9 vector and transforming tobacco, so as to achieve site-directed editing of the tobacco Nta21g00780 gene, and obtaining a line with low expression or non-expression of the Nta21g00780 gene.

5. The method according to claim 3, characterized in that, The sgRNA nucleotide sequence is shown in SEQ ID NO: 3-4.

6. The method according to claim 3, characterized in that, The tobacco is Nicotiana tabacum.

7. A method for cultivating a tobacco mutant with a high content of chlorogenic acid, characterized in that, The method for obtaining the high-chlorogenic acid tobacco mutant is as follows: constructing a target gene Nta21g00780 vector by using the CRISPR / Cas9 gene knockout system, transforming the expression vector into tobacco callus by the agrobacterium-mediated method, carrying out site-directed knockout of the Nta21g00780 gene, and obtaining a tobacco mutant; the nucleotide sequence of the Nta21g00780 gene is shown in SEQ ID NO: 1; the amino acid sequence encoded by the Nta21g00780 gene is shown in SEQ ID NO: 2.

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