Application of tobacco nta07g03160 gene in increasing content of chlorogenic acid in tobacco

Abstract

The application belongs to the technical field of tobacco germplasm improvement, and mainly relates to application of a tobacco Nta07g03160 gene in improving chlorogenic acid content in tobacco. The application takes the tobacco Nta07g03160 gene as a target, designs a gRNA sequence of CRISPR / Cas9, connects a DNA fragment containing the above gRNA into a CRISPR / Cas9 vector, then transforms tobacco, obtains edited materials of the tobacco Nta07g03160 gene, and finds that the edited materials can improve the chlorogenic acid content in tobacco.

Description

Technical Field

[0001] This invention belongs to the field of tobacco germplasm improvement technology, and mainly relates to the application of the tobacco Nta07g03160 gene in increasing the chlorogenic acid content of tobacco. Background Technology

[0002] Chlorogenic acid (CGA), also known as coffee tannin, is a phenylpropanoid compound produced by plants through the phenylalanine pathway during aerobic respiration. Chlorogenic acid has significant medicinal value. Furthermore, as a novel antioxidant, it also has important applications in food and fruit preservation, sun protection, and skincare.

[0003] Current research indicates that plants primarily utilize three secondary metabolic pathways: the phenylpropanoid metabolic pathway, the isoprene metabolic pathway, and the alkaloid synthesis pathway. The main substances affecting tobacco aroma (phenolic compounds, terpenoids, and alkaloids) are also formed through these three pathways. In the phenylpropanoid metabolic 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 process increases the chlorogenic acid content in tobacco, thus improving its quality.

[0004] Studies have shown that recombinant Nta07g03160 possesses similar levels of malg lipase activity to HsMAGL. Neither Nta07g03160 nor HsMAGL exhibits hydrolytic activity against LPC and LPE substrates. Furthermore, the major expression pattern of Nta07g03160 in pollen and germinating seeds, the co-expression of Nta07g03160 with genes related to the decomposition of stored lipids, and the localization of AtMAGL8 on the surface of oil bodies in germinating seeds and leaves accumulating oil bodies suggest that Nta07g03160 may be involved in TAG decomposition and / or membrane lipid remodeling during pollen tube growth, seed germination, and early seedling establishment. Nta07g03160 exhibits specific activity similar to HsMAGL in adipose tissue, and is the best candidate to date for involvement in the decomposition of stored lipids during seed germination; however, existing research focuses on editing Nta07g03160 to increase chlorogenic acid content. Summary of the Invention

[0005] The main technical problem solved by this invention is: how to use genetic engineering to edit the Nta07g03160 gene in tobacco to increase the chlorogenic acid content in tobacco and improve the quality of tobacco.

[0006] The application of the Nta07g03160 gene in increasing the chlorogenic acid content of tobacco, wherein the nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO: 1:

[0007] ATGAGGCCATCTCCAATTGTGATAAGTATTTTGACCAAACTTTGCAACAT

[0008] TATCCCAACCTGGAAACTAATTCCTACTCAAGATGTTGTTGATTCTGCCT

[0009] TCAGAGACCCTGAAGTTAGAAAGAGATAAGAACAATCCATATTGCTAC

[0010] AAGGGACGACCTCGTCTACAAACTGGGTACCAACTGTTTACTGTTAGCAT

[0011] GGATTTGGAGCAAAGACTTGAGGAAGTTACATTGCCATTTCTAATTGTAC

[0012] ATGGAGGAGAGGACACAGTTACTGATCCATCAGTGAGTAAACTTCTATAC

[0013] GAAAAGGCTCCGAGCATCGACAAGAGCTTCAAGTTGTATCCAGGAATGTG

[0014] GCATTCCCTTTCCTATGGTGAATTTCCTGAGAACAGAAACATCGTGTTTT

[0015] CGGACATTATCAGTTGGCTCAATGAGAAAATCAGTATGGGCAATTCAAGG

[0016] CTGGAGAGACAACAAAGCATGCAAATGACAATTTATCAAAGATCAGTAG

[0017] TTAA.

[0018] The amino acid sequence of Nta07g03160 is shown in SEQ ID NO: 2: MRPSPIVISILTKLCNIIPTWKLIPTQDVVDSAFRDPEVRKEIRNNPYCYKGRPRLQTGYQLFTVSMDLEQRLEEVTLPFLIVHGGEDTVTDPSVSKLLYEKASSIDKSFKLYPGMWHSLSYGEFPENRNIVFSDIISWLNEKISMGNSRLERQQKHANDNLSKISS

[0019] A method for increasing the chlorogenic acid content in tobacco, the method comprising: performing site-directed mutation on the Nta07g03160 gene in tobacco using gene editing, wherein the nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO: 1.

[0020] Preferably, the specific method of gene editing is as follows: using the tobacco Nta07g03160 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 Nta07g03160 gene and obtaining strains with low or no expression of the Nta07g03160 gene.

[0021] Preferably, the sgRNA nucleotide sequence is as shown in SEQ ID NO:3-4: SEQ ID NO:3 (knockout target 1)

[0022] TTTGCAACATTATCCCAACCTGG;SEQ ID NO:4(Knockout target 2)GACGACCTCGTCTACAAACTGGG;

[0023] Preferably, the tobacco is cultivated tobacco.

[0024] Preferably, the method for obtaining the tobacco mutant is as follows: a target gene Nta07g03160 vector 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 Nta07g03160 gene is knocked out at a specific site to obtain the tobacco mutant; the nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO: 1; and the amino acid sequence encoded by the Nta07g03160 gene is shown in SEQ ID NO: 2.

[0025] The beneficial effects of this invention are:

[0026] This invention targets the tobacco Nta07g03160 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 edited material of the tobacco Nta07g03160 gene, and finds that it can increase the chlorogenic acid content in tobacco.

[0027] The inventors previously obtained the Nta07g03160 tobacco mutant, planted F1 homologous Nta07g03160 tobacco mutant and K326 tobacco, and when they grew to the 5-6 leaf stage, tobacco tissues from the same leaf position were taken for testing. The chlorogenic acid content was detected by liquid chromatography, and it was found that the chlorogenic acid content of the Nta07g03160 tobacco mutant was significantly higher than that of K326 tobacco. Attached Figure Description

[0028] Figure 1 For the genetic evolution analysis of the MAGL family;

[0029] Figure 2 Identification of Nta07g03160 gene mutant materials; a) Sequencing identification of the five obtained mutants; b) Deletion sites of Nta07g03160 tobacco mutants;

[0030] Figure 3 Liquid chromatography-mass spectra of Nta07g03160 tobacco mutant and K326 tobacco, with bar charts showing chlorogenic acid content. Detailed Implementation

[0031] 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.

[0032] Example 1: Editing the Nta07g03160 gene using CRISPR / Cas9

[0033] 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.

[0034] Using previously reported Arabidopsis genes as query sequences, sequence alignment was performed on the tobacco K326 genome. Genes lacking the Hydrolases domain were then removed using SMART, resulting in the identification of 29 Nta07g03160 genes in tobacco. Multiple sequence alignment was performed between the previously reported MAGL gene in Arabidopsis and the newly identified NtMAGL gene in tobacco to construct a phylogenetic tree for evolutionary analysis. The Nta07g03160 gene in tobacco clustered with AT2G39410.2, AT2G39420.1, AT3G55190.1, AT3G55180.1, and AT2G39400.1 genes in Arabidopsis, suggesting greater evolutionary homology. Figure 1 The nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO.1; the amino acid sequences encoded by the Nta07g03160 gene are shown in SEQ ID NO.2.

[0035] Based on the Nta07g03160 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.

[0036] (1) Construction of CRISPR / Cas9 editing vector

[0037] 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.

[0038] 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.

[0039] (2) Transformation of Escherichia coli

[0040] 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, and place on ice for 2 minutes (do not shake). Add 100 μl of antibiotic-free LB globulin. Incubate at 37°C, 200 rpm for 1 hour. Plate the culture, add LB globulin and bacterial antibiotics, and incubate at 37°C for one day. Select colonies for colony PCR identification, and perform sequencing identification on positive colonies.

[0041] (3) Agrobacterium-mediated transformation

[0042] 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.

[0043] 2: Pick a single clone of bacteria and shake it:

[0044] 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.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] (4) Nta07g03160 editing carrier conversion

[0049] 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 the OD600 approached 0.6. The bacterial suspension was then transferred to a pre-sterilized and pre-chilled 50 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in 20 mL of pre-chilled MS liquid medium and centrifuged again at 4000 rpm for 10 min. The supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in pre-chilled MS liquid medium until the OD600 reached 0.6. 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 infection, with an infection time of 5 min. The infected leaves were removed, and the Agrobacterium tumefaciens bacterial solution was blotted dry on sterile filter paper. The leaves were then laid flat on the co-culture medium (G) with the leaf side down and placed in an artificial climate chamber (temperature 26℃, humidity 40%) for incubation in the dark for 3 days.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] (5) Positive detection of genetically transformed materials

[0055] The six T0 generation transgenic materials obtained by vector-1 transformation were tested. After PCR amplification, they were sent to Qingke Company for sequencing. Several plants with mutations in the T0 generation were harvested and sown to plant the T1 generation.

[0056] Chlorogenic acid detection in genetically modified tobacco

[0057] One-week-old germinated tobacco varieties (Nta07g03160 and wild-type K326) were planted in culture pots and placed in a nursery at 28℃ with a 16-hour light / 8-hour dark cycle. Watering was done weekly, and no fertilizer was applied. After four weeks, samples were 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 Nta07g03160 tobacco was significantly increased.

Claims

1. The application of the Nta07g03160 gene in increasing the chlorogenic acid content of tobacco, characterized in that, Site-directed mutagenesis of the Nta07g03160 gene in tobacco was performed using gene editing. The nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO:

1. The specific method of gene editing is as follows: using the tobacco Nta07g03160 gene as the target, a CRISPR / Cas9-based sgRNA nucleotide sequence was designed. The DNA fragment containing the above-mentioned sgRNA was ligated into a CRISPR / Cas9 vector and tobacco was transformed to achieve site-directed editing of the tobacco Nta07g03160 gene, resulting in strains with low or no expression of the Nta07g03160 gene.

2. The application according to claim 1, characterized in that, The amino acid sequence of Nta07g03160 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 mutation of the Nta07g03160 gene in tobacco is performed using gene editing. The nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO:

1. The specific method of gene editing is as follows: Using the tobacco Nta07g03160 gene as a target, a CRISPR / Cas9-based sgRNA nucleotide sequence is designed. A DNA fragment containing the encoding the sgRNA is ligated into a CRISPR / Cas9 vector and used to transform tobacco, thereby achieving site-directed editing of the tobacco Nta07g03160 gene and obtaining strains with low or no expression of the Nta07g03160 gene.

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

5. The method according to claim 3, characterized in that, The tobacco mentioned is cultivated tobacco.

6. A method for cultivating a tobacco mutant with high chlorogenic acid content, characterized in that, The method for obtaining the high-chlorogenic acid-content tobacco mutant is as follows: a target gene Nta07g03160 vector 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 Nta07g03160 gene is knocked out at a specific site to obtain the tobacco mutant; the nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO: 1; the amino acid sequence encoded by the Nta07g03160 gene is shown in SEQ ID NO: 2.

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