Application of tobacco Nta07g03160 gene in increasing content of chlorogenic acid in tobacco

Abstract

The invention belongs to the technical field of tobacco germplasm improvement, and mainly relates to application of a tobacco Nta07g03160 gene in increasing the content of chlorogenic acid in tobacco. According to the invention, the tobacco Nta07g03160 gene is taken as a target, a gRNA sequence of CRISPR / Cas9 is designed, a DNA fragment containing the gRNA is connected into a CRISPR / Cas9 carrier, then tobacco is transformed, an editing material of the tobacco Nta07g03160 gene is obtained, and it is found that the editing material can increase the content of chlorogenic acid in tobacco.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco germplasm improvement, and mainly relates to the application of tobacco Nta07g03160 gene in improving the content of chlorogenic acid in tobacco. Background Art

[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 important medicinal value. Statistics show that 170 Chinese patent medicines with heat-clearing, detoxifying, antibacterial and anti-inflammatory effects included in the "Drug Standards" all contain chlorogenic acid as the main ingredient. On the other hand, as a new type of antioxidant, chlorogenic acid also has important applications in food and fruit preservation, sun protection and skin care.

[0003] According to current research, there are three main secondary metabolic pathways in plants: phenylpropanoid metabolic pathway, isoprene metabolic pathway and alkaloid synthesis pathway. The main substances that affect the aroma of tobacco (phenolic compounds, terpenoid compounds and alkaloids) are also formed through the above three pathways. In the phenylpropanoid metabolic pathway, shikimic acid from the shikimic acid pathway is converted into phenylalanine through the transamination of branched acid prephenylpropionic acid, thereby entering the phenylpropanoid metabolic pathway, thereby increasing the content of chlorogenic acid in tobacco and improving the quality of tobacco.

[0004] The study showed that recombinant Nta07g03160 had a similar level of magg lipase activity as HsMAGL. Nta07g03160 and HsMAGL also had no hydrolase activity on LPC and LPE substrates. In addition, the major expression pattern of Nta07g03160 in pollen and germinating seeds, the co-expression of Nta07g03160 with genes related to storage oil decomposition, and the localization of AtMAGL8 on the surface of oil bodies in germinating seeds and leaves accumulating oil bodies suggested that Nta07g03160 may be involved in TAG decomposition and / or membrane lipid remodeling during pollen tube growth, seed germination, and early seedling establishment. Nta07g03160 showed similar specific activity to HsMAGL in adipose tissue, and Nta07g03160 is the best candidate to date for involvement in storage lipid decomposition during seed germination, but the existing research on editing Nta07g03160 to increase chlorogenic acid content has not been reported. Summary of the invention

[0005] The main technical problem solved by the present invention is: how to edit the Nta07g03160 gene of tobacco by means of genetic engineering, thereby increasing the content of chlorogenic acid in tobacco and improving the quality of tobacco.

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

[0007] ATGAGGCCATCTCCAATTGTGATAAGTATTTTGACCAAACTTTGCAACAT

[0008] TATCCCAACCTGGAAACTAATTCCTACTCAAGATGTTGTTGATTCTGCCT

[0009] TCAGAGACCCTGAAGTTAGAAAAGAGATAAGAAACAATCCATATTGCTAC

[0010] AAGGGACGACCTCGTCTACAAACTGGGTACCAACTGTTTACTGTTAGCAT

[0011] GGATTTGGAGCAAAGACTTGAGGAAGTTACATTGCCATTCTAATTGTAC

[0012] ATGGAGGAGAGGACACAGTTACTGATCCATCAGTGAGTAAACTTCTATAC

[0013] GAAAAGGCCTCGAGCATCGACAAGAGCTTCAAGTTGTATCCAGGAATGTG

[0014] GCATTCCCTTTCCTATGGTGAATTTCCTGAGAACAGAAACATCGTGTTTT

[0015] CGGACATTATCAGTTGGCTCAATGAGAAAATCAGTATGGGCAATTCAAGG

[0016] CTGGAGAGACAACAAAAGCATGCAAATGACAATTTATCAAAGATCAGTAG

[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 of tobacco, the method comprising: performing site-directed mutation on the Nta07g03160 gene in tobacco by 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: taking the tobacco Nta07g03160 gene as the target, designing a sgRNA nucleotide sequence based on CRISPR / Cas9, and connecting a DNA fragment encoding the above sgRNA into a CRISPR / Cas9 vector to transform tobacco, thereby achieving site-specific editing of the tobacco Nta07g03160 gene and obtaining a strain 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: using the CRISPR / Cas9 gene knockout system to construct a target gene Nta07g03160 vector, using an Agrobacterium-mediated method to transform the expression vector into tobacco callus, and performing site-specific knockout of the Nta07g03160 gene to obtain a 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.

[0025] Beneficial effects of the present invention:

[0026] The present invention takes the tobacco Nta07g03160 gene as the target, designs the gRNA sequence of CRISPR / Cas9, connects the DNA fragment encoding the above gRNA into the CRISPR / Cas9 vector, and then transforms the tobacco to obtain the editing material of the tobacco Nta07g03160 gene, and finds that it can increase the content of chlorogenic acid in tobacco.

[0027] The inventors previously obtained the Nta07g03160 tobacco mutant, planted the first generation pure Nta07g03160 tobacco mutant and K326 tobacco, and took tobacco tissues from the same leaf position for detection when they grew to the 5-6 leaf stage. 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 increased compared with that of the K326 tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 Identification of Nta07g03160 gene mutant materials; a, sequencing and identification of the five mutants obtained; b, deletion site of Nta07g03160 tobacco mutant;

[0030] Figure 3 Liquid chromatography mass spectrometry of Nta07g03160 tobacco mutant and K326 tobacco, and bar graph of chlorogenic acid content. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0033] The tobacco ecotype is K326, the Agrobacterium strain is EHA105, and the vector is pCBSG012-slu-DSG. The main reagents include: restriction endonucleases from Thermo Fisher Biotechnology, DNA polymerase and Infusion ligase from Novazonics; reverse transcription kit from Thermo; RNA extraction kit from Tiangen; plasmid extraction kit and DNA recovery kit from Tiangen; quantitative PCR reagents from Taraka; MS culture medium, agar powder, agarose, ampicillin, kanamycin, rifampicin and other antibiotics were purchased from Sigma; various other chemical reagents used in the examples were imported or domestic analytical reagents; primer synthesis and sequencing were completed by Beijing Qingke Biotechnology Co., Ltd.

[0034] Using the reported Arabidopsis genes as query sequences, sequence alignment was performed from the tobacco K326 genome, and then the genes that did not contain the Hydrolases domain were eliminated by SMART, and a total of 29 Nta07g03160 genes were identified in tobacco. Multiple sequence alignments were performed using the reported MAGLs in Arabidopsis and the newly identified NtMAGLs in tobacco, and a phylogenetic tree was constructed to analyze their evolutionary relationships. Among them, Nta07g03160 in tobacco and AT2G39410.2, AT2G39420.1, AT3G55190.1, AT3G55180.1, and AT2G39400.1 in Arabidopsis were clustered together on the evolutionary tree, suggesting that they are more homologous in evolution ( Figure 1 ). The nucleotide sequence of Nta07g03160 gene is shown in SEQ ID NO.1:; The amino acid sequences encoded by Nta07g03160 gene are shown in SEQ ID NO.2.

[0035] According to the Nta07g03160 gene sequence, the Huazhong Agricultural University CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) was used to design a pair of gRNAs 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 50μL PCR system is as follows: 20μL Nuclease-free Water, 25μL Pfu PCR Mix, 2μL each of 100μM upstream and downstream primers, and 1μL template. Perform 94℃5min, 94℃30s, 50℃45s, 72℃6s in a PCR instrument, and cycle 35 times. Perform 1.5% agarose gel electrophoresis at 5v / cm for 20 minutes, cut out the electrophoresis fragment under ultraviolet light, place it in each system for sol recovery, dissolve the recovered DNA in a total volume of 30μL of water, and connect it to the vector after testing.

[0038] The fragment amplified by PCR was connected to the vector. The connection system was: 1 μL Nuclease-free Water, 2 μL Recombination Buffer, 4 μL PCR product, 2 μL linearized vector template, 1 μL recombinase. The PCR instrument ran the recombination program at 37°C for 30 minutes and then stored on ice or at 4°C.

[0039] (2) Transformation of Escherichia coli

[0040] 10μl or more ligation product + 100μl competent E. coli. After taking the competent DH5α out of the refrigerator, quickly put it on ice. After 5 minutes, wait for the bacterial block to dissolve and add the ligation product. Let it stand on ice for 25 minutes, heat shock at 42℃ for 45s, let it stand on ice for 2 minutes (do not shake), add 100μl LB without antibiotics, shake at 37℃, 200rpm, for 1 hour, spread the plate, LB+bacterial antibiotics, and culture at 37℃ for one day. Select the colonies for colony PCR identification, and the positive colonies are sequenced for identification.

[0041] (3) Agrobacterium transformation

[0042] 1: 20μl Agrobacterium (EHA105-WM) + 1μl plasmid, ice bath for 5min, quick freeze in liquid nitrogen for 5min, 37℃ water bath for 5min, ice bath for 5min. Add 100μl antibiotic-free LB, shake at 28℃ 200rpm for 2h, directly apply on the plate with corresponding bacterial antibiotics + rifampicin, and culture at 28℃ for two days.

[0043] 2: Pick a single clone of bacteria:

[0044] Two days later, pick a single clone and put it into a 5 ml sterile EP tube, add 2 ml of the corresponding bacterial antibiotic + rifampicin in advance, and shake the bacteria overnight.

[0045] 3: Preserve glycerol bacteria: 400ul bacterial solution + 100ul 75% sterile glycerol, mark well, and store in a -70 degree refrigerator. Use the remaining bacterial solution to extract the plasmid and transform E. coli (1ul plasmid + 20μl competent E. coli, stand on ice for 30min, heat shock at 42℃ for 35s, stand on ice for 2min, add 100μl LB without antibiotics, shake at 37℃, 200rpm, for 1h, plate, LB + corresponding bacterial antibiotics, and culture at 37℃ for one day).

[0046] 4: Pick a single clone and shake the bacteria in the morning, send the bacterial solution directly for sequencing in the evening, and select part of it for sequencing.

[0047] 5: The sequencing feedback is correct, verifying that there is no problem with Agrobacterium, and the prepared Agrobacterium can be used for the subsequent transformation experiment

[0048] (4) Transformation of Nta07g03160 editing vector

[0049] Pick the activated Agrobacterium in 50mL Kan+Rif resistant liquid LB medium, shake and culture at 28℃, 180rpm until OD600 is close to 0.6, transfer the bacterial solution to a pre-sterilized and pre-cooled 50mL centrifuge tube, centrifuge at 4000rpm for 10min, discard the supernatant to collect the bacteria. Resuspend the bacteria in 20mL pre-cooled MS liquid medium, centrifuge again at 4000rpm for 10min, discard the supernatant to collect the bacteria. Resuspend the bacteria in pre-cooled MS liquid medium to OD600 to 0.6, then add AS at a final concentration of 20mg / L to prepare for infection. Use scissors to cut off the edges of the sterile leaves on the clean bench, cut the leaves into 1.0×1.0cm leaf discs along the main veins and put them into the Agrobacterium infection solution for infection. The infection time is 5min. The infected leaves were removed, the Agrobacterium bacterial solution was dried on sterile filter paper, and the leaves were spread flat on the co-cultivation medium (G) with the leaf surface facing downward, and placed in an artificial climate chamber (temperature 26°C, humidity 40%) for dark cultivation for 3 days.

[0050] S1 subculture. After 3 days of co-culture, transfer the leaves with the leaf surface facing up to S1 differentiation medium. Wash the explants with sterile water containing 500 mg / L Cef, blot dry with sterile filter paper, and then transfer to S1 medium. Place 5-8 pieces per dish, culture in the dark in an artificial climate chamber for about 1 week, and continue to culture under light until clusters of buds grow on the edge of the leaves and the buds grow to about 0.5 cm.

[0051] S2 subculture. Use tweezers to transfer the buds grown on S1 to the S2 differentiation medium. If the buds can be broken off, inoculate them directly. If they cannot be broken off directly, inoculate them together with the leaves and remove the leaves that have not grown buds. Culture in the light for 1-2 weeks until the buds grow into seedlings.

[0052] S3 subculture. Transfer the seedlings on S2 to S3 differentiation medium and culture them in the light for 1-2 weeks. Rooting culture. Remove the swollen part at the bottom and the yellow leaves at the bottom of the healthy seedlings on S3, and then inoculate them into tissue culture bottles containing rooting medium R and culture them in the light for 1-2 weeks to make the seedlings take root as soon as possible.

[0053] Obtaining transgenic tobacco. When the seedlings have 3-10 roots and the root length is about 2-3 cm, open the lid of the culture bottle to harden the seedlings. After about 3 days, transplant the seedlings into a nutrient pot filled with sterile soil and cover with plastic film to keep it moist. After about a week, remove the plastic wrap according to the growth status of the seedlings, so that they can grow rapidly under natural conditions.

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

[0055] The 6 T0 generation transgenic materials transformed with vector 1 were tested and sent to Qingke Company for sequencing after PCR amplification. Several plants with mutations in the T0 generation were harvested and sown for the T1 generation.

[0056] Detection of Chlorogenic Acid in Transgenic Tobacco

[0057] Nta07g03160 tobacco and wild-type K326 tobacco were planted in culture pots that had been germinated for one week and placed in a seedling room at 28°C, 16h light / 8h dark. They were watered once a week without applying fertilizer. After 4 weeks, samples were taken and sent to Shanghai Zhuocai Biotechnology Co., Ltd. for liquid chromatography testing. The results were as follows: Figure 3 As shown, it was found that the chlorogenic acid content in Nta07g03160 tobacco was significantly increased.

Claims

1. Application of Nta07g03160 gene in increasing the content of chlorogenic acid in tobacco, characterized in that: The nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO:

1.

2. The use 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 content of chlorogenic acid in tobacco, characterized in that: The method comprises: performing site-directed mutation on the Nta07g03160 gene in tobacco by gene editing, wherein the nucleotide sequence of the Nta07g03160 gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that The specific method of gene editing is: taking the tobacco Nta07g03160 gene as the target, designing the sgRNA nucleotide sequence based on CRISPR / Cas9, connecting the DNA fragment containing the encoding of the above sgRNA into the CRISPR / Cas9 vector to transform the tobacco, thereby achieving site-specific editing of the tobacco Nta07g03160 gene and obtaining a strain with low or no expression of the Nta07g03160 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 cultivated tobacco.

7. A method for cultivating a high-chlorogenic acid tobacco mutant, characterized in that: The method for obtaining the high-chlorogenic acid tobacco mutant is as follows: using the CRISPR / Cas9 gene knockout system to construct a target gene Nta07g03160 vector, using an Agrobacterium-mediated method to transform the expression vector into tobacco callus, and performing site-specific knockout of the Nta07g03160 gene to obtain a tobacco mutant; the nucleotide sequence of the Nta07g03160 gene is as shown in SEQ ID NO: 1; the amino acid sequence encoded by the Nta07g03160 gene is as shown in SEQ ID NO: 2.

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