Tobacco peroxidase gene and application thereof in regulating and controlling content of cryptochlorogenic acid in tobacco leaves

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to a tobacco peroxidase gene and application of the tobacco peroxidase gene in regulating and controlling the content of cryptochlorogenic acid in tobacco leaves, the tobacco peroxidase gene for increasing the content of cryptochlorogenic acid in tobacco leaves is an Ntab0890720 gene, and the sequence of the tobacco peroxidase gene is shown as SEQ ID NO.1. After the Ntab0890720 gene is translated, the content of cryptochlorogenic acid in the tobacco leaves is increased, and the content of cryptochlorogenic acid in the tobacco leaves is increased. The sequence of the encoded protein is shown as SEQ ID NO.3, and the content of cryptochlorogenic acid in tobacco leaves in the mature period of an Ntab0890720 gene knockout editing plant is remarkably higher than that of a control plant. The content of cryptochlorogenic acid in tobacco leaves can be effectively regulated and controlled, and materials and references are provided for tobacco peroxidase gene research and tobacco quality regulation and control.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco peroxidase gene and application thereof in regulating the content of cryptochlorogenic acid in tobacco leaves. Background Art

[0002] Chlorogenic acid, also known as 4-caffeoylquinic acid, is an organic acid in plants and a natural polyphenol compound. It has special physiological activities such as clearing heat and detoxifying, antibacterial and anti-inflammatory. It can restore the normal physiological function of cells by enhancing endoplasmic reticulum degradation (ERAD), promoting the degradation of misfolded proteins in cells, improving peptide hormone secretion defects, etc.

[0003] Chlorogenic acid in tobacco can act on smoke together with other polyphenols, and is one of the key components that affect the quality of tobacco products. Usually, the content of chlorogenic acid is positively correlated with the quality of tobacco products. The higher the content of chlorogenic acid, the higher the grade of tobacco products. Not only that, an appropriate amount of chlorogenic acid can also work synergistically with other chemical components to achieve a balance between the aroma and taste of tobacco products. Increasing the content of chlorogenic acid can help improve the aroma of tobacco leaves and make cigarette smoke more mellow and delicate. In addition, by regulating the content of chlorogenic acid, it can also help reduce the impurities in tobacco leaves and improve the overall taste of cigarettes.

[0004] However, the content of cryptochlorogenic acid in wild tobacco plants is relatively low, so the existing technology mainly uses traditional breeding methods to select tobacco varieties with appropriate cryptochlorogenic acid content, but this traditional breeding method is time-consuming and labor-intensive, and the effect is not satisfactory. With the development of molecular biology technology, the regulation technology of cryptochlorogenic acid content in tobacco has brought new hope, but the technology of using genetic engineering to accurately regulate cryptochlorogenic acid polyphenols in tobacco has not been reported.

[0005] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0006] In view of the low content of cryptochlorogenic acid in wild tobacco plants and the problem that traditional breeding methods cannot fundamentally increase the content of cryptochlorogenic acid in tobacco, the purpose of the present invention is to provide a tobacco peroxidase gene, which can effectively regulate the content of cryptochlorogenic acid in tobacco leaves, and provide materials and references for tobacco peroxidase gene research and tobacco quality regulation.

[0007] The first aspect of the present invention provides a tobacco peroxidase gene, which is named as Ntab0890720 gene, and its nucleotide sequence is shown in SEQ ID NO.1, including 2449 bp bases.

[0008] Preferably, the CDS sequence of the Ntab0890720 gene is as shown in SEQ ID No. 2, comprising 1023 bp of bases.

[0009] Preferably, the Ntab0890720 gene sequence is translated, and the encoded protein sequence is shown in SEQ ID No. 3, which includes 340 amino acids in total.

[0010] A second aspect of the present invention provides an application of a tobacco peroxidase gene in increasing the content of cryptochlorogenic acid in tobacco leaves, wherein the gene is the Ntab0890720 gene.

[0011] Preferably, the Ntab0890720 gene knockout plant is obtained by constructing a CRISPR / Cas9 editing vector for knocking out the Ntab0890720 gene through CRISPR / Cas9-mediated gene editing technology, and obtaining a plant with the Ntab0890720 gene knocked out after genetic transformation.

[0012] Preferably, the method for creating a plant with knockout of the Ntab0890720 gene specifically comprises the following method:

[0013] Step (1): Select a more specific 23 nt nucleotide sequence in the Ntab0890720 gene as shown in SEQ ID No. 4 as the guide sequence of CRISPR / Cas9, and connect the sequence fragment with the CRISPR / Cas9 vector, transform and PCR amplify the sequence fragment to obtain a PCR positive clone to obtain a CRISPR / Cas9-Ntab0890720 editing vector;

[0014] Step (2): Using the constructed CRISPR / Cas9-Ntab0890720 editing vector plasmid, genetic transformation is performed to knock out the Ntab0890720 gene in the plant to obtain Ntab0890720 gene knockout plants.

[0015] Preferably, using the above technical solution, the content of cryptochlorogenic acid in the mature tobacco leaves of Ntab0890720 gene knockout edited plants is significantly higher than that of the control plants.

[0016] Preferably, the tobacco variety is Honghua Dajinyuan.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention constructs a CRISPR / Cas9 editing vector for knocking out the Ntab0890720 gene through CRISPR / Cas9-mediated gene editing technology, and obtains tobacco plants with Ntab0890720 gene knockout after editing material creation and molecular detection and identification. Compared with the control tobacco plants, the Ntab0890720 gene knockout edited tobacco plants obtained by the present invention have significantly increased cryptochlorogenic acid content in mature tobacco leaves.

[0019] 2. The present invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out the Ntab0890720 gene to obtain edited tobacco materials with significantly increased cryptochlorogenic acid content in tobacco leaves. This provides a theoretical basis for further clarifying the regulatory mechanism of tobacco polyphenol metabolism and provides new genetic materials for breeding tobacco varieties with significantly changed polyphenol content. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the content of cryptochlorogenic acid in mature tobacco leaves of gene-edited plants (Ntab0890720) and control plants (CK) ((*p<0.05)). DETAILED DESCRIPTION

[0021] The present invention is further described below by way of examples, but is not limited to these examples.

[0022] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0023] Example 1

[0024] This example briefly introduces the process of obtaining the tobacco peroxidase Ntab0890720 gene as follows.

[0025] Using leaves of the cultivated tobacco cultivar Nicotiana tabacum Dajinyuan as samples, Vazyme Plant DNA Isolation Mini Kit was used to extract genomic DNA from tobacco leaves; Total RNA was extracted from tobacco leaves using the Super total RNA extraction kit and then Ⅲlst Strand cDNA Synthesis Kit (+gDNAwiper) reverse transcription kit reverse transcribes RNA into cDNA for later use.

[0026] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0027] F: 5'-TCGTCTTGACAGAAGCCACC-3' (SEQ ID No. 5)

[0028] R: 5'-CCTTCAGTAACCGCACTCGT-3' (SEQ ID No. 6)

[0029] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:

[0030] Amplification system (50 μL):

[0031]

[0032] After mixing and centrifuging, PCR amplification was performed. The PCR reaction conditions were: 95°C for 1 min, 95°C for 15 sec, 56°C for 15 sec, 72°C for 3 min / 1 min, for a total of 35 cycles; 72°C for 10 min; 12°C Hold.

[0033] The amplified product was purified and sequenced, and the tobacco peroxidase gene was obtained as Ntab0890720 gene, whose nucleotide sequence is shown in SEQ ID No.1, including 2449bp bases. The CDS sequence of the Ntab0890720 gene is shown in SEQ ID No.2, including 1023bp bases. The Ntab0890720 gene sequence was translated, and the protein sequence encoded by it was shown in SEQ ID No.3, including 340 amino acids. Further comparative analysis showed that the protein contained a highly homologous sequence and was highly conserved.

[0034] Example 2

[0035] Using the Ntab0890720 gene sequence obtained in Example 1, the present invention further constructed a gene editing vector, and obtained gene-edited plants by leaf disc transformation.

[0036] Using the CRISPR-P 2.0 website, a more specific 23nt nucleotide sequence (SEQID No.4) in the Ntab0890720 gene was selected as the guide sequence of CRISPR / Cas9, and the sequence fragment was connected to the CRISPR / Cas9 vector, transformed and PCR amplified. The PCR-positive clones were sent to a sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-Ntab0890720 editing vector was obtained.

[0037] Using the CRISPR / Cas9-Ntab0890720 editing vector plasmid constructed in the previous step, a genetic transformation experiment was carried out using Honghua Dajinyuan as an example to obtain plants with the tobacco peroxidase gene Ntab0890720 knocked out and edited. The relevant experimental process is briefly described as follows:

[0038] (1) After surface disinfection, tobacco seeds were spotted onto MS culture medium. When they grew to 4 cotyledons (15-20 days), they were transferred into culture bottles containing MS solid culture medium and cultured for 35-40 days at 25±1°C, light intensity of 30-50 μmol / (m2·s), and light duration of 16 h / d.

[0039] (2) Take out the LBA4404 electroporation competent Agrobacterium cells stored at -80°C and place them on ice for freezing and thawing. When the competent cells are just thawed, add 2 μL of CRISPR / Cas9-Ntab0890720 editing vector plasmid, mix well, and place on ice. Then transfer the mixed competent cells to a pre-cooled electroporation cup, place the electroporation cup in an electroporator for transformation, add 0.5 mL of YEB liquid culture medium and mix with the transformation liquid after the transformation is completed, and then place on a shaker at 28°C and 200 rpm for 1.5-2 hours. Centrifuge the cells at 8000 rpm and discard the supernatant, then suspend the cells with 200 μL of YEB liquid culture medium, apply them on YEB solid culture medium containing 50 mg / L rifampicin, 50 mg / L streptomycin and 50 mg / L kanamycin, and culture them in the dark at 28°C for 2-3 days for later use.

[0040] (3) A tobacco leaf disk was made into a square disk with a side length of 1 cm in an ultra-clean workbench, and Agrobacterium colonies containing the CRISPR / Cas9-Ntab0890720 editing vector were prepared into a suspended bacterial liquid (OD600 = 0.6-0.8) using MS liquid.

[0041] The tobacco leaf disks were infected with suspended Agrobacterium solution for 10 min, and then placed on MS solid culture medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA at 28°C in the dark for 3 days.

[0042] Then, subculture was performed and placed in a medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb

[0043] +50mg / LKan on MS solid medium, culture conditions are: 28℃ light culture for 16h / d, light intensity 30-50μmol / (m2·s), 25℃ dark culture for 8h / d, culture for 45-60d, until differentiation buds are formed, replace differentiation culture medium every 7-10d, change 3-4 times; culture until differentiation buds are formed; cut the callus with differentiation buds, place it on MS medium containing 500mg / L carbenicillin and 50mg / L kanamycin, and culture until the differentiation buds on the callus grow to 2-4cm high, culture conditions The conditions were consistent with the differentiation culture conditions and cultured for 8-14 days. For the rooting culture of the regenerated plants, the differentiated buds were cut off and inserted into MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for rooting culture. The culture conditions were consistent with the differentiation culture conditions and cultured for 20-30 days. The regenerated plants were transplanted into pots and cultured. The leaves of the transformed plants were sampled and sent to a sequencing company for molecular testing using Ntab0890720-1F / Ntab0890720-1R primers to determine the homozygous edited plants of the Ntab0890720 gene, which were then self-pollinated for seed collection.

[0044] The homozygous edited T0 generation seeds were planted, and when the plants grew to 5-6 leaves, the leaves of each plant were sampled and molecular detection was performed using Cas9F / Cas9R primers to determine whether T-DNA-free plants with homozygous editing of the Ntab0890720 gene were obtained. After that, self-pollination was performed to obtain T1 generation seeds (T2 generation).

[0045] Ntab0890720-1F: 5'-GATAGAATTACCCGAATCCCATGCA-3' (SEQ ID No. 7);

[0046] Ntab0890720-1R: 5'-AAATGCATCGGATTCGGGTAATT-3' (SEQ ID No. 8);

[0047] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 9);

[0048] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 10);

[0049] The leaves of the transformed gene-edited plants were sampled and sent to BGI for molecular testing, which confirmed that the Ntab0890720 gene-edited plants were obtained.

[0050] The application of the tobacco peroxidase gene Ntab0890720 of the present invention is to reduce the expression of the Ntab0890720 gene in tobacco plants, which can regulate the content of cryptochlorogenic acid in the tobacco maturity stage. The methods of reducing gene expression or gene silencing commonly used in the prior art are applicable to the present invention.

[0051] Example 3

[0052] The seeds obtained by self-pollination of the T1 generation plants with homozygous knockout of the Ntab0890720 gene without T-DNA determined by molecular detection in Example 2. The control safflower Dajinyuan and the edited material T2 generation were planted in a greenhouse. When the plants grew to 5-6 leaves, single leaf samples were taken for molecular detection to determine the T2 generation plants with homozygous edited Ntab0890720 gene without T-DNA. Liquid nitrogen sampling and grinding were performed on the edited material and control tobacco leaves at maturity. The content of cryptochlorogenic acid was determined in accordance with the tobacco industry standard YC / T 202-2006 "Determination of chlorogenic acid, scopoletin and rutin, polyphenolic compounds in tobacco and tobacco products".

[0053] Comparison of the content of cryptochlorogenic acid in leaves of control (unedited) CK and Ntab0890720 gene homozygous edited tobacco plants at mature stage. The results are as follows Figure 1 As shown, compared with the control, the content of cryptochlorogenic acid in the leaves of tobacco plants homozygous for Ntab0890720 gene edited at mature stage was significantly increased by 101.01%.

[0054] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0055]

[0056]

[0057]

Claims

1. Application of a tobacco peroxidase gene in regulating the content of cryptochlorogenic acid in tobacco leaves, characterized in that: The tobacco peroxidase gene for increasing the content of cryptochlorogenic acid in tobacco leaves is the Ntab0890720 gene, and its sequence is shown in SEQ ID NO.

1. After translating the Ntab0890720 gene, the sequence of the protein encoded by it is shown in SEQ ID NO.

3. Among them, the content of cryptochlorogenic acid in the tobacco leaves of the Ntab0890720 gene knockout-edited plants at the mature stage is significantly higher than that of the control plants.

2. The use of a tobacco peroxidase gene according to claim 1 in regulating the content of cryptochlorogenic acid in tobacco leaves, characterized in that: Ntab0890720 gene knockout plants were obtained by using CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the Ntab0890720 gene, and after genetic transformation, plants with Ntab0890720 gene knockout were obtained.

3. The use of a tobacco peroxidase gene in regulating the content of cryptochlorogenic acid in tobacco leaves according to claim 2, characterized in that: The method for creating the plant with the Ntab0890720 gene knocked out specifically includes: Step (1): Select a more specific 23 nt nucleotide sequence in the Ntab0890720 gene as shown in SEQ ID No. 4 as the sgRNA sequence of CRISPR / Cas9, and connect, transform and PCR amplify the sequence fragment with the CRISPR / Cas9 vector to obtain a PCR positive clone and obtain a CRISPR / Cas9-Ntab0890720 editing vector; Step (2): Using the constructed CRISPR / Cas9-Ntab0890720 editing vector plasmid, genetic transformation is performed to knock out the Ntab0890720 gene in the plant to obtain Ntab0890720 gene knockout plants.

4. The use of a tobacco peroxidase gene in regulating the content of cryptochlorogenic acid in tobacco leaves according to claim 2, characterized in that: The variety of tobacco is Honghua Dajinyuan.

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