Tobacco 1-aminocyclopropane-1-carboxylate oxidase gene and application thereof

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to a tobacco 1-aminocyclopropane-1-carboxylate oxidase gene and application thereof, the tobacco 1-aminocyclopropane-1-carboxylate oxidase gene for regulating and controlling the content of reducing sugar and nicotine in tobacco is an Ntab0141030 gene, and the sequence of the gene is shown as SEQ ID NO.1. After the Ntab0141030 gene is translated, the 1-aminocyclopropane-1-carboxylate oxidase gene can be used for regulating and controlling the content of reducing sugar and nicotine in tobacco, and the 1-aminocyclopropane-1-carboxylate oxidase gene can be used for regulating and controlling the content of reducing sugar and nicotine in tobacco. The sequence of the encoded protein is shown as SEQ ID NO.3, the reducing sugar content in tobacco leaves in the mature period of an Ntab0141030 gene knockout editing plant is remarkably higher than that of a control group, and the nicotine content is remarkably lower than that of the control group. The invention provides a new genetic material for cultivating tobacco varieties with remarkably changed nicotine and / or sugar content.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene and application thereof. Background Art

[0002] Improving the aroma quality and taste of tobacco has always been the core of the technical research work of cigarette enterprises. Reducing sugar and nicotine are two important chemical components in tobacco leaves, and their content in tobacco leaves directly affects the quality of cigarette products.

[0003] Reducing sugar can produce pleasant aroma substances. The right amount of reducing sugar can also help neutralize alkaline substances in the smoke and reduce the irritation of the smoke. It can improve the taste of tobacco leaves while making the smoke more mellow. However, when the reducing sugar content is too low, the smoke will appear pungent. Not only that, the reducing sugar content is also closely related to quality indicators such as the color, oil content, and leaf structure of tobacco leaves. Studies have shown that the reducing sugar content in tobacco leaves is positively correlated with the oil score and the leaf structure score. The higher the reducing sugar content, the better the quality of the tobacco leaves. In addition, during the tobacco leaf baking and aging process, reducing sugar can also participate in non-enzymatic browning reactions. The right amount of reducing sugar can improve the appearance and color of tobacco leaves.

[0004] Nicotine content directly affects the smoking quality and strength of tobacco leaves. Moderate nicotine content can give smokers appropriate physiological strength and a pleasant taste experience. Too high or too low nicotine content will have a negative impact on smoke characteristics. For example, too high nicotine content will cause strong smoke, strong irritation, and spicy taste, while too low nicotine content will make the smoke weak and taste bland. In actual production, the overall nicotine content of cigarettes is generally adjusted by mixing tobacco leaves with different nicotine contents in the leaf group formula to control the overall nicotine content of cigarettes.

[0005] The existing technology mainly uses breeding methods to select tobacco varieties with suitable reducing sugar and nicotine 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, new hope has been brought to the technology of regulating the reducing sugar and nicotine content in tobacco, but the technology of accurately regulating the reducing sugar and nicotine content in tobacco by genetic engineering has not been reported.

[0006] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0007] In view of the problem that the reducing sugar content in wild tobacco plants is too low and the nicotine content is too high, the purpose of the present invention is to provide a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene and its application, so as to achieve the synchronous regulation of the reducing sugar content and nicotine content of tobacco, effectively improve the quality of tobacco, and at the same time achieve the purpose of reducing costs and increasing efficiency.

[0008] The present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene, which is named as Ntab0141030 gene, and its nucleotide sequence is shown in SEQ ID NO.1, including 2999bp bases.

[0010] Preferably, the CDS sequence of the Ntab0141030 gene is as shown in SEQ ID No. 2, comprising 963 bp of bases.

[0011] Preferably, the Ntab0141030 gene sequence is translated, and the encoded protein sequence is shown in SEQ ID No. 3, which includes 320 amino acids in total.

[0012] The second aspect of the present invention provides a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene and application thereof, wherein the gene is the Ntab0141030 gene.

[0013] Preferably, the Ntab0141030 gene knockout plant is obtained by constructing a CRISPR / Cas9 editing vector for knocking out the Ntab0141030 gene through CRISPR / Cas9-mediated gene editing technology, and obtaining a plant with the Ntab0141030 gene knocked out after genetic transformation.

[0014] Preferably, the method for creating a plant with knockout of the Ntab0141030 gene specifically comprises the following method:

[0015] Step (1): Select a more specific 23 nt nucleotide sequence in the Ntab0141030 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-Ntab0141030 editing vector;

[0016] Step (2): Use the constructed CRISPR / Cas9-Ntab0141030 editing vector plasmid to perform genetic transformation to knock out the Ntab0141030 gene in the plant and obtain Ntab0141030 gene knockout plants.

[0017] Preferably, using the above technical solution, the Ntab0141030 gene knockout edited tobacco plants have significantly increased reducing sugar content and significantly reduced nicotine content in mature tobacco leaves compared to control tobacco plants.

[0018] Preferably, the tobacco variety is Honghua Dajinyuan.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention uses CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the Ntab0141030 gene, and obtains a tobacco plant with the Ntab0141030 gene knocked out after editing material creation and molecular detection and identification. Compared with the control tobacco plant, the Ntab0141030 gene knocked out edited tobacco plant obtained by the present invention has a significantly increased reducing sugar content and a significantly reduced nicotine content in the mature tobacco leaves.

[0021] (2) The present invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out the Ntab0141030 gene to obtain edited tobacco materials with significantly increased reducing sugar content and significantly reduced nicotine content in mature tobacco leaves. This provides a theoretical basis for further elucidating the regulatory mechanism of tobacco sugar metabolism and nicotine metabolism, and provides new genetic materials for breeding tobacco varieties with significantly altered nicotine and / or sugar content. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison chart of the relative content of reducing sugar in tobacco leaves of gene-edited plants (Ntab0141030) and control plants (CK) at maturity (*p<0.05, **P<0.01);

[0023] Figure 2 Comparison of nicotine content in mature tobacco leaves of gene-edited plants (Ntab0141030) and control plants (CK) (*p<0.05, **P<0.01). DETAILED DESCRIPTION

[0024] The present invention is further described below by way of examples, but is not limited to these examples.

[0025] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0026] In this example, the process of obtaining the tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene Ntab0141030 is briefly introduced as follows.

[0027] 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 (+gDNA wiper) reverse transcription kit reverse transcribes RNA into cDNA for later use.

[0028] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0029] F: 5'-TGCCTTAGGTTACGAAGCGG-3' (SEQ ID No. 5)

[0030] R: 5'-ATACGACGACATTGTGCGGT-3' (SEQ ID No. 6)

[0031] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:

[0032] Amplification system (50 μL):

[0033]

[0034] 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.

[0035] The amplified product was purified and sequenced to obtain the tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene, which was the Ntab0141030 gene, and its nucleotide sequence was shown in SEQ ID NO.1, including 2999bp bases. The CDS sequence of the Ntab0141030 gene was shown in SEQ ID No.2, including 963bp bases. The Ntab0141030 gene sequence was translated, and the protein sequence encoded by it was shown in SEQ ID No.3, including 320 amino acids in total.

[0036] Further comparative analysis showed that the protein contained highly homologous sequences and was highly conserved.

[0037] Example 2

[0038] Using the tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene Ntab0141030 sequence obtained in Example 1, the present invention further constructed a gene editing vector, and used the leaf disc method to transform and cultivate tobacco red flower Dajinyuan to obtain gene-edited plants.

[0039] Using the CRISPR-P 2.0 website, a more specific 23nt nucleotide sequence (SEQID No.4) in the Ntab0141030 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-Ntab0141030 editing vector was obtained.

[0040] Using the CRISPR / Cas9-Ntab0141030 editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, genetic transformation and tissue culture were performed to obtain plants with knockout editing of the tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene Ntab0141030. The relevant experimental process is briefly described as follows:

[0041] (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.

[0042] (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-Ntab0141030 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 to YEB solid culture medium containing 50 mg / L rifampicin, 50 mg / L streptomycin and 50 mg / L kanamycin, and culture in the dark at 28°C for 2-3 days for use.

[0043] (3) A tobacco leaf disk was made into a square disk with a side length of 1 cm in an ultra-clean workbench, and the Agrobacterium colonies containing the CRISPR / Cas9-Ntab0141030 editing vector were prepared into a suspended bacterial liquid (OD600 = 0.6-0.8) using MS liquid.

[0044] The tobacco leaf discs were inoculated with the suspended Agrobacterium solution for 10 minutes, and then placed on MS solid medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA at 28°C in the dark for 3 days.

[0045] Then, subculture was performed and placed on MS solid medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb + 50 mg / L Kan. The culture conditions were: 28°C light culture for 16 h / d, light intensity 30-50 μmol / (m2·s), 25°C dark culture for 8 h / d, culture for 45-60 days, until differentiation buds were formed, the differentiation culture medium was replaced every 7-10 days, and replaced 3-4 times; culture until differentiation buds were formed; the callus with differentiation buds was cut off and placed on MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for culture. When the differentiation buds on the callus grew to 2-4 cm in height, the culture conditions were the same as those for differentiation. The culture conditions were consistent and cultured for 8-14 days. For rooting culture of 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 for culture, and then leaves of transformed plants were sampled and sent to a sequencing company for molecular detection using Ntab0141030-1F / Ntab0141030-1R primers to determine the homozygous edited plants of the Ntab0141030 gene, which were then self-pollinated for seed collection.

[0046] 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 Ntab0141030 gene were obtained. After that, self-pollination was performed to obtain T1 generation seeds (T2 generation).

[0047] Ntab0141030-1F: 5'-GATACGCTTACCCGAATCCCATAGC-3' (SEQ ID No. 7);

[0048] Ntab0141030-1R: 5'-AAATAGCTCGGATATGGGTCGTT-3' (SEQ ID No. 8);

[0049] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 9);

[0050] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 10);

[0051] The leaves of the transformed gene-edited plants were sampled and sent to BGI for molecular testing, which confirmed that the Ntab0141030 gene-edited plants were obtained.

[0052] The application of the tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene Ntab0141030 of the present invention is to reduce the expression of the Ntab0141030 gene in tobacco plants, which can regulate the content of reducing sugar and nicotine in tobacco leaves. The methods of reducing gene expression or gene silencing commonly used in the prior art are applicable to the present invention.

[0053] Example 3

[0054] The seeds obtained by self-pollination of the T1 generation plants with homozygous knockout of the Ntab0141030 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 leaves were sampled for molecular detection to determine the T2 generation plants with homozygous editing of the Ntab0141030 gene without T-DNA. Liquid nitrogen sampling and grinding were performed on the edited material and control tobacco leaves at maturity. The reducing sugar content was determined according to the tobacco industry standard YC / T 159-2019 "Continuous flow method for the determination of water-soluble sugars in tobacco and tobacco products", and the nicotine content was determined according to the gas chromatography method for the determination of nicotine in tobacco and tobacco products.

[0055] Comparison of reducing sugar and nicotine content in leaves of control (unedited) CK and Ntab0141030 gene homozygous edited tobacco plants at maturity (results as shown in Figure 1 , Figure 2 Compared with the control, the reducing sugar content in the leaves of the homozygous Ntab0141030 gene-edited tobacco plants at the mature stage was significantly increased by 17.65%, and the nicotine content was significantly reduced by 85.84%.

[0056] 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.

[0057]

[0058]

[0059]

Claims

1. An application of a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene in regulating the reducing sugar and nicotine content of tobacco, characterized in that: The tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene for regulating the reducing sugar and nicotine content of tobacco is the Ntab0141030 gene, and its sequence is shown in SEQ ID NO.

1. After the Ntab0141030 gene is translated, the sequence of the protein encoded by it is shown in SEQ ID NO.3, wherein the reducing sugar content in the tobacco leaves of the Ntab0141030 gene knockout edited plants at the mature stage is significantly higher than that of the control, and the nicotine content is significantly lower than that of the control.

2. The use of a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene according to claim 1 in regulating the reducing sugar and nicotine content of tobacco, characterized in that: The Ntab0141030 gene knockout plant was produced by using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector was constructed to knock out the Ntab0141030 gene, and plants with the Ntab0141030 gene knocked out were obtained after genetic transformation.

3. The use of a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene in regulating the reducing sugar and nicotine content of tobacco according to claim 2, characterized in that: The method for creating a plant with knockout of the Ntab0141030 gene specifically includes: Step (1): Select a more specific 23 nt nucleotide sequence in the Ntab0141030 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-Ntab0141030 editing vector; Step (2): Use the constructed CRISPR / Cas9-Ntab0141030 editing vector plasmid to perform genetic transformation to knock out the Ntab0141030 gene in the plant and obtain Ntab0141030 gene knockout plants.

4. The use of a tobacco 1-aminocyclopropane-1-carboxylic acid oxidase gene in regulating the reducing sugar and nicotine content of tobacco according to claim 2, characterized in that: The variety of tobacco is Honghua Dajinyuan.

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