Application of tobacco biosynthetic enzyme gene NtBIO3-BIO1 in regulating tobacco waist leaf length, waist leaf width and polyphenol content

Abstract

The application discloses application of a tobacco biotin synthetase gene NtBIO3-BIO1 in regulation of tobacco waist leaf length, waist leaf width and polyphenol content. The nucleotide sequence of the NtBIO3-BIO1 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the NtBIO3-BIO1 coding protein is shown as SEQ ID NO. 3. The application constructs a CRISPR / Cas9 editing vector for knocking out the NtBIO3-BIO1 gene through a CRISPR / Cas9 mediated gene editing technology, and obtains tobacco plants with the NtBIO3-BIO1 gene knocked out after editing material creation and molecular detection identification. The edited tobacco plants with the NtBIO3-BIO1 gene knocked out obtained by the application have significantly increased waist leaf length and waist leaf width at a mature stage, and significantly reduced chlorogenic acid and total polyphenol content in the waist leaf compared with control tobacco plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of a tobacco biotin synthase gene NtBIO3-BIO1 in regulating the length, width, and polyphenol content of tobacco leaflets. Background Technology

[0002] Biotin (vitamin B7, coenzyme R, or vitamin H) is a water-soluble vitamin essential for the normal metabolism of fats and proteins. In plants, the process of converting desulfurized biotin into biotin occurs in the mitochondria and is a crucial step in biotin synthesis. In Arabidopsis thaliana, diaminononanoate aminotransferase (dapa-at) and desulfurized biotin synthase (DTBS) catalyze the penultimate and penultimate steps of biotin synthesis, respectively. These enzymes are encoded by a bifunctional gene, a chimeric gene named BIO3-BIO1. Mutants of bio1 in Arabidopsis thaliana exhibit chlorosis and fail to produce fertile flowers, while the bio3 mutant displays a phenotype similar to the bio1 auxotrophic form. However, further research revealed a chimeric BIO3-BIO1 transcript, confirming that BIO3-BIO1 possesses a bifunctional site catalyzing two sequential reactions within the same metabolic pathway. All studies on biotin synthesis gene mutants have confirmed that biotin is an essential vitamin for plant growth.

[0003] The area of ​​tobacco leaf lobes and the content of polyphenols are important indicators affecting the quality of tobacco leaves, as well as the appearance and curing properties of tobacco leaves. Therefore, it is of great significance to study the genes that affect the length, width and polyphenol content of tobacco leaf lobes. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide an application of the tobacco biotin synthase gene NtBIO3-BIO1 in regulating the length, width and polyphenol content of tobacco leaflets, providing materials and reference for the study of tobacco biotin synthase protein and the regulation of tobacco quality.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] Application of a tobacco biotin synthase gene NtBIO3-BIO1 in regulating the length, width, and polyphenol content of tobacco leaflets.

[0007] Preferably, the nucleotide sequence of the NtBIO3-BIO1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by NtBIO3-BIO1 is shown in SEQ ID NO.3.

[0008] Preferably, the NtBIO3-BIO1 gene editing is carried out using CRISPR / Cas9-mediated gene editing technology. A CRISPR / Cas9 editing vector for knocking out the NtBIO3-BIO1 gene was constructed, and after genetic transformation, homozygous tobacco plants with T-DNA-free NtBIO3-BIO1 gene editing were obtained.

[0009] Preferably, the method for creating tobacco plants with NtBIO3-BIO1 gene editing includes:

[0010] (1) Select a relatively specific 20nt nucleotide sequence in the NtBIO3-BIO1 gene as the guide sequence for CRISPR / Cas9, and ligate, transform and PCR amplify the sequence fragment with the CRISPR / Cas9 vector (pOREU3TR) to obtain PCR positive clones and obtain the pOREU3TR-NtBIO3-BIO1 editing vector.

[0011] (2) Using the constructed pOREU3TR-NtBIO3-BIO1 editing vector, genetic transformation and tissue culture were carried out. After self-pollination, T2 generation tobacco plants with T-DNA knockout editing of the tobacco NtBIO3-BIO1 gene were obtained.

[0012] Preferably, the more specific 20nt nucleotide sequence in the NtBIO3-BIO1 gene in step (1) is shown in SEQ ID No. 4.

[0013] Preferably, agronomic traits and polyphenol content were investigated in the edited plants and control plants at maturity. The results showed that the length and width of the leaf blades of the NtBIO3-BIO1 gene-edited plants were significantly greater than those of the control, while the contents of chlorogenic acid and total polyphenols in the leaf blades were significantly lower than those of the control.

[0014] The above-described technical solution of the present invention has the following beneficial effects:

[0015] (1) This invention utilizes CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the NtBIO3-BIO1 gene. After the creation of the editing material and molecular detection and identification, tobacco plants with the NtBIO3-BIO1 gene knocked out were obtained. Compared with control tobacco plants, the edited tobacco plants with the NtBIO3-BIO1 gene knocked out by this invention showed significantly increased length and width of the middle leaves at maturity, and significantly reduced chlorogenic acid and total polyphenol content in the middle leaves.

[0016] (2) The gene-edited tobacco material obtained by knocking out the NtBIO3-BIO1 gene using CRISPR / Cas9-mediated gene editing technology provides a theoretical basis for further elucidating the regulatory mechanism of tobacco leaf length, leaf width and metabolites such as polyphenols, and provides new genetic material for breeding tobacco varieties with altered leaf area and aroma flavor. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0018] Figure 1 The length and width of the middle leaf of a mature tobacco plant are shown in Figure 1 (*, P<0.05). Figure 2 The polyphenol content of mature tobacco plants is shown in Figure 1 (*, P<0.05; **, P<0.01). Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0020] Unless otherwise specified, all experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] Unless otherwise stated, in this invention, the percentage sign refers to volume percentage, and the ratio refers to volume ratio.

[0022] The tobacco variety used in this application is Honghua Dajinyuan, a commercially available tobacco variety.

[0023] Example 1

[0024] The sequence information of the biotin synthase gene NtBIO3-BIO1 of the tobacco cultivar 'Honghua Dajinyuan' was obtained by searching the Chinese Tobacco Genome Database using the gene function "BIO3-BIO1".

[0025] Its DNA sequence, as shown in SEQ ID No. 1, comprises 10076 bp, and its CDS sequence, as shown in SEQ ID No. 2, comprises 1566 bp. The protein sequence encoded by this gene sequence, as shown in SEQ ID No. 3, comprises 521 amino acids.

[0026] Example 2

[0027] Using the NtBIO3-BIO1 sequence of the tobacco biotin synthase-related gene obtained in Example 1, this invention further constructed a gene editing vector and used the leaf disc method to transform cultivated tobacco safflower to obtain gene-edited plants.

[0028] Using the CRISPR-P 2.0 website, a relatively specific 20nt nucleotide sequence (SEQ ID No. 4) from the NtBIO3-BIO1 gene was selected as the CRISPR / Cas9 guide sequence. This sequence fragment was then ligated into the CRISPR / Cas9 vector (pOREU3TR), transformed, and amplified by PCR. PCR-positive clones were sent to a sequencing company for sequencing confirmation, and finally, the pOREU3TR-NtBIO3-BIO1 editing vector was obtained.

[0029] Using the pOREU3TR-NtBIO3-BIO1 editing vector plasmid constructed in the previous step, taking safflower 'Da Jin Yuan' as an example, genetic transformation and tissue culture were carried out to obtain plants with the NtBIO3-BIO1 gene related to tobacco biotin synthase knocked out. The relevant experimental process is briefly described below.

[0030] After surface sterilization, tobacco seeds are sown on MS medium. Once they have grown to 4 cotyledons (15-20 days), they are transferred to culture flasks containing MS solid medium and cultured for 35-40 days at 25±1℃, light intensity of 30-50 μmol / (m2·s), and light duration of 16 h / d.

[0031] Remove LBA4404 electroporation competent Agrobacterium cells stored at -80℃ and freeze-thaw them on ice. When the competent cells are just thawed, add 2 μL of the pOREU3TR-NtBIO3-BIO1 editing vector plasmid, mix well, and place on ice. Then transfer the mixed competent cells to pre-chilled electroporation cuvettes and perform transformation in an electroporator. After transformation, add 0.5 mL of YEB liquid medium and mix with the transformation solution, then incubate on a shaker at 28℃ and 200 rpm for 1.5-2 h. Centrifuge the cells at 8000 rpm and discard the supernatant. Resuspend the cells in 200 μL of YEB liquid medium and plate them onto YEB solid medium containing 50 mg / L rifampin, 50 mg / L streptomycin, and 50 mg / L kanamycin. Incubate in the dark at 28℃ for 2-3 days.

[0032] Tobacco leaf discs were prepared in a clean bench, forming rectangular discs with a side length of 1 cm. Agrobacterium colonies containing the pOREU3TR-NtBIO3-BIO1 editing vector were prepared as a suspension using MS liquid (OD600 = 0.6-0.8). The tobacco leaf discs were then immersed in the Agrobacterium suspension for 10 min. Afterward, the discs were placed on MS solid medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA and co-cultured at 28°C in the dark for 3 days. Subculture was then performed on medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb + 50 mg / L... On Kan's MS solid medium, the culture conditions were as follows: 28℃ light for 16 h / d, light intensity 30-50 μmol / (m2·s), 25℃ dark for 8 h / d, for 45-60 days until differentiated shoots formed. The differentiation culture medium was changed every 7-10 days, for 3-4 times. Callus tissue with differentiated shoots was excised and placed on MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for further culture. When the differentiated shoots on the callus tissue grew to 2-4 cm in height, the culture conditions were the same as before. Differentiation culture conditions were consistent, with a culture period of 8-14 days. For rooting culture of regenerated plants, differentiated buds were cut and inserted into MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin, under the same conditions as differentiation culture, for 20-30 days. Regenerated plants were then transplanted into pots and cultured. Leaf samples were taken from the transformed plants, and molecular analysis was performed using NtBIO3-BIO1F / NtBIO3-BIO1R primers. This confirmed the acquisition of homozygous NtBIO3-BIO1 gene-edited plants, which were then self-pollinated for seed collection. The homozygous edited T0 generation seeds were planted, and when the plants had 5-6 leaves, leaf samples were taken from individual plants. Molecular analysis was performed using Cas9F / Cas9R primers to confirm the acquisition of plants without T-DNA due to homozygous NtBIO3-BIO1 gene editing. These plants were then self-pollinated for seed collection to obtain T1 generation seeds (T2 generation).

[0033] NtBIO3-BIO1F: 5'-GGGGCATGCTGTAACTCCAT-3' (SEQ ID No. 5);

[0034] NtBIO3-BIO1R: 5'-TAAGCCCCAGTTCGCAGATG-3' (SEQ ID No. 6);

[0035] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 7);

[0036] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 8);

[0037] The application of the tobacco biotin synthase gene NtBIO3-BIO1 described in this invention involves reducing the expression of the NtBIO3-BIO1 gene in tobacco plants, which can regulate the length and width of the middle leaves and the content of polyphenols at the tobacco maturity stage. Commonly used methods in the prior art for reducing gene expression or silencing genes are applicable to this invention.

[0038] Example 3

[0039] Seeds were obtained from self-pollination of T1 generation plants (identified by molecular detection in Example 2 as homozygous knockout of the NtBIO3-BIO1 gene without T-DNA). The control plant, *Sedum rubrum*, and the edited material, T2 generation, were planted in a greenhouse. When the plants reached 5-6 leaves, leaf samples were taken from individual plants for molecular detection, confirming them as T2 generation plants with homozygous editing of the NtBIO3-BIO1 gene without T-DNA. At maturity, the main agronomic and botanical traits of the edited material and control were investigated according to the industry standard *YCT 142-2010 Tobacco Agronomic Trait Survey and Measurement Methods*, including plant height, internode distance, stem circumference, number of effective leaves, mid-leaf length, and mid-leaf width. Liquid nitrogen samples were taken from the mid-leaf of both the edited material and control plants for polyphenol content detection.

[0040] The results of the comparison between the length and width of the lateral leaves at maturity in control (unedited) and NtBIO3-BIO1 gene plants are as follows: Figure 1 As shown in the figure, compared with the control, the width and length of the middle leaves of tobacco plants edited with the NtBIO3-BIO1 gene were significantly increased at maturity.

[0041] Example 4

[0042] The content of polyphenols in the leaves of *Phyllostachys edulis* was determined using the samples collected in Example 3 by high performance liquid chromatography-ultraviolet detection. The specific method is as follows.

[0043] Accurately weigh 0.2 g of each phenolic substance standard, accurate to 0.0001 g, dissolve and dilute to 100 mL in a brown volumetric flask, mix well to obtain a mixed standard stock solution with a concentration of 2 mg / mL. Store in a sealed container at -20℃ for later use. Dilute the mixed standard stock solution to obtain a series of phenolic substance standard working solutions with concentrations of 400, 200, 100, 80, 60, 40, 20, and 10 mg / mL.

[0044] After freeze-drying the collected samples, they were ground into powder. 50 mg of fresh tobacco powder was accurately weighed and added to 5 mL of extraction solution (methanol / water (4:1, v / v)). The mixture was vortexed for 5 s and then ultrasonically extracted for 20 min. The mixture was centrifuged at 10,000 rpm for 7 min. The supernatant was filtered through a 0.22 mm organic filter membrane and analyzed by HPLC-UV.

[0045] Reference conditions for high performance liquid chromatography-ultraviolet detection:

[0046] —Chromatographic column: C18 column, with specifications of [250mm (length) × 4.6mm (inner diameter) × 5mm (particle size)];

[0047] —Mobile phase A: 10% methanol - 2% acetic acid - 88% water;

[0048] —Mobile phase B: 88% methanol - 2% acetic acid - 10% water;

[0049] —Column temperature: 30℃;

[0050] —Column flow rate: 0.8 mL / min;

[0051] —Injection volume: 5 mL.

[0052] —Detection wavelength: 340nm.

[0053] —The elution gradient of the mobile phase is shown in Table 1.

[0054] Table 1 Gradient elution program

[0055]

[0056] Polyphenol content in mature leaves of tobacco plants (unedited) and NtBIO3-BIO1 gene-edited plants is as follows: Figure 1 As shown in the figure. Compared with the control, the length and width of the middle leaves of NtBIO3-BIO1 gene-edited tobacco plants (bio3-bio1) at maturity were significantly increased, while the contents of chlorogenic acid and total polyphenols in the middle leaves were significantly decreased, as was the content of rutin.

[0057] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A tobacco biotin synthase gene NtBIO3-BIO1 Its application in regulating the length and width of tobacco leaflets, as well as the content of chlorogenic acid and total polyphenols, is characterized by... NtBIO3-BIO1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. NtBIO3-BIO1 The amino acid sequence encoding the protein is shown in SEQ ID NO.3; the regulation is knockout. NtBIO3-BIO1 The gene knocked out resulted in tobacco plants with increased leaf length and width at maturity, and decreased chlorogenic acid and total polyphenol content in the leaves.

2. The application according to claim 1, characterized in that, Knockout NtBIO3-BIO1 The gene was constructed using CRISPR / Cas9-mediated gene editing technology to knock out... NtBIO3-BIO1 Gene editing vectors, after genetic transformation, yielded homozygous T-DNA-free genes. NtBIO3-BIO1 Gene knockout tobacco plants.

3. The application according to claim 2, characterized in that, Knockout NtBIO3-BIO1 The methods for creating genetically modified tobacco plants include: (1) selection NtBIO3-BIO1 The specific 20nt nucleotide sequence in the gene is the guide sequence of CRISPR / Cas9. This sequence fragment was ligated to the CRISPR / Cas9 vector, transformed, and amplified by PCR to obtain PCR-positive clones, resulting in the pOREU3TR-NtBIO3-BIO1 editing vector. (2) Using the constructed pOREU3TR-NtBIO3-BIO1 editing vector, genetic transformation and tissue culture were performed, and homozygous tobacco without T-DNA was obtained through self-crossing. NtBIO3-BIO1 T2 generation tobacco plants that were knocked out.

4. The application according to claim 3, characterized in that, In step (1), the CRISPR / Cas9 vector is pOREU3TR.

5. The application according to claim 3, characterized in that, In step (1) NtBIO3-BIO1 The gene-specific 20nt nucleotide sequence is shown in SEQ ID No. 4.

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