Application of a tobacco alcohol dehydrogenase-related gene NtADH1 in regulating tobacco waist leaf width

Abstract

The present invention discloses an application of tobacco alcohol dehydrogenase-related gene NtADH1 in regulating tobacco waist leaf width. The nucleotide sequence of the NtADH1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the NtADH1 encoded protein is shown in SEQ ID NO.3. The present invention constructs a CRISPR / Cas9 editing vector for knocking out the NtADH1 gene through CRISPR / Cas9-mediated gene editing technology, and obtains tobacco plants with NtADH1 gene knockout after editing material creation and molecular detection and identification. Compared with control tobacco plants, the edited tobacco plants with NtADH1 gene knockout obtained by the present invention have significantly increased average waist leaf width at maturity. It provides a theoretical basis for further clarifying the mechanism of tobacco waist leaf width regulation and provides new genetic materials for cultivating tobacco varieties with significantly changed waist leaf width.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to an application of a tobacco alcohol dehydrogenase-related gene NtADH1 in regulating tobacco waist leaf width. Background Art

[0002] Alcohol dehydrogenase (ADH) belongs to the medium-chain dehydrogenase / reductase protein family. It is a zinc-binding enzyme that can catalyze the reversible reaction between ethanol and acetaldehyde with NAD as a coenzyme. ADH is widely distributed in plants and plays an important role in plant growth and development and stress resistance. With the deepening of research, the role of ADH in plant aroma synthesis has gradually received attention. Plant aroma substances mainly include esters, alcohols, aldehydes and terpenes. Aliphatic, branched or aromatic aldehydes in fatty acid metabolism pathways and amino acid metabolism pathways generate corresponding alcohols under the action of ADH, and finally synthesize carbonyl compounds, acids and esters. Previous studies on ADH1 mainly focused on the resistance function in biological and abiotic stresses (salt stress, drought stress, cold stress and pathogen infection) and the role of aroma components in plant fruits, while its influence on the agronomic traits of plants has been less studied.

[0003] The length and width of tobacco loin are important indicators affecting tobacco yield, and are also factors affecting the appearance quality and curing properties of tobacco leaves. Therefore, it is of great significance to study the related genes that affect the area of ​​tobacco loin. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of tobacco alcohol dehydrogenase related gene NtADH1 in regulating tobacco waist leaf width, so as to provide materials and references for tobacco alcohol dehydrogenase protein research and tobacco quality regulation.

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

[0006] Application of a tobacco alcohol dehydrogenase-related gene NtADH1 in regulating tobacco waist leaf width.

[0007] Preferably, the nucleotide sequence of the NtADH1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the NtADH1 encoded protein is shown as SEQ ID NO.3.

[0008] Preferably, NtADH1 gene editing is carried out through CRISPR / Cas9-mediated gene editing technology, a CRISPR / Cas9 editing vector for knocking out the NtADH1 gene is constructed, and after genetic transformation, homozygous T-DNA-free NtADH1 gene-edited tobacco plants are obtained.

[0009] Preferably, the method for creating a tobacco plant with edited NtADH1 gene comprises:

[0010] (1) Selecting a more specific 20 nt nucleotide sequence in the NtADH1 gene as the guide sequence of CRISPR / Cas9, and connecting the sequence fragment with the CRISPR / Cas9 vector, transforming and PCR amplification detection, obtaining PCR positive clones, and obtaining the CRISPR / Cas9-NtADH1 editing vector;

[0011] (2) The constructed CRISPR / Cas9-NtADH1 editing vector was used for genetic transformation and tissue culture, and after self-pollination, T2 generation tobacco plants with homozygous tobacco NtADH1 gene knockout editing without T-DNA were obtained.

[0012] Preferably, the more specific 20 nt nucleotide sequence in the NtADH1 gene in step (1) is as shown in SEQ ID No.6.

[0013] Preferably, agronomic and botanical traits of the edited plants and control plants are investigated at the budding stage and the late topping stage, and the results show that the waist leaf width of the NtADH1 gene homozygous edited tobacco plants at maturity is significantly widened.

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

[0015] (1) The present invention uses CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the NtADH1 gene, and obtains a tobacco plant with the NtADH1 gene knocked out after editing material creation and molecular detection and identification. The NtADH1 gene knocked out edited tobacco plant obtained by the present invention has a significantly increased average waist leaf width at maturity compared to the control tobacco plant.

[0016] (2) The present invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out the NtADH1 gene to obtain edited tobacco materials with significantly increased waist leaf width, which provides a theoretical basis for further clarifying the regulation mechanism of tobacco waist leaf width and provides new genetic materials for breeding tobacco varieties with significantly changed waist leaf width. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 The length and width of waist leaves of mature tobacco plants (p<0.01). DETAILED DESCRIPTION

[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 the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0020] All experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0021] Unless otherwise specified in the present invention, percentages refer to volume percentages and ratios refer to volume ratios.

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

[0023] Example 1

[0024] 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 1st Strand cDNA Synthesis Kit (+gDNA wiper) reverse transcription kit reverse transcribes RNA into cDNA for later use.

[0025] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0026] NtADH1F: 5'-ATGTCAAGCAATACTGCTGGG-3' (SEQ ID No. 4);

[0027] NtADH1R: 5'-TCAATGCTCCATAGTGATCATG-3' (SEQ ID No. 5);

[0028] Using the DNA and cDNA prepared above as templates, PCR amplification was performed using the above primers:

[0029] Amplification system (50 μL):

[0030]

[0031] 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; and 12°C Hold.

[0032] The amplified product was purified and sequenced to obtain the sequence of tobacco alcohol dehydrogenase-related gene NtADH1, whose DNA sequence is shown in SEQ ID No.1, including 3355bp bases, and its CDS sequence is shown in SEQ ID No.2, including 1146bp bases. After translating the gene sequence, the encoded protein sequence is shown in SEQ ID No.3, including 381 amino acids. Further comparative analysis shows that the protein contains highly homologous sequences and is highly conserved.

[0033] Example 2

[0034] Using the gene NtADH1 sequence of tobacco alcohol dehydrogenase obtained in Example 1, the present invention further constructed a CRISPR / Cas9 vector, and obtained gene-edited plants by leaf disc transformation.

[0035] Using the CRISPR-P 2.0 website, a more specific 20nt nucleotide sequence (SEQ ID No. 6) in the NtADH1 gene was selected as the guide sequence of CRISPR / Cas9, and the sequence fragment was connected, transformed and PCR amplified with the CRISPR / Cas9 vector (provided by Southwest University). The PCR-positive clones were sent to a sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-NtADH1 editing vector was obtained.

[0036] Using the CRISPR / Cas9-NtADH1 editing vector plasmid constructed in the previous step, genetic transformation and tissue culture were performed using Honghua Dajinyuan as an example to obtain plants with knockout editing of the tobacco amino acid transport-related gene NtADH1. The relevant experimental process is briefly described as follows.

[0037] After surface disinfection, tobacco seeds were spotted on MS culture medium. When they grew to 4 cotyledons (15-20 days), they were transferred into culture bottles containing MS solid culture medium. They were cultured for 35-40 days at 25±1°C, with a light intensity of 30-50 μmol / (m2·s) and a light duration of 16 h / d.

[0038] Take out the LBA4404 electroporation competent Agrobacterium cells stored at -80℃ and place them on ice for freezing and thawing. When the competent cells are just thawed, add 2μL of CRISPR / Cas9-NtADH1 editing vector plasmid, mix well, and place on ice. Then transfer the mixed competent cells to the pre-cooled electroporation cup, place the electroporation cup in the electroporator for transformation, add 0.5mL of YEB liquid medium and mix with the transformation liquid after the transformation is completed, and then place it on a shaker at 28℃ and 200rpm for 1.5-2h. Centrifuge the cells at 8000rpm and discard the supernatant, then suspend the cells with 200μL of YEB liquid medium, apply it on YEB solid medium containing 50mg / L rifampicin, 50mg / L streptomycin and 50mg / L kanamycin, and culture it in the dark at 28℃ for 2-3d.

[0039] In the clean bench, a tobacco leaf disc was made into a square leaf disc with a side length of 1 cm, and the Agrobacterium colonies containing the CRISPR / Cas9-NtADH1 editing vector were prepared into a suspended bacterial solution (OD600 = 0.6-0.8) using MS liquid. The tobacco leaf disc was soaked and infected with the suspended Agrobacterium bacterial solution for 10 minutes. The leaf disc was then placed on an MS solid medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA, 28°C, dark, and co-cultured for 3 days. After subculture, it was placed on a medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb + 50 mg / L Kan's MS solid medium, culture conditions are: 28 ℃ light culture 16h / d, light intensity 30-50μmol / (m2·s), 25 ℃ dark culture 8h / d, culture 45-60d, until differentiation buds are formed, change 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, The culture conditions are consistent with the differentiation culture conditions, and the culture is carried out for 8-14 days; the regenerated plants are cultured for rooting, the differentiated buds are cut off, and the rooting culture is carried out on the MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin. The culture conditions are consistent with the differentiation culture conditions, and the culture is carried out for 20-30 days. After the regeneration is transplanted to the flower pot, it is cultured, and then the leaves of the transformed plants are sampled, and the NtADH1-1F / NtADH1-1R primers are used to send the sequencer company for molecular detection to determine that the NtADH1 gene homozygous edited plants are obtained, and then self-pollination is performed to obtain seeds of the T0 generation. The homozygous edited T0 seeds are planted, and when the plants grow to 5-6 leaves, the leaves of the individual plants are sampled, and the Cas9F / Cas9R primers are used for molecular detection to determine that the NtADH1 gene is homozygous edited and T-DNA-free plants are obtained, and then self-pollination is performed to obtain seeds of the T1 generation (T2 generation).

[0040] NtADH1-1F: 5'-GCATGGTCTGTTTGGTGTGTG-3' (SEQ ID No. 7);

[0041] NtADH1-1R: 5'-TTTCTGTGGTGGTGCAACCT-3' (SEQ ID No. 8);

[0042] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 9);

[0043] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 10);

[0044] The application of the tobacco alcohol dehydrogenase gene NtADH1 of the present invention is to reduce the expression of the NtADH1 gene in tobacco plants, which can regulate the width of tobacco leaves at maturity. The commonly used methods for reducing gene expression or gene silencing in the prior art are applicable to the present invention.

[0045] Example 3

[0046] The seeds obtained by self-pollination of the T1 generation plants with homozygous knockout of the NtADH1 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 plant leaves were sampled and molecular detection was performed to determine that the T2 generation plants were homozygous edited by the NtADH1 gene without T-DNA. In the budding stage and the late topping stage, the main agronomic traits and botanical traits of the edited materials and controls were investigated with reference to the industry standard "YCT 142-2010 Tobacco Agronomic Traits Survey and Measurement Method". Including systematic measurement of the main agronomic traits (plant height, internode distance, stem girth, number of effective leaves, waist leaf length, waist leaf width, etc.) and botanical traits (plant type, leaf shape, leaf color, etc.) of the expanded population.

[0047] Comparison of waist leaf length and width of control (unedited) and NtADH1 gene homozygous edited tobacco plants at maturity (results as shown in Figure 1 Compared with the control, the width of the waist leaves of the homozygous NtADH1 gene-edited tobacco plants at maturity was significantly wider.

[0048] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. An application of tobacco alcohol dehydrogenase-related gene NtADH1 in regulating tobacco waist leaf width, characterized in that: The nucleotide sequence of the NtADH1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the NtADH1 encoded protein is shown in SEQ ID NO.

3. The application is to increase the width of tobacco waist leaves by knocking out the NtADH1 gene in tobacco.

2. The use according to claim 1, characterized in that: Through CRISPR / Cas9-mediated gene editing technology, a CRISPR / Cas9 editing vector for knocking out the NtADH1 gene was constructed, and after genetic transformation, homozygous T-DNA-free NtADH1 gene-edited tobacco plants were obtained.

3. The use according to claim 2, characterized in that: The method for creating tobacco plants with edited NtADH1 gene includes: (1) Selecting a more specific 20 nt nucleotide sequence in the NtADH1 gene as the guide sequence of CRISPR / Cas9, and connecting the sequence fragment with the CRISPR / Cas9 vector, transforming and PCR amplification detection, obtaining PCR positive clones, and obtaining the CRISPR / Cas9-NtADH1 editing vector; (2) The constructed CRISPR / Cas9-NtADH1 editing vector was used for genetic transformation and tissue culture, and after self-pollination, T2 generation tobacco plants with homozygous tobacco NtADH1 gene knockout editing without T-DNA were obtained.

4. The use according to claim 3, characterized in that: The more specific 20 nt nucleotide sequence in the NtADH1 gene in step (1) is shown as SEQ ID No.

6.

5. The use according to claim 4, characterized in that: The agronomic and botanical traits of the edited plants and control plants were investigated at the budding stage and the late topping stage. The results showed that the waist leaf width of the homozygous edited tobacco plants with NtADH1 gene knockout was significantly widened at maturity.

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