Tobacco dihydrodiphosphate synthase gene and application thereof

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to a tobacco dehydrogenation diphosphate monoglyceride synthase gene and application thereof, the tobacco dehydrogenation diphosphate monoglyceride synthase gene for regulating and controlling the tobacco plant height and the tobacco leaf total sugar and reducing sugar content is an At5g58770 gene, and the sequence of the gene is shown as SEQ ID NO.1. After the At5g58770 gene is translated, the content of the tobacco leaf total sugar and reducing sugar in the tobacco leaf total sugar and reducing sugar in the tobacco leaf total sugar and reducing sugar in the tobacco leaf total sugar and reducing sugar in the tobacco leaf total sugar and reducing sugar in the tobacco leaf total sugar can be regulated and controlled. And the sequence of the protein coded by the gene is shown as SEQ ID NO.3. A CRISPR / Cas9 editing carrier used for knocking out the At5g58770 gene is constructed through a CRISPR / Cas9 mediated gene editing technology, and a tobacco plant with the At5g58770 gene knocked out is obtained after editing material creation and molecular detection and identification. Compared with a control tobacco plant, the editing tobacco plant with the At5g58770 gene knocked out has the advantages that the normal plant height and the topped plant height are remarkably reduced, and the content of total sugar and reducing sugar in tobacco leaves in the mature period is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene and an application thereof. Background Art

[0002] Plant height is an important agronomic trait in tobacco growth and development. In modern cultivation, moderate plant height can improve the efficiency and adaptability of mechanical harvesting, making production more scaled and standardized, thereby indirectly affecting the overall quality and cost control of tobacco leaves. In the process of tobacco cultivation, by affecting photosynthesis and nutrient distribution, it can indirectly affect the content and distribution of chemical components in tobacco leaves, such as total sugar, nicotine and other compounds, and further affect the combustibility and taste of tobacco leaves. The carbohydrate components in tobacco include total sugar and reducing sugar. High-sugar tobacco leaves will produce more aroma substances when burned, which can improve the taste and aroma of tobacco, making the smoke softer and sweeter. Appropriate sugar can reduce the irritation of smoke. During the tobacco leaf baking and aging process, sugar can also participate in non-enzymatic browning reactions, affecting the color and appearance of tobacco leaves.

[0003] The existing technology mainly uses breeding methods to select tobacco varieties with moderate plant height and appropriate leaf total sugar and reducing sugar content, but the traditional breeding methods are time-consuming and labor-intensive, and the results are not satisfactory. With the development of molecular biology technology, new hope has been brought to the technology of regulating tobacco plant height and sugar content, but there is no report on the technology of accurately regulating tobacco plant height and sugar content by genetic engineering.

[0004] The present invention is proposed to solve at least one of the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene and its application, so as to achieve the regulation of tobacco plant height, total sugar and reducing sugar content of tobacco leaves, effectively improve tobacco quality and achieve cost reduction and efficiency improvement.

[0006] The present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a tobacco dehydrogenated dihydrogen glycerol diphosphate synthase gene, which is named At5g58770 gene, and its nucleotide sequence is shown in SEQ ID NO.1, including 2157bp bases.

[0008] Preferably, the CDS sequence of the At5g58770 gene is as shown in SEQ ID No. 2, comprising 915 bp bases.

[0009] Preferably, the At5g58770 gene sequence is translated, and the encoded protein sequence is shown in SEQ ID No. 3, which includes 304 amino acids in total.

[0010] The second aspect of the present invention provides a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene and its application, wherein the gene is the At5g58770 gene.

[0011] Preferably, the At5g58770 gene knockout plant is obtained by constructing a CRISPR / Cas9 editing vector for knocking out the At5g58770 gene through CRISPR / Cas9-mediated gene editing technology, and obtaining a plant with At5g58770 gene knockout after genetic transformation.

[0012] Preferably, the method for creating a plant with At5g58770 gene knockout specifically includes the following method:

[0013] Step (1): Select a more specific 23 nt nucleotide sequence in the At5g58770 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-At5g58770 editing vector;

[0014] Step (2): Using the constructed CRISPR / Cas9-At5g58770 editing vector plasmid, genetic transformation is performed to knock out the At5g58770 gene in the plant to obtain At5g58770 gene knockout plants.

[0015] Preferably, using the above technical solution, the edited tobacco plants with At5g58770 gene knockout have significantly reduced normal plant height (i.e., plant height without topping) and topping plant height compared to the control tobacco plants, and the total sugar and reducing sugar contents in the tobacco leaves at maturity are significantly increased.

[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 At5g58770 gene through CRISPR / Cas9-mediated gene editing technology, and obtains tobacco plants with At5g58770 gene knockout after editing material creation and molecular detection and identification. Compared with the control tobacco plants, the normal plant height and topping plant height of the At5g58770 gene knockout edited tobacco plants obtained by the present invention are significantly reduced, and the total sugar and reducing sugar content in the mature tobacco leaves are significantly increased.

[0019] (2) The present invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out the At5g58770 gene to obtain edited tobacco materials with significantly reduced normal plant height and topping plant height, and significantly increased total sugar and reducing sugar contents in mature tobacco leaves. This provides a theoretical basis for further elucidating the tobacco plant height regulation mechanism and sugar metabolism regulation mechanism, and provides new genetic materials for breeding tobacco varieties with significantly changed plant height and / or sugar content. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of plant height test between gene-edited plants (At5g58770) and control plants (CK) (*p<0.05, **P<0.01);

[0021] Figure 2 This is a comparison of the relative contents of total sugar and reducing sugar in tobacco leaves of gene-edited plants (At5g58770) and control plants (CK) at maturity (*p<0.05, **P<0.01). DETAILED DESCRIPTION

[0022] The present invention is further described below by way of examples, but is not limited to these examples.

[0023] 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.

[0024] Example 1

[0025] This example briefly introduces the process of obtaining the tobacco dehydrogenated dihydrogen phosphate glycerol synthase gene At5g58770 as follows.

[0026] 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.

[0027] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0028] F: 5'-TGCCTTAGGTTACGAAGCGG-3' (SEQ ID No. 5)

[0029] R: 5'-ATACGACGACATTGTGCGGT-3' (SEQ ID No. 6)

[0030] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:

[0031] Amplification system (50 μL):

[0032]

[0033] 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.

[0034] The amplified product was purified and sequenced to obtain the tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene, which is the At5g58770 gene, and its nucleotide sequence is shown in SEQ ID NO.1, including 2157bp bases. The CDS sequence of the At5g58770 gene is shown in SEQ ID No.2, including 915bp bases. The At5g58770 gene sequence was translated, and the protein sequence encoded by it is shown in SEQ ID No.3, including 304 amino acids in total.

[0035] Further comparative analysis showed that the protein contained highly homologous sequences and was highly conserved.

[0036] Example 2

[0037] Using the tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene At5g58770 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.

[0038] Using the CRISPR-P 2.0 website, a more specific 23nt nucleotide sequence (SEQ ID No. 4) in the At5g58770 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-At5g58770 editing vector was obtained.

[0039] Using the CRISPR / Cas9-At5g58770 editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, genetic transformation and tissue culture were carried out to obtain plants with knockout editing of tobacco dehydrogenated dihydrogen phosphate glycerol synthase gene At5g58770. The relevant experimental process is briefly introduced as follows.

[0040] (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.

[0041] (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-At5g58770 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 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 medium, apply to YEB solid 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.

[0042] (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-At5g58770 editing vector were prepared into a suspended bacterial liquid (OD600 = 0.6-0.8) using MS liquid.

[0043] 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.

[0044] 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. Cultivate until the differentiated buds on the callus tissue grow to 2-4 cm in height, the culture conditions are consistent with the differentiation culture conditions, and culture for 8-14 days; for rooting culture of regenerated plants, cut off the differentiated buds, insert them into MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for rooting culture, the culture conditions are consistent with the differentiation culture conditions, culture for 20-30 days, regenerate and transplant to flower pots for culture, and then take samples of the transformed plant leaves, use At5g58770-1F / At5g58770-1R primers, send them to a sequencing company for molecular detection, determine that the At5g58770 gene homozygous edited plants are obtained, and then self-pollinate for seed collection.

[0045] The homozygous edited T0 generation seeds were planted, and when the plants grew to 5-6 leaves, the leaves of individual plants were sampled and molecular detection was performed using Cas9F / Cas9R primers to determine whether plants with homozygous editing of the At5g58770 gene and no T-DNA were obtained. After that, self-pollination was performed to obtain T1 generation seeds (T2 generation).

[0046] At5g58770-1F: 5'-GATACGCTTACCCGAATCCCATAGC-3' (SEQ ID No. 7);

[0047] At5g58770-1R: 5'-AAATAGCTCGGATATGGGTCGTT-3' (SEQ ID No. 8);

[0048] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 9);

[0049] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 10);

[0050] The leaves of the transformed gene-edited plants were sampled and sent to BGI for molecular testing, which confirmed that the At5g58770 gene-edited plants were obtained.

[0051] The application of the tobacco dehydrogenated dihydrogen phosphate glycerol synthase gene At5g58770 described in the present invention is to reduce the expression of the At5g58770 gene in tobacco plants, which can regulate tobacco plant height, total sugar and reducing sugar content in tobacco leaves. The methods for reducing gene expression or gene silencing commonly used in the prior art are applicable to the present invention.

[0052] Example 3

[0053] The seeds obtained by self-pollination of the T1 generation plants with homozygous knockout of the At5g58770 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 At5g58770 gene without T-DNA. The plant height was measured during the flowering period and one week after topping, and the liquid nitrogen sampling and grinding of the edited material and the control tobacco leaves were carried out at maturity. The total sugar and reducing sugar content were determined in accordance with the tobacco industry standard YC / T 159-2019 "Continuous Flow Method for Determination of Water-soluble Sugars in Tobacco and Tobacco Products".

[0054] Comparison of normal plant height and topping plant height, and total sugar and reducing sugar content in leaves at maturity between control (unedited) CK and homozygous At5g58770 gene-edited tobacco plants (results shown in Figure 1 , Figure 2 Compared with the control, the normal plant height and topping plant height of At5g58770 gene homozygous edited tobacco plants were significantly reduced, and the total sugar and reducing sugar contents in mature leaves were significantly increased.

[0055] 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.

[0056]

[0057]

[0058]

Claims

1. An application of a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene in regulating tobacco plant height, total sugar and reducing sugar content in tobacco leaves, characterized in that: The tobacco dehydrogenated dihydrogen phosphate diglyceride synthase gene for regulating tobacco plant height, total sugar and reducing sugar content in tobacco leaves is the At5g58770 gene, and its sequence is shown in SEQ ID NO.

1. After the At5g58770 gene is translated, the sequence of the protein encoded by it is shown in SEQ ID NO.

3. Among them, the normal plant height and topping plant height of the At5g58770 gene knockout edited plant are significantly lower than those of the control, and the total sugar and reducing sugar contents in the tobacco leaves at maturity are significantly higher than those of the control.

2. The use of a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene according to claim 1 in regulating tobacco plant height, total sugar and reducing sugar content in tobacco leaves, characterized in that: At5g58770 gene knockout plants were obtained by constructing a CRISPR / Cas9 editing vector for knocking out the At5g58770 gene through CRISPR / Cas9-mediated gene editing technology, and obtaining plants with At5g58770 gene knockout after genetic transformation.

3. The use of a tobacco dehydrogenated dihydrogen phosphate glycerol synthase gene according to claim 2 in regulating tobacco plant height, total sugar and reducing sugar content in tobacco leaves, characterized in that: The method for creating a plant with At5g58770 gene knockout specifically includes: Step (1): Select a more specific 23 nt nucleotide sequence in the At5g58770 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-At5g58770 editing vector; Step (2): Using the constructed CRISPR / Cas9-At5g58770 editing vector plasmid, genetic transformation is performed to knock out the At5g58770 gene in the plant to obtain At5g58770 gene knockout plants.

4. The use of a tobacco dehydrogenated dihydrogen diphosphate glycerol synthase gene in regulating tobacco plant height, total sugar and reducing sugar content in tobacco leaves according to claim 2, characterized in that: The variety of tobacco is Honghua Dajinyuan.

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