Tobacco helicase gene and application thereof

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to a tobacco helicase gene and application thereof, the tobacco helicase gene is an RECQL1 gene, the sequence of the tobacco helicase gene is as shown in SEQ ID NO.1, and after the RECQL1 gene is translated, the sequence of a protein coded by the tobacco helicase gene is as shown in SEQ ID NO.3. A CRISPR / Cas9 editing carrier used for knocking out the RECQL1 gene is constructed through a CRISPR / Cas9 mediated gene editing technology, a tobacco plant with the RECQL1 gene knocked out is obtained after editing material creation and molecular detection and identification, and compared with a control tobacco plant, the obtained RECQL1 gene knocked out editing tobacco plant has the advantages that the yield of the tobacco plant is increased, and the yield of the tobacco plant is increased. And the contents of chlorogenic acid, total sugar and reducing sugar in the tobacco leaves in the mature period are obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco helicase gene and application thereof. Background Art

[0002] Chlorogenic acid is one of the important active substances in tobacco. In addition to its unique aroma, it will also decompose into pyrazine, pyridine and other chemicals with specific flavors when participating in the combustion of cigarettes, increasing the aroma volume and aroma quality of cigarette products. In addition, chlorogenic acid also has an important influence on improving the color of tobacco leaves and maintaining the stability of tobacco leaf quality. Studies have shown that chlorogenic acid is positively correlated with the grade of tobacco and its products. The higher the chlorogenic acid content, the better the quality of tobacco leaves and cigarettes. The content of carbohydrates has a significant effect on the appearance quality and sensory quality of tobacco, which mainly includes total sugar and reducing sugar. Within a certain range, the higher the content of carbohydrates in cigarette products, the better the taste and fuller the aroma. At the same time, the smoke is softer and sweeter. The appropriate content of carbohydrates can also reduce the irritation of smoke. Not only that, carbohydrates will also participate in non-enzymatic browning reactions during the baking and aging of tobacco leaves, and their content directly affects the color and appearance of tobacco leaves. Therefore, regulating the content of chlorogenic acid, total sugar and reducing sugar in tobacco is crucial to improving tobacco quality.

[0003] The existing technology mainly uses breeding methods to select tobacco varieties with appropriate chlorogenic acid, total sugar and reducing sugar content, but this traditional breeding method is time-consuming and labor-intensive, and the effect is unsatisfactory. With the development of molecular biology technology, new hope has been brought to the regulation technology of chlorogenic acid, total sugar and reducing sugar content in tobacco, but the technology of using genetic engineering to accurately regulate the content of chlorogenic acid polyphenols and sugar substances in tobacco has not been reported.

[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 helicase gene and its application to achieve the regulation of chlorogenic acid, total sugar and reducing sugar content in tobacco leaves, and to provide materials and references for tobacco helicase gene research and tobacco quality regulation.

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

[0007] The first aspect of the present invention provides a tobacco helicase gene, which is named RECQL1 gene, and its nucleotide sequence is shown in SEQ ID NO.1, including 5429bp bases.

[0008] Preferably, the CDS sequence of the RECQL1 gene is as shown in SEQ ID No. 2, comprising 1884 bp of bases.

[0009] Preferably, the RECQL1 gene sequence is translated, and the protein sequence encoded thereby is as shown in SEQ ID No. 3, which comprises 627 amino acids in total.

[0010] A second aspect of the present invention provides an application of a tobacco helicase gene in increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves, wherein the gene is a RECQL1 gene.

[0011] Preferably, the RECQL1 gene knockout plant is obtained by constructing a CRISPR / Cas9 editing vector for knocking out the RECQL1 gene through CRISPR / Cas9-mediated gene editing technology, and obtaining a plant with RECQL1 gene knockout after genetic transformation.

[0012] Preferably, the method for creating the plant with knockout of RECQL1 gene specifically comprises the following method:

[0013] Step (1): Select a more specific 23 nt nucleotide sequence in the RECQL1 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 and obtain a CRISPR / Cas9-RECQL1 editing vector;

[0014] Step (2): Using the constructed CRISPR / Cas9-RECQL1 editing vector plasmid, genetic transformation is performed to knock out the RECQL1 gene in the plant to obtain RECQL1 gene knockout plants.

[0015] Preferably, using the above technical solution, the contents of chlorogenic acid, total sugar and reducing sugar in the mature tobacco leaves of RECQL1 gene knockout edited plants are significantly higher than those 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 RECQL1 gene through CRISPR / Cas9-mediated gene editing technology, and obtains tobacco plants with RECQL1 gene knockout after editing material creation and molecular detection and identification. Compared with the control tobacco plants, the RECQL1 gene knockout edited tobacco plants obtained by the present invention have significantly increased contents of chlorogenic acid, total sugar and reducing sugar in mature tobacco leaves.

[0019] 2. The present invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out the RECQL1 gene to obtain edited tobacco materials with significantly increased chlorogenic acid, total sugar and reducing sugar contents in tobacco leaves. This provides a theoretical basis for further clarifying the regulation mechanism of tobacco polyphenol metabolism and sugar metabolism, and provides new genetic materials for breeding tobacco varieties with significantly changed polyphenol content and / or sugar content. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of chlorogenic acid content in tobacco leaves of gene-edited plants (RECQL1) and control plants (CK) at maturity ((*p<0.05));

[0021] Figure 2 This is a comparison chart of the relative contents of total sugar and reducing sugar in tobacco leaves of gene-edited plants (RECQL1) and control plants (CK) at maturity (*p<0.05). 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 helicase RECQL1 gene 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'-TCGTCTTGACAGAAGCCACC-3' (SEQ ID No. 5)

[0029] R: 5'-CCTTCAGTAACCGCACTCGT-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, and the tobacco helicase gene was obtained as RECQL1 gene, whose nucleotide sequence is shown in SEQ ID No.1, including 5429bp bases. The CDS sequence of the RECQL1 gene is shown in SEQ ID No.2, including 1884bp bases. The RECQL1 gene sequence was translated, and the protein sequence encoded by it was shown in SEQ ID No.3, including 627 amino acids. Further comparative analysis showed that the protein contained a highly homologous sequence and was highly conserved.

[0035] Example 2

[0036] Using the RECQL1 gene sequence obtained in Example 1, the present invention further constructed a gene editing vector, and obtained gene-edited plants by leaf disc transformation.

[0037] Using the CRISPR-P 2.0 website, a more specific 23nt nucleotide sequence (SEQ ID No. 4) in the RECQL1 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-RECQL1 editing vector was obtained.

[0038] Using the CRISPR / Cas9-RECQL1 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 knockout editing of the tobacco helicase gene RECQL1. The relevant experimental process is briefly described as follows:

[0039] (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.

[0040] (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-RECQL1 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.

[0041] (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-RECQL1 editing vector were prepared into a suspended bacterial liquid (OD600 = 0.6-0.8) using MS liquid.

[0042] 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.

[0043] 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. The callus was cultured on the same medium until the differentiated buds on the callus grew to 2-4 cm in height. The culture conditions were consistent with the differentiation culture conditions and the culture was carried out for 8-14 days. The regenerated plants were cultured for rooting. The differentiated buds were cut off and inserted into the MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for rooting. The culture conditions were consistent with the differentiation culture conditions and the culture was carried out for 20-30 days. The regenerated plants were transplanted into pots for culture. The leaves of the transformed plants were sampled and sent to a sequencing company for molecular detection using RECQL1-1F / RECQL1-1R primers to determine the homozygous edited plants of the RECQL1 gene. After that, self-pollination was performed for seed collection.

[0044] 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 RECQL1 gene and no T-DNA were obtained. After that, self-pollination was performed to obtain T1 generation seeds (T2 generation).

[0045] RECQL1-1F: 5'-GATAGAATTACCCGAATCCCATGCA-3' (SEQ ID No. 7);

[0046] RECQL1-1R: 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 to confirm that RECQL1 gene-edited plants were obtained.

[0050] The application of the tobacco helicase gene RECQL1 of the present invention is to reduce the expression of the RECQL1 gene in tobacco plants, which can regulate the content of chlorogenic acid, total sugar and reducing sugar 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 RECQL1 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 RECQL1 gene without T-DNA. Liquid nitrogen sampling and grinding were performed on the edited material and control tobacco leaves at maturity. The total sugar and reducing sugar contents were determined according to the tobacco industry standard YC / T159-2019 "Continuous flow method for determination of water-soluble sugars in tobacco and tobacco products", and the chlorogenic acid content was determined according to 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 chlorogenic acid, total sugar and reducing sugar contents in leaves of control (unedited) CK and RECQL1 gene homozygous edited tobacco plants at mature stage. Figure 1 , Figure 2 As shown, compared with the control, the contents of chlorogenic acid, total sugar and reducing sugar in the leaves of RECQL1 gene homozygous edited tobacco plants at mature stage were significantly increased.

[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]

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[0059]

[0060]

Claims

1. An application of a tobacco helicase gene in increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves, characterized in that: The tobacco helicase gene for increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves is the RECQL1 gene, and its sequence is shown in SEQ ID NO.

1. After the RECQL1 gene is translated, the sequence of the protein encoded by it is shown in SEQID NO.

3. Among them, the content of chlorogenic acid, total sugar and reducing sugar in the tobacco leaves of RECQL1 gene knockout edited plants at the mature stage is significantly higher than that of the control plants.

2. The use of a tobacco helicase gene according to claim 1 in increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves, characterized in that: RECQL1 gene knockout plants were obtained by using CRISPR / Cas9-mediated gene editing technology to construct a CRISPR / Cas9 editing vector for knocking out the RECQL1 gene, and plants with RECQL1 gene knockout were obtained after genetic transformation.

3. Use of a tobacco helicase gene according to claim 2 in increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves, characterized in that: The method for creating the plant with knockout of the RECQL1 gene specifically comprises: Step (1): Select a more specific 23 nt nucleotide sequence in the RECQL1 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-RECQL1 editing vector; Step (2): Using the constructed CRISPR / Cas9-RECQL1 editing vector plasmid, genetic transformation is performed to knock out the RECQL1 gene in the plant to obtain RECQL1 gene knockout plants.

4. The use of a tobacco helicase gene according to claim 2 in increasing the content of chlorogenic acid, total sugar and reducing sugar in tobacco leaves, characterized in that: The variety of tobacco is Honghua Dajinyuan.

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