Application of tobacco Ntdedol-PP synthase gene in regulation and control of conventional chemical components and terpenoid content of tobacco

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to application of a tobacco Ntdedol-PP synthase gene in regulation and control of conventional chemical components and terpenoid content of tobacco, and the nucleotide sequence of the Ntdedol-PP synthase gene is shown as SEQ ID NO.1. The invention also relates to a preparation method of the Ntdedol-PP synthase gene. According to the invention, the CRISPR / Cas9 technology is utilized to obtain the tobacco plant edited by the Ntdedol-PP synthase gene with obvious metabolite content difference, and a new way is provided for regulating and controlling redistribution of the whole terpenoids in the tobacco and improving the aroma quality of the tobacco.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and particularly relates to an application of a tobacco Ntdedol-PP synthase gene in regulating the content of conventional chemical components and terpenoid compounds in tobacco. Background Art

[0002] Tobacco aroma is one of the important indicators to measure tobacco quality and availability. There are more than 700 compounds related to tobacco aroma, and most of the aroma substances in these more than 700 aroma substances are mainly derived from the secondary metabolites of tobacco plants. Among them, terpenoids are widely present in tobacco secondary metabolites, and most terpenoids play an important role in the formation of tobacco aroma substances. For example, pigments, as a terpenoid substance in tobacco, mainly include chlorophyll, carotenoids, etc. During the process of tobacco leaf modulation and aging, most of these pigments will decompose to produce aroma substances. These aroma substances have good aroma quality and low irritation, which can increase the aroma of tobacco products and are important components for forming the unique aroma of tobacco products. However, not all secondary metabolites can produce aroma substances, and not all secondary metabolites have a higher content, the better. For example, chlorophyll, the decomposed part will form aroma substances such as leaf alcohol and furan, while the undecomposed part will bring green smell to tobacco, affecting the quality of tobacco. Therefore, the higher the chlorophyll, the heavier the green smell of tobacco.

[0003] However, the content and types of secondary metabolites of wild tobacco plants are often not completely distributed in a direction that is beneficial to tobacco quality, which seriously affects the formation of the main aroma substances of tobacco products and further affects the quality of tobacco products. Therefore, overall regulation of the distribution of secondary metabolites in tobacco plants, especially the distribution of terpenoids, has become an urgent problem to be solved in order to improve the aroma quality of tobacco.

[0004] In the prior art, the content distribution of conventional chemical components and terpenoid compounds in tobacco is mainly adjusted through breeding and aging regulation methods, but such adjustment methods are time-consuming and labor-intensive, and the final adjustment results are often unsatisfactory.

[0005] The present invention is proposed to solve at least one of the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a Ntdedol-PP synthase gene, which is closely related to the tobacco leaf metabolic pathway. The present invention obtains a tobacco plant with a knockout of the Ntdedol-PP synthase gene by using CRISPR / Cas9-mediated gene editing technology. Compared with the control tobacco plant, the distribution of conventional chemical components and terpenoid compounds in the leaves of the gene-knockout tobacco plant of the present invention at the mature stage changes significantly, and the overall aroma quality of tobacco products is greatly improved. The present invention provides a new way to improve the aroma quality of tobacco.

[0007] The present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a tobacco Ntdedol-PP synthase gene, the nucleotide sequence of the Ntdedol-PP synthase gene is shown as SEQ ID NO.1.

[0009] The second aspect of the present invention provides tobacco plants edited with the Ntdedol-PP synthase gene according to the first aspect.

[0010] The third aspect of the present invention provides an application of the Ntdedol-PP synthase gene described in the first aspect in regulating the content of conventional chemical components and terpenoid compounds in tobacco.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) The present invention creates tobacco plants edited with the Ntdedol-PP synthase gene by genetic technology. Compared with wild-type tobacco plants, the tobacco plants edited with the Ntdedol-PP synthase gene obtained by the present invention show significant differences in the overall content distribution of terpenoids and the content distribution of main chemical components.

[0013] (2) The present invention uses CRISPR / Cas9 technology to obtain Ntdedol-PP synthase gene-edited tobacco plants with significant differences in metabolite content, providing a new way to regulate the redistribution of the entire terpene compounds in tobacco and improve the aroma quality of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Compile material sequence alignment for Ntdedol-PP of the present invention;

[0015] Figure 2 The photos of tobacco seedlings positive for Ntdedol-PP were edited for the present invention. DETAILED DESCRIPTION

[0016] The present invention is further described below by way of examples, but is not limited to these examples.

[0017] 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.

[0018] Example 1

[0019] The process of obtaining the tobacco Ntdedol-PP synthase gene is briefly described as follows:

[0020] 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 StrandcDNA Synthesis Kit (+gDNAwiper) reverse transcription kit reverse transcribes RNA into cDNA for later use.

[0021] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0022] F: 5'-TCGTCTTGACAGAAGCCACC-3' (SEQ ID No. 2)

[0023] R: 5'-CCTTCAGTAACCGCACTCGT-3' (SEQ ID No. 3)

[0024] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:

[0025] Amplification system (50 μL):

[0026]

[0027] 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.

[0028] The amplified product was purified and sequenced to obtain the tobacco Ntdedol-PP synthase gene, whose nucleotide sequence is shown in SEQ ID No.1.

[0029] Furthermore, the gene function "BGLU" was used to search the Chinese tobacco genome database to obtain the sequence information of the Ntdedol-PP synthase gene of the tobacco cultivar Honghua Dajinyuan. Using the CRISPR-P 2.0 website, a more specific 23nt nucleotide sequence (SEQ ID No. 4) in the Ntdedol-PP synthase 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, and the PCR positive clones were sent to a sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-Ntdedol-PP editing vector was obtained.

[0030] Example 2

[0031] Using the CRISPR / Cas9-Ntdedol-PP editing vector constructed in the previous step, genetic transformation and tissue culture were performed on Honghua Dajinyuan to obtain the edited plants with the Ntdedol-PP synthase gene knocked out in tobacco. The PCR sequencing results showed that the editing material inserted a base T at the 435th position of the gene (see Appendix Figure 1 ), resulting in frameshift mutations, indicating that the material has indeed undergone gene editing. Observation of plant growth showed that the edited plants grew and developed normally (see attached for photos of seedlings) Figure 2 ).

[0032] The relevant experimental process is briefly introduced as follows:

[0033] (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.

[0034] (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-Ntdedol-PP 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.

[0035] (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-Ntdedol-PP editing vector were prepared into a suspended bacterial liquid (OD600 = 0.6-0.8) using MS liquid.

[0036] 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.

[0037] Then, subculture was performed and placed in a medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb

[0038] +50mg / L Kan 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, culture The conditions were consistent with the differentiation culture conditions, and the culture was carried out for 8-14 days. For the rooting culture of the regenerated plants, the differentiated buds were cut off and inserted into the 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 the plants were cultured for 20-30 days. The regenerated plants were transplanted into pots and cultured. The leaves of the transformed plants were sampled and sent to a sequencing company for molecular testing using Ntdedol-PP-1F / Ntdedol-PP-1R primers to determine the homozygous edited plants of the Ntdedol-PP gene, which were then self-pollinated and harvested.

[0039] 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).

[0040] Ntdedol-PP-1F: 5'-CCCCAATTGCAACACAGCTG-3' (such as SEQ ID No. 5);

[0041] Ntdedol-PP-1R: 5'-CCACCCTCTAATCTCCCTCAAAC-3' (SEQ ID No. 6);

[0042] Cas9F: 5'-GATCTCCCAGTCACGACGTT-3' (SEQ ID No. 7);

[0043] Cas9R: 5'-TGTAACGGCGTCTGGCGGTGCGCTTC-3' (SEQ ID No. 8);

[0044] The leaves of the transformed gene-edited plants were sampled and sent to BGI for molecular testing to confirm that the Ntdedol-PP gene-edited plants were obtained.

[0045] Example 3

[0046] Liquid nitrogen sampling and grinding were performed on the leaves of the Ntdedol-PP synthase gene knockout plants and wild-type plants at the small cross stage and the large cross stage obtained in Example 2, and conventional chemical composition determination and LC-MS detection were performed. The test results are as follows:

[0047] As shown in Table 1, compared with the wild-type material of Honghua Dajinyuan, the total sugar content, reducing sugar content and potassium content of fresh tobacco leaves of the Ntdedol-PP synthase gene knockout material increased, while the total alkaloid content and chloride content showed a downward trend.

[0048] Table 1 Comparison of conventional chemical components of wild-type and gene knockout tobacco leaves

[0049]

[0050] Conclusion: It can be seen that the conventional chemical composition of the gene knockout material tobacco leaves in this example has obvious changes compared with the wild-type material, among which the total sugar, reducing sugar and potassium that have a positive effect on tobacco quality have increased to a certain extent, while the total alkaloids and chlorine that have a negative effect on tobacco quality have decreased to varying degrees.

[0051] Terpenoids are important aroma precursors in tobacco. In addition, the content of terpenoids in wild-type and gene knockout materials was measured. Compared with wild-type materials, 22 terpenoids in gene knockout materials showed significant changes, of which 14 were significantly upregulated and 8 were significantly downregulated.

[0052] Table 2 Comparison of significant differences in terpenoids between wild type and gene knockout materials

[0053]

[0054]

[0055] Furthermore, a smoking evaluation team consisting of 7 smoking evaluation experts was organized to conduct sensory evaluation on the tobacco leaves edited with the Ntdedol-PP synthase gene of the present invention and the tobacco leaves of the control tobacco strain CK (wild-type plants) according to Q / YNZYJ07.30-2015, and the average values ​​of the 8 items with significant changes were taken and recorded as follows:

[0056]

[0057] It can be seen that the aroma quality and aroma quantity of the tobacco leaves of the Ntdedol-PP synthase gene-edited strain of the present invention are significantly improved compared with the control, while the impurities are significantly reduced. It can be seen that the tobacco quality of the tobacco leaves of the Ntdedol-PP synthase gene-edited strain of the present invention is significantly improved.

[0058] 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.

[0059]

[0060]

Claims

1. A tobacco Ntdedol-PP synthase gene, characterized in that: The nucleotide sequence of the Ntdedol-PP synthase gene is shown in SEQ ID NO.

1.

2. A tobacco plant edited with the Ntdedol-PP synthase gene according to claim 1.

3. Use of the Ntdedol-PP synthase gene according to claim 1 in regulating the content of conventional chemical components and terpenoid compounds in tobacco.

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